POSTER ABSTRACTS 40th Annual Oak Ridge …clinchem.aaccjnls.org/content/suppl/2008/10/09/54.11...40th Annual Oak Ridge Conference Breakthrough Technologies for Clinical Diagnostics

POSTER ABSTRACTS

40th Annual Oak Ridge Conference Breakthrough Technologies for Clinical Diagnostics

April 17 & 18, 2008 ‐ San Jose, California

Posters 1 & 2 Withdrawn

Poster 3 Ultra‐sensitive time‐resolved luminescent lateral flow assay technology. Song X1, Knotts M2, Huang L1. 1Kimberly Clark, Roswell, Ga. (2Georgia Institute of Technology, Atlanta, Ga.) Membrane-based lateral flow immunoassay (LFIA) technology has seen amazing commercial success because of its simplicity, low cost and user friendliness. The technology is particularly suitable for point-of-care and over-the-counter test markets. Much effort has been made to integrate different detect techniques into lateral flow immunoassays for analyte quantification. The reported techniques include absorbance (colored particles), fluorescence (fluorescent particles), magnetic field (magnetic particles) and surface enhanced Raman scattering (nanoparticles). For instance, fluorescence-based LFIA technology has been recently commercialized that can accomplish analyte quantification with good detection sensitivity and ease of use. However, the system is relatively expensive and can not meet detection sensitivity requirements for some analytes. Here we report a detection technology that integrates highly sensitive time-resolved luminescence technique into LFIA platform to achieve remarkable detection performance with low cost. We have developed very bright, surface-functionalized, and mono-dispersed phosphorescent nanoparticles of long lifetime under ambient conditions. The phosphorescent nanoparticles have been used to conjugate with monoclonal antibody for C-reactive protein (CRP), an inflammatory biomarker. Lateral flow immunoassay devices have been developed using the conjugate for highly sensitive detection of CRP. We have also developed a low cost time-resolved luminescent reader for the time-resolved luminescent LFIA devices. The reader does not use expensive band pass filter and still provide very low detection background and high detection sensitivity on solid substrates such as nitrocellulose membranes. The technology should find a number of applications, ranging from clinical diagnostics, detection of chemical and biological warfare agents, to food and environmental monitoring.

Poster 4 Specificity and sensitivity of a novel anti‐citrullinated peptide antibody assay in a four‐analyte multiplexed Quantispot™ rheumatoid arthritis test system. Biessels P, Mudumba S, Kahama A, Ding, S‐F, Ling M, Lea P. (QI Diagnostics, Toronto, Ontario, Canada.) Rheumatoid Arthritis affects about 1% of the general population. Early detection and monitoring are advantageous to patients for better management of this disease. A sensitive anti-CCP antibody assay along with the simultaneous detection assays of all three rheumatoid factor (RF) analytes, i.e. RF-IgA, -IgG and -IgM have been developed. Using a very small amount of serum sample from a patient, SQI’s multiplex microarray fluorescent immunoassay offers simultaneous quantitative /semi-quantitative and positive/negative test results for 4 analytes, i.e. anti-CCP antibody, rheumatoid factor IgA, IgG and IgM for each micro-array sample well on a 96-well microtiter-formatted. Detection is achieved by sensitive fluorescent tagged anti-bodies captured on micro-array spots for reading in a micro-array scanner. Each of the results comes with full reference standard quality control applied in every sample well. In studying the performance of the anti-CCP antibody assay of this test system, we report that in one of the validation studies conducted by SQI scientists, of human serum samples obtained from 158 European and North American subjects, 118 Rheumatoid Arthritis sufferers and 40 normal subjects were tested using SQI’s anti-CCP antibody diagnostic test. The results demonstrated that the test achieved

78% sensitivity and 97.5% specificity, a set of performance outcomes equivalent to currently accepted single analyte anti-CCP tests (Bizarro et al., Clin Chem 2007,53(8):1527-33). Comparable analytical and diagnostic characteristics of eleven commercial, second and third generation immuno-enzymatic methods for the detection of antibodies to citrullinated proteins were also compared. In a follow up study by SQI scientists, the sensitivity and the specificity were further compared with a leading competitor’s commercial CCP assay. With specificity pegged at 95%, the SQI anti-CCP assay was 4.2% more sensitive relative to the compared method. Similar method concordance has also demonstrated full comparability between each of the simultaneous rheumatoid factors as tested in this multiplexed test and with the corresponding predicate test. This novel anti-CCP antibody assay, together with the three other assays that constitute this multiplex profile testing system, provide quantitative and calibrated detection of rheumatoid arthritis with high sensitivity and specificity, while maintaining significant cost-effectiveness and time-savings.

Poster 5 Microfluidic immunoassays in polymeric chips. Chang WWP1, Li C1, Bousse L1, Kawabata T1, Shih Y1, 2Kagebayashi C, 2Kurosawa T, Wada HG1, 2Watanabe M, and 2Satomura S. (1Wako Pure Chemical Industries, Mountain View, Calif., and 2 Wako Pure Chemical Industries, Osaka Research Laboratories, Amagasaki, Japan.) A quantitative method for performing rapid immunoassays was recently developed utilizing microfluidic channels made on quartz substrate. Instead of pipetting, the reagent and sample volumes are quantitatively aliquoted by microfluidic disposition in zones of a chip channel. After loading reactants by pressure, an electrokinetic analyte transport method (EATA1) enables on-chip reaction and concentration of reaction products prior to separation by capillary gel electrophoresis. For the clinical chemistry application of this method, a disposable microfluidic device was fabricated in thermo-plastic polymer to deliver high sensitivity, precision, and accuracy at a fraction of the cost of the quartz chip. Here, we demonstrate the performance of a diagnostic application using the AFP-L3 assay in the polymeric chips. AFP-L3 is an isoform specific for liver cancer (HCC)2. All isoforms of alpha-fetoprotein (AFP) in human serum are sandwiched by two specific monoclonal antibodies, one coupled with DNA and the other with fluorescent dye. The highly charged DNA conjugate quickly migrates through the channel and reacts with the complex of AFP and dye-labeled conjugate. The DNA-coupled antibody complex concentrates and separates from unreacted components via isotachophoresis (ITP), and after ITP stacking of the immunocomplexes, the system switches to gel electrophoresis. Utilizing differential binding affinity to Lens culinaris agglutinin (LCA), the HCC specific isoform (AFP-L3) is separated from AFP-L1 by capillary affinity gel electrophoresis. The ratio of AFP-L3 to the total AFP, expressed in %L3, has been established as a sensitive marker for patients at risk for HCC3. A detection limit of 1 pM AFP, linear concentration response up to 10 nM, and reproducibility in a range of 3 to 10% CV were achieved in pre-clinical studies using pooled human sera spiked with AFP-L1 and L3 as the sample. These results show that disposable polymeric chips can achieve similar performance to quartz chips for a clinically relevant, microfluidic immunoassay. 1Kawabata T, et al, manuscript submitted to Electrophoresis 2007, 2Taketa K, et al., Gastroenterology (1990) 99:508-518, 3Oka H, et al., J Gastroenterol Hepatol (2001) 16:1378-1383.

40th Annual Oak Ridge Conference – April 17–18, 2008 Poster Abstracts

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Poster 6 Flow‐enhanced spread of localized infections in microchannels. Zhu Y1, Warrick J2, Haubert K2, Beebe DJ2, Yin J1. (1Department of Chemical and Biological Engineering, University of Wisconsin‐Madison, Madison, Wis., and 2Department of Biomedical Engineering, University of Wisconsin‐Madison, Madison, Wis.)

Although the plaque assay has long served as the "gold standard" to measure virus infectivity and test antiviral drugs, the assay is labor-intensive, lacks sensitivity, uses excessive reagents, and is hard to automate. Recent modification of the assay to exploit flow-enhanced virus spread with quantitative imaging has increased its sensitivity. Here we perform flow-enhanced infection assays in microscale channels, employing passive fluid pumping to load host cells into the channel, inoculate cell monolayers with virus, and drive infection spread. We tested an antiviral drug (5-fluorouracil) against vesicular stomatitis virus infections of BHK cells and attained a 2-fold improvement in sensitivity using 80-fold less drug, relative to the plaque assay. These advantages, together with the reduction in scale, simplified fluid handling, and image-based quantification, will facilitate infection measurements for high-throughput drug screening, seroconversion testing for epidemiology, and patient-specific diagnosis and treatment of infectious diseases.

Poster 7 Circulating levels of CAIX oncoprotein in human serum and plasma and elevations in cancer patient serum. Hamer PJ, Pierce K, Brown‐Shimer S, Ramirez J, Trivedi D, Newman D, Carney WP, (Siemens HealthCare Diagnostics/Oncogene Science Group, Cambridge, Mass.)

Background: The transmembrane protein known as CAIX (CA9) is a member of the enzyme family of carbonic anhydrases, which have the ability to catalyze the reversible hydration of carbon dioxide to carbonic acid, leading to a decrease in pH. CAIX gene expression is up-regulated under hypoxic conditions and is under the control of the transcription factor HIF-1. In tumors, an increase of CAIX expression is believed to contribute to the acidic microenvironment. Normal expression of CAIX protein in humans is restricted to primarily gastric, intestinal and liver mucosa. Using immunohistochemistry (IHC) and RT-PCR, increased levels of CAIX protein have been detected in a variety of tumors, including kidney, cervix, lung, bladder, colon, breast, liver, gall bladder, and pancreas. Methods: The ELISA that we have developed is a sandwich-type microtiter plate immunoassay that uses a monoclonal capture antibody (V10) and a biotinylated monoclonal antibody as the detector (M75). Diluted samples are incubated in the wells to allow binding of the antigen. Biotinylated detector antibody is then added, followed by a streptavidin–HRP conjugate. The intensity of colored product generated from the catalysis of substrate is proportional to the amount of CAIX oncoprotein present in the sample. Results: An ELISA recently developed by Oncogene Science is able to measure circulating levels of CAIX oncoprotein and can be used to evaluate the clinical value of CAIX. We have evaluated serum and plasma samples both from normal individuals and from a variety of cancer patients. Our preliminary studies show the mean level of circulating CAIX oncoprotein in male serum (n = 145) to be 571 pg/mL and, in male plasma (n = 67), 374 pg/mL. In female serum (n = 52), CAIX levels were 506 pg/mL and, in female plasma (n = 32), 247 pg/mL. Combined male and female data show a serum cutoff of 555 pg/mL, (n = 197), while the total for plasma (n = 99) is 341 pg/mL. Analysis of various cancer specimens showed CAIX elevations in significant percentages of specimens: 44% of the renal cell carcinoma specimens (n = 129 tested), 42% in ovarian cancer (n = 50 tested), 24% in colon cancer, (n = 34 tested), 12% in non-small-cell lung cancer (n = 133 tested), and 22% in metastatic breast cancer (n = 147 tested). Conclusion: This is the first commercially available ELISA to measure circulating levels of the CAIX oncoprotein.* Since elevated levels of CAIX were observed in samples from patients with various cancers, this indicates that this ELISA may have clinical utility. With the development of CAIX-targeted therapies, the ELISA may be useful in identifying patients for therapy as well as in monitoring the rise and fall of circulating CAIX. This may assist in assessing the efficacy of CAIX-targeted therapies or the efficacy of traditional therapies in CAIX-positive tumors. * For research use only. Not for use in diagnostic procedures.

Poster 8 Withdrawn

Poster 9 Digital magnetic beads for multiplexed assays. Ho WZ, Collins J, Tajbakhsh J, Low P, Vera L. Maxwell (Sensors Inc., Santa Fe Springs, Calif.)

Bead technology is ideally suited to a wide range of multiplexed assays throughout the drug-discovery and diagnostics fields, as well as basic research. Digital Magnetic Beads (DMB) technology, which combines two advanced technologies, lithographic digital barcodes and molecular chemistry, is designed to significantly improve the isolation and identification capacities of molecular diagnostics. The polymer-based digital microbeads (50-300µm), fabricated by photolithography, have 10-digit (1,024) multiplexed barcodes. The beads’ paramagnetic property makes the technology well suited for washing, purification, and automation protocols using a 96-well microplate format. The beads can be functionalized with nucleic acids, proteins or other probe molecules, allowing large multiplexed assays to be carried out in homogeneous or heterogeneous media. Because of its simplicity, scalability, and low cost, DMB technology enables new probes to be added quickly to multiplexed assays in a single tube. The digital readout system includes a microfluidic transducer, a bar-code reader and fluorescence detection. The microfluidic transducer consists of a sheath flow system to provide steady and stable high speed bead flow (0.5 m/sec) through the optical detection area. Due to the high-contrast barcode signal, decoding optics is relatively simple. The beads support both high speed and high throughput (< 30 seconds for thousands of beads) for sensitive target detection. Reactions are all-in-one and can be carried out in small volumes, saving labor, reagents and consumables. The digital beads can also be decoded and fluorescently detected by image processing in a microplate.

Poster 10 A sensitive, Raman‐scattering based method for peroxidase activity detection as applied to enzyme‐linked immunoassay. Siegel N, Kundu S, Ginsburgh C. (Sword Diagnostics, Chicago, IL.)

Raman spectroscopy has not enjoyed substantial application as a biochemical detection technique compared with other methodologies such as absorbance, fluorescence, etc. For Raman to become a practical and broadly based application it is necessary to demonstrate its ability to be easily adapted by existing applications, that it is quantifiable and performs as well or better than techniques currently available. A system is presented here that has been developed for detecting peroxidase (including antibody-conjugated) activity lending it to broad application in existing immunoassay systems. The chemistry system for peroxidase detection consists of three parts, 5-aminosalicylic acid, 4-hydroxybenzyl alcohol in the presence of peroxidase (horseradish) and Sodium Hydroxide as a developing agent. A 532 nm Raman spectrometer is used to collect signal from a 250 microliter sample volume. A quantitative relationship is shown between Raman signal and Peroxidase content applying logistic curve fitting. This quantitative system has resulted in greater sensitivity than other Peroxidase detection. For example, the Raman based detection system was substituted into a commercially available 96-well plate immunoassay for human IL-2. Both the TMB substrate recommended by the kit manufacturer and the Raman chemistry system were run together and the results of the calibration compared as shown below. As can be seen, this kit without further optimization of the reagents, substituting the Raman chemistry alone showed a 25-fold increase in sensitivity of detection and larger signal to blank ratio across the entire calibration curve. In summary a novel and practical, sensitive detection method for peroxidase-linked immunoassays using Raman spectroscopy is demonstrated.

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Poster 11 Withdrawn

Poster 12 Demonstration of improved light harvesting with novel silver plated magnetic particles in luminescent applications. Zhelev P1, He J2, Allard M1, Thorogood S2, Carville D1, Gawad Y1. (1CardioGenics Inc., Mississauga, ON, and 2LuXSpheres Inc., Mississauga, ON.) Due to the ease of manipulation and large surface area, paramagnetic particles are currently the solid phase of choice in automated clinical lab analyzers. In the quest for an improved system and immunoassay sensitivities, developers employ light measurements with large light output and improved detection. However, paramagnetic particles represent the largest source of light signal loss in binding assays that employ them. We have developed paramagnetic particles optimized for light measurements as a solid phase for binding assays. This includes improved process for silver plating and following polymer encapsulation of the particles in order to improve the light harvesting abilities. We compared the developed particles to non-silver coated black counterparts in binding assays that employ light measurements. Antibodies were conjugated to the surface carboxyl groups by using 2 step carbodiimide conjugation procedure. Paramagnetic particles of various sizes were used. The conjugated antibodies were detected by bioluminescent immunoassay and commercial automated injection luminometer. Magnetic particles were used at 1% concentration. Results (in Relative Light Units) are summarized in the table as follows: Conclusions: In immunoassay bioluminescent measurements, silver coated magnetic particles showed 5-7 fold improvement in the collected light signal, although the antibody coating efficiency of both was the same. This improvement in the measured light signal was evident using color-converted paramagnetic particles of various sizes in comparison with the black counterparts. The differential effect was maintained along the calibration curve. The improved light harvesting effect of color-converted paramagnetic particles will enable assay developers to improve sensitivity as well as the dynamic range of assays that employ the novel magnetic particles in light measurements.

Poster 13 Comparison of novel silver plated magnetic particles versus commercially available magnetic particles by chemiluminescent assay. Zhelev P1, He J2, Allard M1, Thorogood S2, Carville D1, Gawad Y1. (1CardioGenics Inc., Mississauga, ON, and 2LuXSpheres Inc., Mississauga, ON.) Paramagnetic particles are widely used as a solid support in automated clinical lab analyzers, giving the advantages of large surface area and high level of automation. In the race for improved immunoassay sensitivities and system performance most developers are using light as a signal in the form of chemiluminescence. However, currently used paramagnetic particles were not historically optimized for light detection, being the largest source of light signal loss in binding assays that employ them. Employing light measurement binding immunoassays, we compared the measured light signal of several commercial paramagnetic particles to recently developed silver-coated paramagnetic particles. Antibodies were actively conjugated to the surface carboxyl groups of various beads by using a 2 step

carbodiimide conjugation procedure as recommended by various suppliers as optimal. The conjugated antibodies were detected by chemiluminescent assay that employs Lumigen PS-Atto substrate and commercial automated injection luminometer. Particles were tested at 0.25% concentration. Results are summarized in the table with the collected light expressed in relative light units (RLU). Conclusions: Although commercial particles of various sizes and density of carboxyl groups were employed, in immunoassay chemiluminescent measurements, the silver coated magnetic particles shows a 3-7 fold improvement in the collected light signal over the paramagnetic particles from various commercial sources. This improvement in the measured light signal was evident, regardless of the amount of generated light. A clear correlation exists between the amount of paramagnetic material used and the collected light signal indicating that the major effect is due to light harvesting effect rather than antibody density.

Poster 14 Novel NMR‐based platform for in vitro diagnostics assays. Taktak S, Wong S, Mozeleski B, Kumar S, Lowery T, Prado P, and Foster K. (T2 Biosystems, Cambridge, Mass.) T2’s proprietary NanoDxTM technology for in vitro diagnostics is based on the ability of modified superparamagnetic nanoparticles to assemble in the presence of a target analyte thus inducing a change in NMR parameters. The NanoDx technology eliminates the need for complex preparation of a sample. The nanoparticles typically have a superparamagnetic metal core surrounded by a polymer layer decorated with multiple analyte-specific binding agents. The nanoparticle platform can be used with a variety of binding agents such as antibodies, nucleic acids and small molecules.

The relationship between T2 and nanoparticle clustering is understood from a theoretical perspective and is unique for this type of nanoparticles. For functional assays, both dispersed and clustered states of nanoparticles remain suspended in solution. The effect of clustering on surrounding water molecules induces a change in the observed transverse relaxation time of the water protons; clustered nanoparticles have short T2 values and dispersed nanoparticles have long T2 values (Scheme 1). The approach for sensing with analyte-induced nanoparticle clustering reported via T2 relaxation rates has been termed magnetic relaxation switch (MRSw) technology and has been demonstrated for a wide variety of analytes and assay configurations (1).

Recent advances in the readout methodology and data analysis as well as in the formulation of the particles and assays have led to enhanced performance of the assays and enabled its use in clinical diagnostics. Single-step measurement of multiple samples in a single coil can be achieved by, for example, bi-exponential analysis of the observed T2 relaxation time curve. Data analysis and internal calibration of the sensor can be achieved by looking at multiple NMR parameters, including T2/T1 and T2 replication interrogation methods.

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Scheme 1. Magnetic relaxation switches detect analyte (green) by transitioning between a dispersed state and clustered state in the presence of analyte. The nanoparticles (30 nm DIA) consist of a monocrystalline iron oxide core (orange) with a cross-linked dextran coating (gray) and attached moieties that selectively bind the desired analyte. (1) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Hogemann, D.; Weissleder, R. Magnetic relaxation switches capable of sensing molecular interactions. Nature Biotech (2002), 20(8), 816-820.

Poster 15 Neutrophil gelatinase‐associated lipocalin (ngal) as an early marker of acute renal injury in ICU multitrauma patients. Makris K1, Evodia E2, Ntetsika K1, Markou N2, Dimopoulou E2, Drakopoulos I1, Baltopoulos G2, Rizos D3, Haliassos A4.(1Clinical Biochemistry Department KAT General Hospital, Kiffissia, Athens Greece, 2University of Athens, Intensive Care Unit, KAT General Hospital, Kiffissia, Athens Greece, 3University of Athens, Hormons Laboratory, Aretaieion Hospital, Athens Greece, 4Diamedica SA, Athens, Greece.) Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, is readily excreted and detected in urine, due to its small molecular size (25kDa) and resistance to degradation. NGAL is highly accumulated in the human kidney cortical tubules, blood and urine, after nephrotoxic and ischaemic injuries. Thus, NGAL might represent an early, sensitive, non-invasive biomarker for acute renal injury. The aim of this study was to correlate the urinary NGAL levels with the state of acute kidney damage of multitrauma patients as reflected by RIFLE classification. Thirty five multitrauma nonoliguric, non septic patients without cardiac or chronic kidney disease were evaluated along with 10 healthy subjects who served as controls. Serum creatinine and urinary NGAL were measured upon admission in ICU, on all patients and RIFLE, ISS, APACHE-II and MDRD eGFR was calculated. NGAL was measured in urine by ELISA technique, using a commercially available kit (ANTIBODYSHOP Gentofte, Denmark) and serum creatinine with a Jaffe method on Aeroset autoanalyzer (Abbott Diagnostics, Chicago, Il.). Data were analysed using the NCSS statistical program (Kaysville, Ut.). Statistical analysis involved analysis of variance (ANOVA) or t-test with P<0.05 considered statistically significant when appropriate. The following table summarizes our results along with the statistical evaluation.

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0.80 (0.15)

0.90 (0.15)

1.26 (0.13)

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not available

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Urine NGAL was significantly higher in the non kidney disease group compared to controls (t-test <0.005) probably due to inflammation caused by trauma, but a nine fold increase occurred when patients are in the risk or injury category, and another five fold increase in failure category, while the creatinine was increased 71% and 78% respectively. These preliminary results show that we can use the urinary NGAL as early predictive marker (from the 1st day of hospitalization) of acute kidney failure that will require renal replacement therapy in multitrauma patients.

Poster 16 The use of antibody coated paramagnetic particles as a label for the rapid detection of intracellular PSA in LNCAP cells. Sharif E, Kiely J, Hawkins P , Luxton R. (University of the West of England, Bristol, UK.) The objective of this study is to investigate the measurement of intracellular prostate specific antigen (PSA) as a model to demonstrate dual functionality of paramagnetic particles. When energised with ultrasound antibody coated particles perforate and disrupt the cells and captured PSA, bound to the antibody, is quantified using a rapid magneto-immunoassay based on measuring the magnetic properties of material present in the particle (1,2). The majority of current diagnostic testing performed in a clinical biochemical laboratory uses blood sample for investigating disease. The development of genomics and proteomics over the past 10 years, has given a greater understanding of the pathogenesis of many disease such as cancers and inborn–errors of metabolism. Currently the analysis of intracellular proteins is complicated and laborious involving extraction, electrophoretic separation, blotting technique and densitometric analysis for quantitation. A rapid test, using cells as the sample, could be developed for intra-cellular proteins associated with disease, such as cancer or inborn-errors. The prostate cancer cell line LNCAP was chosen as a model for a rapid intracellular protein assay, as they are known to produce PSA. A PSA magneto-immunoassay was developed by coating tosylated activated magnetic particles with monoclonal antibody against PSA. A magnetic sensor was coated with a second antibody directed against PSA. Antibody coated paramagnetic particles were mixed with washed LNCAP cells and subjected to a brief pulse of ultrasound to disrupt the cells. An applied magnetic field attracted the antibody coated paramagnetic particles to the sensor surface, PSA released from the cells acted as a bridge between the antibodies on the particles and sensor surface resulting in the capture of the paramagnetic particles on the sensor surface. A sensitive magnetometer measured the quantity of immobilised particles which was related to the amount of PSA produced by the cells. The detection limit of the PSA assay was 1.0 ng/ml and showed a dose dependent response with cell number. LNCAP cells grown in the presence of testosterone demonstrated increased levels of PSA production. The assay was compared with an ELISA method for free PSA which also demonstrated increasing levels of free PSA in the growth media with increasing concentrations of testosterone ( 0 – 50 ng/ml). The results demonstrate a proof-of-principal that paramagnetic particles can be used as a label in a rapid magneto-immunoassay and, when energised with ultrasound, they are able to disrupt cells allowing quantification of intracellular proteins. 1. Luxton, R, Badesha, J., Kiely, J., Hawkins P., 2004. Use of External Magnetic Fields to Reduce Reaction Times in an Immunoassay Using Micrometer-Sized Paramagnetic Particles as Labels (Magneto-immunoassay). Analytical. Chemistry. 76, pp 1715-1719. 2. Richardson, J., Hawkins, P., Luxton, R., 2001. The use of coated paramagnetic particles as a physical label in a magneto-immunoassay. Biosensors and Bioelectronics. 16 pp 989-993.

Poster 17 Surface nanomanipulation for enhanced microplate assay performance. Hopkins SL1, Qin DJ1, Pan J1, Milunic D1, David JM1, Kimos MC1, Uzdilla LA1, Gibbs SK1, Pokhrel PS1, Vallejo RP1, Yin R1.( 1ANP Technologies, Inc., Newark, Del.) The Nano-Intelligent Detection System (NIDS®) technology uses nanomanipulation to orient bioactive molecules on surfaces in order to enhance the performance of diagnostic tests in various formats. Previously, the NIDS chemistry has been successfully applied to various materials such as gold, latex, quantum dot, and other signal producing nano- and micro-particles for lateral flow assays. The NIDS technology has now been applied to 96-well ELISA plates, enabling a dramatic improvement in the efficiency of coating specific protein and nucleic acid reagents. ELISA plates coated with NIDS-functionalized streptavidin show a five- to ten-fold increase in binding capacity for biotinylated capture antibody compared to commercially available streptavidin-coated ELISA plates. Lower concentrations of capture reagent generate stronger signals with these treated plates, an


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important consideration when these reagents are rare or expensive. These plates exhibit very low non-specific binding and no detectable leaching of coated reagent through 12 washes. As examples of the enhanced assay performance attainable with NIDS-functionalized streptavidin plates, an ELISA for Thyroid Stimulating Hormone (TSH) and a nucleic acid assay for B. anthracis Sterne strain mRNA, both using colorimetric horseradish peroxidase detection, are presented. The TSH assay achieves high sensitivity needing only 20% of the capture antibody used in conventional coating. In the latter case, a detection limit of 460 attomoles of mRNA is achieved without the benefit of sample amplification.

Poster 18 A novel detection technology mediated by Multi‐component Nucleic Acid enzymes (MNAzymes) allows rapid development of multiplex real time PCR assays using generic probes. Mokany E, Doan TB, Young PE, Suwandi R, Lomas M, Wang L and Todd AV. (Johnson & Johnson Research Pty Limited, Eveleigh, Australia.) The engineering of novel non-protein enzymes known as “MNAzymes” has allowed the development of a new real-time PCR technology with multiple advantages over other real-time PCR methods. MNAzymes comprise two DNA “partzymes” which come together to create catalytically active enzymes only when target amplicons are present. The enzymatic MNAzyme complexes cleave generic reporter probes and generate fluorescent signals. The MNAzyme approach has enhanced specificity compared to other real-time PCR protocols. This arises from the requirement for the binding of two partzymes and two PCR primers to confer four levels of specificity. The use of generic probes, as opposed to target specific probes such as those employed by TaqMan and Molecular Beacon assays, provides multiple advantages. Generic probes give reliable, consistent performance when coupled to any target or used for any PCR application. This reduces the amount of time required for development of assays for new targets. Further, generic probes reduce the cost of assays due to scale of manufacture, ease of QA and their use eliminates probe waste, since unused probe can be used for new targets. A series of generic probes has been successfully used for multiplex PCR analysis. Simultaneous quantification of five target transcripts in a single tube multiplex RT-PCR format has been demonstrated using five unique probes. The same probes have been incorporated in real-time assays for RNA or DNA quantification, methylation analysis and single nucleotide polymorphism detection. The MNAzyme technology is highly precise as evidenced by low inter- and intra-assay variation in PCR replicates and it has been shown to perform well on several real-time platforms. MNAzyme PCR is a new approach to real-time PCR that allows target-specific interrogation of amplicons while using generic probes. It has the advantages of greater specificity, reduced cost, reduced time for development of assays for new targets and is more amenable than other technologies to the rapid development of multiplex assays.

Poster 19 Comparison of multivariate calibration models for non‐invasive blood glucose measurement based on the novel method of Pulse Glucometry. Yamakoshi Y1, 2, Ogawa M2, 3, Tamura T1, Yamakoshi K3. (1Graduate School of Engineering, Chiba University, Chiba, Japan, 2yu.sys Corporation, Kanazawa, Japan, 3Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan.) Objectives: We have previously reported a novel optical technique for non-invasive in vivo blood glucose concentration (BGL) measurement, named "Pulse Glucometry" (1). This involves interrogation of a tissue segment with near infra-red (NIR) radiation and collection of a large number of transmittance spectra. Analysis of the spectra involves the use of a suitable calibration model and the study described here was carried out to compare four such multivariate calibration models. Methods: The total transmitted radiation intensity at wavelength λ (Iλ) and the cardiac-related pulsatile changes superimposed on Iλ in human adult fingertips were measured over the wavelength range from 900 to 1700 nm with resolution of 8 nm, at a sampling rate of 100 spectra/s using a fast spectrophotometer, obtaining differential optical densities (ΔODλs). The pulsatile changes relate to

blood volume changes and thus ΔODλs bear a relationship with arterial blood constituents including BGL. In this study data sets of a family of measured ΔODλs and the corresponding known BGLs were combined using four multivariate calibration methods; Principal Component Regression (PCR), Partial Least Squares Regression (PLS), Artificial Neural Networks (ANN) and Support Vector Machines Regression (SVMsR). Results: A 5-fold cross validation for a total of 95 data sets, in which the BGLs ranged from 100.7-246.3 mg/dl, was used. The results were evaluated by the Clarke error grid analysis and almost all of data points from four models fell within the clinically acceptable regions (region A or B). Among them, ANN and SVMsR calibration provided the best plot distributions (shown in the figure). Conclusions: These data provide preliminary evidence that Pulse Glucometry with ANN and SVMsR can be applied effectively to measure BGLs non-invasively. References 1. Yakmakoshi K, Yamakoshi Y, Pulse glucometry: a new approach for noninvasive blood glucose measurement using instantaneous differential near-infrared spectrophotometry. Journal of Biomedical Optics, 2006;11:054028(1-9).

Poster 20 Improved blood glucose measurements using a thin layer cell and tri‐pulse electrochemical detection. Hodges AM, Chatelier RC. (Universal Biosensors, Victoria, Australia.) We have developed a glucose test system based on a thin-layer cell design with opposing electrodes and tri-pulse electrochemical detection that is fast (~5 sec analysis time), requires a small sample (~0.4 µL), and can provide improved reliability and accuracy of blood glucose measurements. In the reaction cell, glucose in the sample is oxidized to gluconolactone using glucose dehydrogenase and an electrochemically active mediator is used to shuttle electrons from the enzyme to a palladium working electrode. A potentiostat applies a tri-pulse potential waveform to the working and counter electrodes, resulting in three current transients used to calculate the glucose concentration. Additional information gained from the three current transients may be used to discriminate between sample matrices and correct for variability in blood samples due to hematocrit, temperature variation, or electrochemically active components. The analytical capabilities of the technology were tested in preclinical experiments conducted using human blood samples. The figure below shows the effect of whole blood samples having a wide range of hematocrit on the accuracy and precision of the new test system. The bias of the sensor response with respect to the YSI 2700 (Yellow Springs Instruments, Yellow Springs, Ohio) is plotted against the plasma glucose concentration. The data were obtained with 3 batches of sensors and 4 blood donors. The hematocrit was adjusted to 20% (squares), 37-45% (circles) or 60% (triangles) prior to spiking the samples with glucose. These data suggest that the thin layer cell and tri-pulse approach for electrochemical measurement offers the opportunity for improved analytical performance with blood glucose test systems.

Poster 21 Withdrawn

Poster 22 Synthetic biotinylated glycoconjugates for the detection of shiga toxins and influenza viruses. Iyer SS1, Flagler MJ2, Fuller‐Schaefer C2, Harris JF,3 Hatch DM1, Kale RR1, Lewallen DM,1 McGannon MC2, Mukundan H4, Price DN4, Schmidt JG3, Swanson BI4, Weiss AA2 (1Center for Sensors and Biosensors, University of Cincinnati, Cincinnati, Ohio, 2Department of Molecular Genetics, University of Cincinnati, Cincinnati, Ohio, 3Bioscience Division (B‐7 and B‐9), Los Alamos National Laboratory, Los Alamos, N.M., 4Physical Chemistry and Applied Spectroscopy (C‐PCS), Los Alamos National Laboratory, Los Alamos, N.M.) Most point-of-care diagnostics use antibodies as the capture and recognition elements. However, there is a great need to develop synthetic ligands that exhibit antibody-like selectivity and sensitivity and additionally, are robust, inexpensive, amenable to scale up and require no refrigeration. Unlike antibodies, oligosaccharides are robust and not prone to facile decomposition. However,


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carbohydrates have not been used as recognition motifs in biosensor technologies because of their "apparent" lack of selectivity. We have recently demonstrated that it is possible to tailor carbohydrates to achieve high selectivity and sensitivity towards specific analytes. Using a modular synthetic approach, we have synthesized a panel of high affinity designer glycoconjugates that differentiate between closely related serotypes of Shiga toxin. Synthetic variants of Gb3, the Shiga toxin receptor, can distinguish Stx1 from the more potent variant, Stx2. We also demonstrate the ability of these glycoconjugates to capture toxins from clinical human stool samples with minimal interference. Using the same modular approach, we have synthesized robust, biantennary biotinylated stable sialyl glycoconjugates that are impervious to the cleavage of viral Neuraminidase. These robust, synthetic recognition elements can be readily incorporated onto existing biosensor platforms. We demonstrate the ability of these glycoconjugates to capture and detect Influenza virus and differentiate between Influenza variants using a novel optical waveguide biosensor developed at the Los Alamos National Laboratory, New Mexico. This work is novel because the glycoconjugates described in this presentation are robust, inexpensive, require no refrigeration, can be scaled up, applied to any sensor platform and bind to toxins and viruses present in real-world samples. Also, this detection strategy relies on receptor-mediated recognition events, as opposed to antigen-based recognition. Receptor-binding strategies are particularly useful to detect antigenic variants since amino acid changes may change antibody binding without changing function; however, receptor binding cannot be varied without loss of function. Thus, the recognition event can be correlated to function. The design, synthesis of the glycoconjugates and their ability to capture toxins and viruses will be presented. The work on Shiga toxins has been published online in Angew. Chem. Int. Ed. Engl. ( DOI: 10.1002/anie.200703680 ) and has also been highlighted in C&E News, the weekly chemistry news magazine published by the American Chemical Society. ("Fighting Foodborne Illness" www.http://pubs.acs.org/cen/news/86/i03/8603news3.html).

Poster 23 A superior analytical technique for the isolation and detection of biological compounds using micro‐fluidic technology coupled with tandem mass spectrometry. Yang DD, Yin H, Killeen K, Van‐Cleve M, George MP. (Agilent Technologies, Inc., Santa Clara, Calif.) Introduction: Tandem Mass Spectrometry has gained momentum for small biological compounds such as testosterone and 25-OH Vitamin D in both the research and clinical applications (1,2,3). These applications employ High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (LC/MS/MS). We have conducted a study using the µ-fluidic chips (LC-Chip) technology coupled with tandem mass spectrometry for the analysis of testosterone and 25-OH Vitamin D in biological fluid. Our study demonstrates that the LC-Chip coupled with tandem mass spectrometry is a superior analytical technique for the analysis of these biological compounds compared to the conventional LC/MS/MS. Methods: The µ-fluidic chips are made of laser-ablated and laminated inert polymer films. The chip consists of a sample loading structure, a packed LC separation column as well as an integrated Electro-Spray (ES) tip. Both sample enrichment and LC separation columns are packed with experimental C18 modified silica beads. The sample enrichment column has a total volume of 40nL. The LC column dimension is 75µm (W) x 50µm (D) x 43mm (L). The liquid chromatographic separation is achieved under water/ACN gradient or water/methanol condition at 300nL/min. The analysis is performed using the deuterium labeled internal standard. The LC-chip is interfaced to an Agilent 6410 Tandem Mass Spectrometry. The tandem mass spectrometry is operated in multiple reaction monitor (MRM) mode. Experimental Data: Samples are prepared for tandem mass spectrometry analysis by protein precipitation using 0.3M ZnSO4:methanol 50:50 ratio, and centrifugation. The chromatographic separation is run under acidic condition with 0.1% formic acid in both aqueous and organic solvents. For testosterone the limit of detection is 20pg/ml and the linearity of the assay is from 20pg/ml to 50ng/ml. The limit of detections for 25-OH Vitamin D3 and 25-OH Vitamin D3 are 3ng/ml and the linear dynamic ranges are from 3ng/ml to 150ng/ml. Our results demonstrate that LC-chip / Tandem Mass Spectrometry can be used for high sensitivity testosterone and vitamin D isomer detections in human serum.

References R.L. Taylor, S.K. Grebe, and R.J. Singh, Clinical Chemistry, 50(12), 2345-2352, 2004 M.L. Cawood, H.P. Field, C.G. Ford, S. Gillingwater, A. Kicman, D. Cowan, and J.H. Barth, Clinical Chemistry, 51(18), 1471-1479, 2005 Z. Maunsell, D.J. Wright, and S.J. Rainbow, Clinical Chemistry, 51(9), 1683-1690, 2005

Poster 24 Application of exciton coupling effect on primers for rapid detection of tamiflu‐resistant H3N2 viruses using the Smart Amplification Process. Lezhava A1, Ishizu Y1, Nogawa C1, Murakami K1, Hayashizaki Y1, Kogo Y2, Mitani Y2, Cizdziel P2, Okamoto A3, KawaokaY4, Kiso M4. (1Genome Exploration Research Group, RIKEN, Yokohama, Japan, 2K.K. Dnaform, Yokohama, Japan, 3Frontier Research System, RIKEN, Wako, Japan, 4Division of Virology, Institute of Medical Science, University of Tokyo, Japan.) We have developed a sensitive, accurate, rapid, and simple DNA amplification technique which can be used in various applications ranging from pharmacogenomics-based drug discovery to point-of-care diagnostics. This procedure, called the SMart Amplification Process (SMAP), employs a unique primer design and background suppression technology that amplifies target sequences from crude cell lysates without thermocycling. RT-SMAP was successfully employed to detect single mutations in influenza A H3N2 strains which lead to Tamiflu resistance. Here we report a detection system for three such point mutations located at E119V, R292K and N294S positions of the neuraminidase gene. Newly designed primers with conceptually new on–off fluorescent DNA modified nucleotides have been successfully applied to SMAP. Design of primers with dyes that emit fluorescence only when they recognize the target molecule give number of advantages in molecular diagnostics field, which is lower background, higher specificity, two/multicolor assay in one tube and easy visual detection.

Poster 25 & 26 Withdrawn

Poster 27 Dynabeads® MyOneTM Silane: a platform bead for automated nucleic acid capture. Bosnes M1, Keiserud A1, Bremnæs C1, Lindstrom H1, Finne E, Ellis D1. (1Invitrogen Dynal, Oslo, Norway.) The introduction of automated platforms has streamlined workflows for nucleic acid (NA) isolation from patient samples. A larger number of samples can be processed faster, with greater reproducibility while using less reagents and hands-on time. The successful development of a reliable protocol on an automated platform is facilitated by choosing a solid phase which can meet the demands of automation. The two most important contributions from the solid phase to this development process are functionality and reproducibility. Dynal Invitrogen has developed Dynabeads MyOne Silane to be ideally suited to the development of automated protocols for the isolation of total NA from a variety of sample types. These magnetic beads are monosized, monodispersed spheres produced using validated manufacturing techniques producing excellent in-batch and between batch reproducibility. The beads have an increased magnetic strength compared to the main Dynabeads portfolio. This ensures rapid magnetic mobility and excellent performance in viscous fluids such as blood. The 1 um beads present a silica-like surface optimized for the capture of NA and their small size and defined surface area offer excellent capture kinetics. The beads offer a very large dynamic range: suitable for high sensitivity applications such as the capture of viral NA present at a few copies per ml of patient serum – typically femtograms of NA - all the way up to high capacity applications where tens of micrograms of genomic DNA or total RNA may be isolated from patient cell samples. As a development case study this poster highlights automated protocols developed on both IPrep (Invitrogen) and Tecan Genesis (Tecan, Switzerland)

http://www.http//pubs.acs.org/cen/news/86/i03/8603news3.html


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that cover the full spectrum of standard NA isolation procedures. As a clinically relevant test system for high sensitivity NA isolation we present data to show the isolation of viral NA from patient serum infected with HBV, with downstream characterization by quantitative PCR which shows improved performance over competitor technology. From the high capacity end of the spectrum of NA isolation procedures we show data for the isolation of genomic DNA from human blood and tissue culture cells (Daudi) as well as total RNA. Isolated DNA/RNA was characterized spectrophotometrically and by gel to determine purity, yield and integrity. These procedures developed with Dynabeads are reliable, robust, and have minimal batch to batch variation.

Poster 28 Carbohydrate deficient transferrin testing by determination of N‐glycosylation site occupancy. O’Brien JF , Bergen HR , Turgeon C , Benson LM . (Mayo Clinic, Rochester, Minn.) Carbohydrate deficient transferrin (CDT) has been used for identification of alcohol abuse and the detection of glycosylation congenital disorders - for well over a decade. When mass spectrometry was introduced to estimate under-glycosylated transferrin in these conditions, it was found that whole oligosaccharide omission from one or both of the two N-glycosylation sites was the basis of the occurrence of CDT and not the absence of sialic acid residues. We hypothesized that analysis of the N-glycosylation site tryptic peptides derived from transferrin would result in a higher level of precision in quantitating CDT by allowing the use of internal standards. The amino acid sequence of transferrin is given below. The peptides identified by bold script are:* asparagine linkage sites (N1 and N2) and a ** “proteotypic transferrin peptide.”

MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGK*CGLVPVLAENYNK(N1)SDNCEDTPEAGYFAVAVVKK**SASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILR*QQQHLFGSNVTDCSGNFCLFR(N2)SETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP Isotopically labeled peptides corresponding to the tryptic peptides containing the transferrin N-glycosylation sites Asn413 (N1) and Asn611 (N2), as indicated above, were synthesized with and without isotopic labels. Appropriate MRM transitions were identified for each N-linked peptide and internal standard. The N2 peptide has a lower MS response compared to N1 at identical concentrations. The current LOD and LOQ for each peptide were determined to be 25 fmol, 250 fmol for N1 and 250 fmol, 2.0 pmol for N2, respectively, for the pure standards. Reduction, alkylation, and trypsinization of 5 μL whole serum and LC/MS/MS analysis results in the quantitation of the two glycosylation sites N1 and N2 and the proteotypic peptide T46 that can be utilized as an internal standard for normalization of transferrin concentration. Serum from positive adult patients identified by whole protein MS as having normal, marginal and increased CDT were analyzed to determine site occupancy. The results were in agreement with whole protein MS. The possibility of absolute quantitation of unoccupied glycosylation sites offers a more sensitive and specific method to detect CDT. This is necessary for detection of alcohol exposure since unlike the Congenital Deficiencies of Glycosylation the quantity of unoccupied glycosylation sites is far more subtle.

Poster 29 DNA‐programmed chemistry for the detection of BCR‐ABL fusion protein in expressing cells. Haff, LA, Chang, Y, Huang, Y, Seigal, B. (Ensemble Discovery Corporation, Cambridge, Mass.) The fusion protein BCR-ABL is a tyrosine kinase formed by a chromosomal translocation in chronic myeloid leukemia (CML. BCR-ABL has constitutive kinase activiy and activate numerous signaling pathways leading to resistan ce to apoptosis and clonal expansion in CML. This protein is present in all patients with CML and its presence is sufficient to cause the disease. Quantitation of the number of cells containing the BCR-ABL protein, is highly desirable, particularly for tracking patients exhibiting minimal residual disease (MRD) during treatment. Quantitation of these cells, however, is very difficult. An estimate of the frequency of the BCR-ABL mutation can be obtained through quantitative PCR but this is difficult to relate to the number of individual cells expressing the mutation. Quantitation through antibody-mediated FACS (fluorescent activated cell sorting) is not possible since, while there are antibodies available against BCR and ABL, there are no satisfactory antibodies for BCR-ABL. We developed an assay for detection and quantitation of cells expressing the BCR-ABL fusion protein based upon DNA Programmed Chemistry (DPC). A DPC assay employing developing a fluorescent signal employing aptamer binders to the A and B subunits of platelet derived growth factor (PDGF-AB) was previously described. An extension of this technology employed antibodies to BCR and ABL proteins. These antibodies were conjugated to zip-coded oligonucleotides which complemented anti-zipcoded oligonucleotides conjugated to fluorophore precursors. Upon simultaneous binding of the antibody conjugates with their annealed antizipcode conjugates to BCR-ABL fusion protein, the fluorophore precursors become localized at an elevated concentration and react, producing a fluorophore from the non-fluorescent precursors. This assay was first demonstrated using purified BCR-ABL as a target, and it was shown that the assay required BCR-ABL, both antibody conjugates, and both fluorophore precursors to produce a fluorescent signal. The assay was then modified to detect BCR-ABL in preparations of fixed, permeabilized cultured KY01 cells. Cells exhibiting the characteristic fluorescence of an indolinium fluorophore could be readily distinguished by FACS assay from cells not expressing BCR-ABL. This assay will be used to demonstrate detection of MRD in samples from CML patients. Further extensions of this technology show that is also applicable to the immunohistochemical detection of protein dimers in fixed tissue sections.

Poster 30 Europium as fluorescent marker for detection of specific IgE antibodies in a protein microarray. Harlin A1, Eriksson AK2, Ljung A, Phadia AB1, Nystrand M1. 1Phadia AB, (Research & Development, Uppsala, Sweden, 2Swedish University of Agricultural Science, Department of Plant Biology and Forest Genetics, Uppsala, Sweden.) Introduction: Specific IgE is an established marker of the in vitro diagnosis of allergy. As there is a need to screen a number of different allergens in order to establish or exclude the cause of the symptoms, an experimental multiplex protein microarray assay for detection of a low concentration analyte, specific IgE antibodies has been developed. The prime attraction of multiplexed allergy testing is the vast amount of serological information that can simultaneously obtained from a small volume of sample. This assay uses anti-IgE antibody conjugated FarRed particles as detecting reagent. Other commonly used fluorescent markers are Alexa, FarRed, Cy3 and Cy5. The advantageous fluorescent characteristics of Europium chelate particles; large Stokes shift, high emission capacity and light stability, simplifies the development of detecting instrumentation using CCD camera technique, instead of more complex and expensive laser scanners. Objective: The objective of this study was to investigate if the test performance of the microarray assay using Europium particles and CCD camera for detection of specific IgE is comparable to FarRed particles and laser scanner. Materials and Methods: The allergen reagents were arrayed at a density of 200 spots/cm2 on a nitrocellulose membrane strip sized 5 x 40 mm, each spot being 250 µm in diameter. The assay uses a sequential series of liquid flows driven by capillary forces. In following order 30 µl serum sample, 20 µl detecting antibody


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conjugated fluorescent particles and 2x30 µl washing buffer is added, which results in a total assay time of about 30 minutes. A commercial laser scanner and CCD camera instrument as well as an inhouse developed CCD camera instrument were used for detection. ImmunoCAP® was used as a sIgE reference method. Results: The background signal for Europium was higher than for FarRed resulting in a decreased signal range and less sensitivity with Europium. The average intra-assay precision was in the same range for both fluorescent markers. The sensitivity of the experimental microarray test system using FarRed was comparable to the ImmunoCAP® system. FarRed signals detected with a commercial CCD camera instrument are comparable to the signals detected with a laser scanner. Conclusion: The microarray assay described here exhibits the performance including analytical sensitivity sufficient to make it possible to develop a multiplexed allergy test. A CCD camera can be used in a simple and non-expensive instrumentation for the detection of specific IgE. Although Europium-particles have great advantage compared to other fluorescent particles the assay has to be further optimized to fully take advantage of Europium properties. The extreme high sensitivity of the inhouse developed CCD-camera could not be turned to account as the background generated by the assay was far much higher.

Poster 31 A fully automated enzyme immunoassay for the detection of the Marburg I‐polymorphism of factor VII‐activating protease in human citrated plasma and its correlation to an amplification created restriction site PCR. Schwarz H, Vitzthum F, Stephan S, Quak E, Simshaeuser‐Knaub B, Teigelkamp S. (Dade Behring Marburg GmbH, Marburg, Germany.) Factor VII-activating protease (FSAP) is a plasma serine-protease that is also localized within atherosclerotic lesions and can bind to growth factors and inhibit vascular smooth muscle cell (VSMC) proliferation and migration. The Marburg I (MRI) single nucleotide polymorphism of the FSAP gene is characterized by an exchange of guanine by adenine at position 1601, leading to an exchange of glycine to glutamic acid at amino acid 511 of the proenzyme and exhibits much lower proteolytic activity towards prourokinase as compared to wild-type FSAP. The prevalence of the heterozygous FSAP genotype (1601 G/A) has been reported to be in a range from 4 to 9% in Caucasian populations. The MRI polymorphism has been associated with incidence and progression of carotid stenosis and venous thromboembolism. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large study populations it is beneficial to rely on a cost effective, reliable, easy-to-use, and rapid to perform assay method that can eventually be automated. In addition, it appears advantageous to directly determine antigen levels as it may more closely relate to the phenotype than the genotype. After the development of a suitable and specific monoclonal antibody a fully automated ELISA for the qualitative determination of the MRI FSAP variant in human citrated plasma was established. The prototype MRI assay* employs an anti-MRI FSAP capture mouse monoclonal antibody immobilized on microwell strips. Bound MRI FSAP is detected with a horseradish peroxidase labeled anti-FSAP conjugate. The measured OD of the converted 3,3’,5,5’-tetramethylbenzidine (TMB) is proportional to the MRI FSAP protein concentration. The prototype assay was automated using a Dade Behring ELISA Processor (BEP®). Results are available within 1.5 hours. Precision of the test was determined in five independent runs with 5 different samples, 3 from subjects with the FSAP genotype 1601 G/A and 2 with 1601 G/G. Each sample was determined 8fold in each run. Repeatability (within-run) and within-lab (total) precision estimates were determined by ANOVA 1R analysis. Repeatability was less than 8% and within-lab precision was below 10%. No significant interference (<10% bias) was seen from lipemia (3000 mg/dl TRIG), hemoglobin (1000 mg/dl Hb), icterus (60 mg/dl TBIL), selected serine-protease inhibitors (aprotinin, C1-esterase inhibitor, alpha-2-antiplasmin for the maximum concentrations applied which were 473 KIU/ml, 37 µg/ml, and 100 µg/ml respectively) or heterophilic antibody containing samples. No significant cross-reactivity was observed with structural homologous (coagulation factor XII, prourokinase, urokinase for the maximum concentrations applied, which were 340 µg/ml, 100 µg/ml, and 20 µg/ml respectively). The MRI FSAP ELISA correctly identified the FSAP genotypes of 131 samples, which were determined by an amplification created restriction site PCR test. The immunoassay prototype is very convenient for the testing of large study populations and represents a valuable tool for laboratories that do not have the

equipment to perform probe or SYBR Green I based PCR. * Product not available for sale

Poster 32 Development of an NT‐proBNP point‐of‐care test based on a novel microfluidic platform. Rundström G, Peterson C, Mendel‐Hartvig I, Lindström T, Bergman D, Lundbladh L, Read M, Öhman O, Larsson O. Åmic AB, (Uppsala, Sweden.) Åmic is developing the Forecast technology platform with broad applications in the field of clinical diagnostics from immunoassays, through cell-based assays to molecular diagnostics. The Forecast technology comprises an injection molded microfluidic plastic chip (4castchip®) and a unique fluorescence detection scheme. The 4castchip consists of a dense array of micropillars which drive the flow of sample and reagent in a highly controllable way in an open capillary channel. By designing the pillar density and flow-path, complex protocols such as integrated two-step assays can be developed with sequential flow of sample and reagent and tailored flow speeds. We will demonstrate that the technology enables immunoassay performance on par with central laboratory systems in a convenient and rapid format suitable for professional point-of-care (POC) applications. In this study we report the first results from development, using clinical samples, for the quantitative determination of N-terminal pro-brain natriuretic peptide (NT-proBNP) in whole blood samples using the Forecast technology. The NT-proBNP test will be one of the key cardiac markers developed for the Forecast POC system, and is intended as an aid in the diagnosis and management of suspected heart failure (HF), and risk stratification of patients with acute coronary syndrome (ACS). We have examined the analytical performance of the Forecast NT-proBNP assay in an early prototype system, evaluating its sensitivity, precision, and measuring range. We assessed the assay’s clinical performance using fresh whole blood samples collected from patients presenting at the emergency department (ED) and intensive care unit (ICU), as well as from healthy individuals, at Uppsala University Hospital. In a comparison study, with the laboratory reference method (Elecsys proBNP, Roche Diagnostics), preliminary data show a wide measuring range from 10 – 37000 pg/mL for heparinised venous blood (HVB) patient samples, using a single-step protocol in the Forecast POC system. Passing-Bablock linear regression analysis gave a slope of 0.83, intercept 4.28 with a Spearman correlation r = 0.95 for n = 116 samples. In the low region, <1000 pg/mL, we have a slope of 0.97, intercept -2.24 with correlation r = 0.92 for n = 98 samples. Analytical sensitivity has been determined to <5 pg/mL using a high plasma sample serially diluted in analyte free serum. The functional sensitivity of the assay, with serum standards, was <20 pg/mL (CV 20%) and < 50 pg/mL (CV 10%), respectively. Aspects of the Forecast technology will be presented that enable further performance improvements, including on-chip micro-optical surface reliefs for enhanced optical detection, and multiplex capabilities for multimarker assays.

Poster 33 Isolation, sorting and improvements in differentiation yield of human mesenchymal stem cells by tag‐less, flow‐assisted fractionation. Roda B1, Zattoni A1, Alviano F2, Lanzoni G2, Costa R2, Marchionni C2, Di Tullio A2, Reschiglian P1, Bagnara GP2. 1Department of Chemistry “G. Ciamician”, Via Selmi 2,( 2Department of Histology, Embryology, and Applied Biology, Bologna, Italy.)

The interest in stem cell (SC) isolation from easily accessible clinical specimens is booming. However, the lack of homogeneity in pluri/multipotent SC preparation blurs their definition and standardization for a successful therapy. Multipotent mesenchymal SCs (MSCs) isolated from human tissues such as bone marrow, fetal membranes, Wharton’s Jelly, adipose tissue and dental pulp, express a broad panel of surface antigens and the isolation procedure frequently implies a contamination by undesired cell populations. This limits possibility to precisely define and to sort homogeneous MSC preparations just on an immuno-phenotypical basis [1,2]. At 2007’s Oak Ridge Conference, we presented a proprietary tag-less sorting method for human stem cells, able to preserve cell integrity and viability for an in


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vivo cell expansion, which is based on differences of the biophysical cell properties [3]. Cells are suspended in a transport fluid (PBS or cell culture media), and injected into a capillary device by means of a continuous flow of the transport fluid. In a relatively short time (about 30 minutes), sorted cells are collected (as high as 105 cells/fraction per run) and subjected to characterization, expanded and/or committed to a specific differentiation, or stored. Here we present improvements of the developed method as for the isolation, sorting and differentiation yield of human MSCs. We have obtained baseline separation between MSCs and epithelial cells that represent an important contaminant of the MSCs isolated from clinical specimens, fetal membranes above all. These findings indicate that our technology may allow to directly obtain, with limited cell culture treatments, a well-selected stem cell preparation from heterogeneous cell populations isolated from clinical specimen. We have obtained different and characteristic fractionation profiles with high reproducibility (run-to-run and day-to-day %CV below 15%). for each source. Such differences indicate different MSC morphology distribution, as observed in ultrastructure by transmission electron microscopy. Moreover, MSCs deriving from different sources display peculiar pluripotency marker expression levels and differentiation potentials, in relation to the tissue origin: correlation between potency differences and MSC fractograms can be assessed. Along with that, the heterogeneity of each MSC population was investigated: the differentiation potential of sorted fractions from a single MSC source appears different. In particular, when adipose tissue-derived MSCs were separated into distinct fractions, the subpopulations in each fraction displayed consistent differences in adipogenic differentiation yield (ranging from 10% to 100%): notably, a characteristic fraction showed a differentiation efficiency close to 100%. This was not only the highest commitment yield among the fractions, but also higher than the one obtained in the unfractionated MSCs (30%). These findings show the high purity that can be obtained by MSC subpopulation sorting. Altogether, these results indicate that our tag-less method is able to isolate, characterize and purify MSCs directly from clinical preparations, with a high throughput of selected and well-characterized viable cells for their effective application. Due to the reduced operation and maintenance costs, to its full biocompatibility and high reproducibility, the method may be easily integrated in a clinical laboratory. Its automation will also support the highest throughput of sorted cells. [1] E.M. Horwitz et al., Cytotherapy (2005) 7, 393- [2] Parolini O et al., Stem Cells (2007) 11, Epub [3] P. Reschiglian et al., patent pending PCT/EP2007/054226

Poster 34 Tag‐less isolation of endothelial cells from human cord blood by gravitational field‐flow fractionation. Roda B1, Reschiglian P1, Zattoni A1, Lattuada D2, Magni R2, Pignatari C2, Cetin I2,3. (1Department of Chemistry “G. Ciamician”, Bologna, Italy, 2Istituto di Clinica Ostetrica Ginecologica I, Università degli Studi di Milano "Laboratorio di ricerca molecolare per lo studio e la cura delle patologie riproduttive", 3Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Clinica Mangiagalli U.O. Ostetricia e Ginecologia I, Milano.)

Human umbilical vein endothelial cells (HUVECs) are a model system for investigating on certain patho-physiological conditions of different types of endothelial cells, showing also a particular potential in predicting endothelial dysfunction in adult life. The most common approach for the isolation of endothelial cells is the enzymatic (e.g. by collagenase) removal of the cells from

human umbilical vein. This method allows for a good sample yield, but so-obtained HUVECs are often contaminated with red blood cells (RBCs) and fibroblasts, which need to be negatively selected by in vitro passages [1]. Otherwise, monolayers of cultured cells are used to obtain a reasonable amount of cells able to simulate a model for studying endothelial cells. Nevertheless, they can not reproduce real physiological conditions. In fact, some biological factors necessarily change in culture conditions. As a consequence, it would be useful to improve preparation of the cellular starting materials from real samples using different techniques, especially when gene expression studies are further required on the cells. In this work we present a method for the easy isolation of HUVECs from raw cord blood, which is based on gravitational field-flow fractionation (GrFFF) [2]. GrFFF is a low-cost, fully biocompatible, tag-less method for flow-assisted cell

fractionations. In GrFFF, cell fractionation is performed in an empty, flat capillary channel. Cells separation is structured by the combined action of Earth’s gravity field and of mobile phase flow. Cell fractionation occurs because of biophysical differences existing between the cells. Sterile conditions and cell viability are fully preserved after fractionation, and so-sorted cells can be collected and reused. A 1:2 dilution in PBS of raw cord blood derived from umbilical vein treated with collagenase was prepared. Aliquots of 50 µl of cell suspension were then injected into the GrFFF system by means of a continuous flow of transport fluid. The fractionation process was performed at a flow rate of 0.5 mL/min, and it was completed in approximately 20 minutes. An UV/Vis detector operating at 600 nm was placed at the fractionator outlet to detect cell elution. A fraction collector was connected downstream to the detector to recover the fractionated cells that were found to be as high as 105 per fraction. The high difference in cell morphology between HUVECs (20 µm as mean cell diameter) and RBCs, which represent the main contaminant (7 µm as mean cell diameter) in raw cold blood, allowed to collect purified HUVECs in specific fractions. Purity of the fractionated HUVECs was determined through flow cytometric (FC) analysis via cell labeling with the surface antigen CD31, which specifically tags HUVECs. To supply FC with a sufficient amount of cells, cells were accumulated from 4/5 repeated runs. High enrichment levels (approximately 70%) of viable HUVECs were obtained in the fractions. This first results indicate promising use of GrFFF as a method to highly enrich HUVECs cell population from raw clinical specimens, with no needs of cell culture treatments. The method and relevant technology can be easily implemented in biomedical laboratories with low instrumental and training investments. [1] Baudin B., Brunnel A., Bosselut N., Vaubourdolle M. Nature Protocols (2007) 2(3), 481-485 [2] Tong, X. and Caldwell, K.D. J. Chromatogr. B Biomed. Appl. (1995) 674, 39–47

Poster 35 Novel biomarker discovery and clinical diagnostic applications using aptamer arrays. Ostroff R, Brody E, Schneider D, Wilcox S, Zichi D, Gold L.( SomaLogic Inc., Boulder, Colo.)

Next generation medical diagnostics based on multi‐analyte protein signatures can lead to improved early detection of disease, when treatment is most likely to be successful. A novel proteomics technology developed by SomaLogic uses aptamer microarrays to simultaneously analyze hundreds to thousands of proteins from a single blood sample and identify unique protein expression profiles. Aptamers are single-stranded DNA and RNA molecules that assume sequence-dependent three-dimensional shapes and can specifically bind target molecules with high affinity. When an aptamer interacts with a target protein, the result is a tightly bound complex analogous to an antibody-antigen interaction. We have developed in vitro aptamer selection methods that result in specific analyte binding with slow dissociation rates (>30 minutes), while nonspecific proteins dissociate in seconds. We are able to employ this difference in dissociation rates to provide the specificity necessary to perform specific analyte detection without the need for sandwich-type assays. The resulting aptamers are combined in a multiplex assay capable of detecting 1pM analyte in plasma or serum samples. Multiplex aptamer assays have been conducted for numerous biomarker discovery studies, including lung cancer. Lung cancer is the leading cause of cancer death in both men and women in the United States, and it is estimated that 160,390 lung cancer deaths will occur in the US during 2007. In this study, plasma was analyzed from patients newly diagnosed with lung cancer and the protein profiles were compared to samples from matched control plasma donors who were smokers with no evidence of lung cancer. The data were examined for protein markers that discriminated between cancer and non-cancer samples using the Kolmogorov-Smirnov (K-S) test, a non-parametric test that can be applied to marker identification. Of the 91 proteins analyzed, 11 were found to between differentially expressed between cancer and non-cancer samples. Aggregation of these protein markers into a diagnostic signature resulted in 95% sensitivity and 95% specificity for discriminated between cancer and non-cancer samples. Analysis of a larger set of lung cancer and control samples will be conducted to test the validity of this diagnostic algorithm.


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Poster 36 Using multiplex TaqMan® assays to profile a prognostic signature for breast cancer. Sigua C1, Santini C1, Gillett C2, Springall R2, Iverson A1, Sninsky J1, Tutt A2,3, Wang A1, Chang S‐Y1.( 1Celera, Alameda, Calif., 2Guy’s Breast Tissue and Databank, Guy’s Hospital, London, UK, and 3Breakthrough Breast Cancer Research Center, Institute of Cancer Research, London, UK.) OBJECTIVE: In order to reduce the required RNA amount recovered from formalin-fixed, paraffin-embedded sections (FFPE) and decrease the number of assays for a multi-gene assay, we developed research prototypes for five multiplex TaqMan® assays to provide equivalent results obtained by a previously reported SYBR® Green-based 14-gene prognostic signature for breast cancer. The comparative performance of the multiplex TaqMan® and SYBR® Green-based 14-gene assays was evaluated in cell lines, and research subjects. METHODS: Five multiplex RT-PCR TaqMan® assays were designed to quantitatively measure the mRNA levels of a prognostic signature which consists of 14 genes of interest and 3 housekeeping (HSK) genes. In addition, we also included assays to quantitate mRNA levels of hormonal receptors, ESR1, PGR, and growth factor receptor, ERBB2. Twenty genes were divided into five 4-plex assays with 4 fluorescent reporters in each multiplex. Total RNA was extracted from FFPE sections of 35 breast cancer research subjects from Guy’s Hospital in the United Kingdom. The gene expression levels were quantified using the 7500 Real-time PCR System (Applied Biosystems). A control sample, Universal Human Reference RNA (Stratagene), was included in each run. The ��CT (the difference between HSK genes normalized CT of the sample and HSK genes normalized CT of the control) for each of the 14 genes were first calculated for each sample, and then the sum of all 14 ��CT (SDD) of each sample. Two predetermined cutoffs were used to determine three categories of prognostic risk: low, moderate, and high. The SDD results and risk calls from the multiplex TaqMan® assays and the simplex SYBR® Green assays were compared. RESULTS: The five 4-plex TaqMan® assays were first evaluated with RNA from five commonly used breast cancer cell lines. There was a significant correlation between the SYBR® Green assay and the multiplex TaqMan® assays. The correlation coefficient, R2, for SDD was 0.984. The status of ESR1 and PGR genes of 5 cell lines were consistent with those reported in the literature. For the 35 subject samples, the correlation coefficient, R2, was 0.977. Thirty-one of 35 (89%) risk category calls were identical to those determined by the SYBR® Green assays. Discordance mainly occurred in the intermediate category. The correlation coefficients, R2, between the SYBR® Green and the multiplex TaqMan assays for ESR1 and PGR were 0.991 and 0.915, respectively. CONCLUSIONS: We have developed five 4-plex TaqMan® prototype assays to profile a 14-gene prognostic signature plus critical hormonal and growth factor receptors for breast cancer. These TaqMan® assays allow the measurement of specimens with low RNA yield and facilitate high throughput testing. This high throughput RT-PCR platform is compatible with adoption in diagnostic laboratories.

Poster 37 Simple binding, competitive binding and sandwich assay web‐based simulation system. Tetin SY1, Ruan Q1, Adamchik VS2.( 1Diagnostics Research, Abbott Diagnostics Division, Abbott Park, 2Computer Science Department, Carnegie Mellon University, Pittsburgh, Pa.) Biophysical characterizations of antibodies and antigens that are used for research and development of diagnostic immunoassays require correct understanding of the binding mechanisms. We developed mathematical models and relevant solving algorithms that can be applied to antibody binding interactions at equilibrium and also to study antibody-antigen binding kinetics. Such models include systems of polynomial and non-linear differential equations. The computational complexity of the systems arises from the non-linear behavior of the kinetics processes. The models that cover all possible ways to conduct non-competitive and competitive binding assays were translated into web-based computational applications written in Java. Using these modeling tools it is easy to calculate optimal reagent concentrations, incubation times and predict assay sensitivity and performance. It is also easy to simulate the assay calibration curve and perform error propagation analysis.

The programs require input of the experimentally determined dissociation constants and kinetics rates. The assay specific equilibrium binding constants were measured using traditional steady-state fluorescence measurements (fluorescence quenching, fluorescence polarization, and Föster Resonance Energy Transfer) and new experimental developments such as fluorescence correlation spectroscopy and dual color fluorescence cross-correlation spectroscopy. The solution phase association rates were measured using a stopped-flow module mounted on a spectrofluorimeter. The binding kinetics of antibody-coated microparticles was determined on a flow-cytometer and /or using a fluorescence microscope. On examples of several immunoassays we found a good agreement in the calculated and experimentally determined assay calibration curves. Moreover, the modeling tools provides the prerequisites for reagent characteristics and concentrations that are required to achieve desired assay performance. Initially developed for modeling antibody-antigen interaction, this suite of computational applications can be applied for any binding studies including protein and/or small molecule characterizations or analytical assay design.

Poster 38 A novel, rapid, highly sensitive, and specific nucleic acid detection method. Thai HT, Keating K, O’Neil DS, (Pachowicz KA, Rangwala S, Nazarenko I. Qiagen, Gaithersburg, Md.) The value in detecting specific nucleic acid sequences from biological samples can be seen in multiple applications, including diagnosing infectious disease, studying genetic susceptibility to disease, and measuring response to various types of treatment. Utilizing the capabilities of Qiagen’s proprietary RNA-DNA hybrid-specific antibodies with synthetic RNA (synRNA) probes and Whole Genome Amplification (WGA) we have developed a method that comprises target enrichment, amplification, and detection to produce an ultra-sensitive, specific assay. The assay implements the technology of hybrid specific antibodies in both the target enrichment step and the detection step. During the target enrichment or sample processing step, use of the antibodies with synRNA produces an amplification-ready sample that is free of inhibitors while specifically concentrating the target of interest. In the detection step, utilizing multiple hybrid-specific antibodies per target produces a signal amplification that increases assay sensitivity by approximately 2-logs. The addition of a target amplification component, WGA, provides enhanced sensitivity. Our prototype assay has shown the capability of detecting 10 copies of HPV plasmids or 10 SiHa cells consistently and accurately. The test has also demonstrated robust analytical specificity, reproducibly showing the ability to distinguish HPV 16 or HPV18 plasmid from all other high- and low-risk HPV types.

Poster 39 Development of a multiplex SNP detection assay for warfarin dosing. Rai AJ, Udar N, (Fleisher M. Memorial Sloan Kettering Cancer Center, New York, N.Y.) Warfarin is a commonly used anticoagulant prescribed to over one million patients in the US in 2006. It serves to effectively prevent coagulation and is given to patients at increased risk of thromboembolic events. It is well known that individuals can vary greatly in their response to warfarin therapy and that this difference has a genetic basis. Sixty percent of this difference can be ascribed to two genes: cytochrome P450 2C9 (2C9) and vitamin K epoxide reductase subunit protein 1 (VKORC1). There are two clinically important alleles of 2C9 (*2 and *3) and one of VKORC1 (-1639 G->A). We designed an assay for the simultaneous detection of these three alleles in a single reaction. Our assay entails four steps: (1) PCR amplification of the target gene fragment, (2) hybridization of primer oligonucleotide upstream of the SNP of interest, (3) single base extension to allow incorporation of a fluorophore-specific chain terminating nucleotide, followed by (4) capillary electrophoresis separation of the fragments (see Figure). The entire assay can be performed within an eight hour day by a single technologist with minimal hands-on effort. We observe 100% concordance on twenty samples when our assay is compared to traditional DNA sequencing on an automated ABI sequencer. We have optimized this assay for high-throughput screening of patient samples, allowing for analysis of two 96 well plates on a single overnight run. Our multiplex SNP panel can be used as a


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stand-alone test for patients starting warfarin therapy, or its results can be combined in an algorithm with additional parameters (e.g. weight, age, sex, etc.) to provide dosing recommendations for initial warfarin administration.

Poster 40 Sonorheometry—a novel, non‐contact, point‐of‐care method for dynamic evaluation of thrombosis. Viola F1,2, Mauldin FW1, Lawrence MB1,2, Walker WF1,2,3. (1Department of Biomedical Engineering, University of Virginia, Va., 2HemoSonics LLC, Charlottesville, Va., and 3Department of Electrical & Computer Engineering, University of Virginia, Va.) Inappropriate blood coagulation is a significant contributing factor in the onset of potentially fatal thrombotic events such as myocardial infarction (MI), stroke, deep vein thrombosis (DVT), and pulmonary embolism (PE). Being able to recognize and quantify thrombotic disorders is thus critical to reduce mortality and implement appropriate treatment. Moreover, the ability to evaluate the dynamics of clot formation may improve patient outcomes by optimally directing therapy. We have developed a novel technique, termed sonorheometry, which can identify an increased or decreased clotting propensity by observing the coagulation rate and mechanical characteristics of the developing thrombus at any time during formation. Sonorheometry utilizes acoustic radiation force to produce small and localized displacements within the sample. Returning echoes are processed using ultrasound motion tracking so that the response of the sample to the induced force can be evaluated. These responses hold combined information about the viscoelastic properties of the blood sample being analyzed. We can then generate graphs depicting parameters such as maximum displacement and other mechanical model parameters in a temporal pattern. Here we present results describing the reproducibility and sensitivity of sonorheometry. The first set of experiments was performed on blood samples obtained from 10 healthy volunteer subjects. Fresh blood samples of 1mL were placed into a modified polystyrene cuvette and 100μL of 0.5% kaolin (W/V) was immediately added to each sample with inversion. Sonorheometry measurements were initiated within 1 min and were repeated every 6 sec over a 7 min period to characterize the dynamics of thrombogenesis. Coefficients of variation based on the observed time to clot (defined as the time when maximum displacement reaches the value of zero) were <10% for this data set. We also performed intra-subject experiments using both fresh and citrated blood, and found the coefficients of variation to be 4.9% and 5.2%, respectively. Citrated samples were analyzed by adding 100μL of 0.5% kaolin and 62μL of 0.2M CaCl2 with inversion. Additionally, we analyzed citrated blood samples obtained from the central coagulation lab at the UVA hospital. In this case we observed a 25% increase in clotting time for a DVT patient when compared to a control subject (no known thrombotic disorders) and a 46% decrease in clotting for a hemophiliac patient compared to the same control subject. These data demonstrate the feasibility of sonorheometry to assess the dynamics of thrombogenesis by characterizing the viscoelastic properties of thrombi during clot formation. Ongoing studies should provide better assessment of the potential to identify patients at high risks of thrombosis. Furthermore, sonorheometry does not require any moving mechanical parts, and can be implemented in compact,

readily available electronics, thus being an ideal technology for point-of-care settings. This work was supported by grant R01 EB005433 from the NIH.

Poster 41 The handheld NIDS® system for quantitative whole blood lateral flow immunoassays and rapid nucleic acid detection. Li J, Pan J, Small TJ, Uzdilla LA, David JM, Qin DJ, Knapp HK, Vallejo RP, Yin R. (ANP Technologies Inc., Newark, Del.) The Nano-Intelligent Detection System (NIDS®) technology uses nanomanipulation of bioactive molecules on surfaces to enhance the sensitivity of diagnostic tests. Combined with a simple handheld reader with stored standard curves, the NIDS system enables quantitative lateral flow assays to be performed for POCT and on-site testing. The versatility of this technology is demonstrated by a fully validated quantitative rapid lateral flow test for high sensitivity C-Reactive Protein (hsCRP) and a rapid nucleic acid test for the detection B. anthracis mRNA. C-Reactive Protein (CRP) is a well-studied acute phase inflammation marker that is also a promising predictive risk factor for coronary heart disease. Current recommendations are to classify patient risk into tertiles of <1.0 mg/L for low risk, 1-3 mg/L for moderate risk and >3 mg/L for high risk. The hsCRP lateral flow assay, which utilizes NIDS-enhanced gold particles, delivers a test result within 10 minutes with 5 uL of a fingerstick whole blood sample. This allows the routine testing of patients for hsCRP to be performed in POC settings instead of the centralized laboratory. The sensitivity of the assay was measured at 0.17 mg/L with a wide linearity range from 0.2 to 10 mg/L. The assay is calibrated against IFCC CRM 470. Total precision over 21 days of testing with 3 levels and 2 runs/day showed a CV of <10% at all levels. No interferences were observed with 14 substances commonly found in whole blood samples. The assay was >98% accurate in classifying 75 patient serum samples measured with the Beckman Immage CRP Assay. A rapid lateral flow assay for the detection of B. anthracis Sterne strain mRNA has also been developed for the NIDS system. The mRNA fragments are detected by annealing with a capture sequence that is immobilized on a membrane and a detector sequence that is attached to NIDS-enhanced gold particles in situ. The limit of detection is 125 pg or <5 femtomoles of mRNA without benefit of sample amplification. This test can also be used for the rapid detection of samples amplified by PCR or similar methods.

Poster 42 Multiplexed microfluidic assay chip for profiling sepsis‐related biomarkers in patient’s blood. Lee SW, Kupfer K, Parker E, Avalos B, Hou A, De Sana C, Banerjee R, Grondek J, Parson R, Reilly I, (McPherson P. Biosite Inc., San Diego, Calif.) Background: Sepsis is the leading cause of death in medical and surgical intensive care units and the mortality rate for severe sepsis ranges from 28% to 50%. Patients with symptoms indicative of sepsis are difficult to assess, often resulting in delay in initiation of treatment. Patients with sepsis present with a wide variety of symptoms resulting from infection, inflammation, hypercoagulation and organ dysfunction. Methods: A fluorescent microfluidic protein chip for measuring nine biomarkers (BNP, CRP, D-dimer, IL-1Ra MIP3, NGAL, PGRP-S, Protein C and TNF-R1a) was developed for measuring plasma specimens from patients suspected of sepsis. The biomarkers were measured using standard immunoassay techniques involving the use of recombinant mouse antibodies to specifically detect the protein targets. The 9-assay chip was used in conjunction with the Triage® MeterPlus (Biosite, San Diego, CA). Assays were performed using either sandwich or competitive assays. Assay results were subjected to multivariate analysis to derive multi-marker panel scores for use in the assessment for risk of sepsis progression within 72 hours of subjects presenting in the Emergency Department and meeting the diagnostic criteria for sepsis who might be considered for hospital admission, including the ICU. Results: The most significant biomarkers from stepwise logistic regression analysis were MIP3, CRP and NGAL (p < 0.05). The odds ratios for a multi-marker panel score consisting of these three biomarkers are significantly greater


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than 1 in the 2nd, 3rd and 4th quartiles (p < 0.05). The score exhibited greater odds ratios than any of the single biomarkers including CRP and PCT (p < 0.05). Conclusion: A biomarker panel approach may provide useful information in conjunction with other laboratory findings and clinical assessments as an aid in the assessment of sepsis progression.

Poster 43 Point‐of‐care HbA1c test using a novel direct enzymatic method. Chen X, Liu L. Hood S. and Yuan C. Diazyme (Laboratory, Poway, Calif.)

The American Diabetes Association (ADA) recommends the hemoglobin A1C test, or HbA1c, as the best way to determine a patient's blood glucose control over the past 2-3 months. The Diabetes Control and Complications Trial (DCCT) confirmed the importance of the HbA1c test and that lowering the HbA1c number can potentially reduce the development of complications from diabetes and improve a patient’s chance of staying healthy. The HbA1c test is usually done in a clinical laboratory by sending a patient blood sample to the laboratory and forwarding the test results to the patient’s physician in a few days. Recent studies have shown that an instant HbA1c result at the time of the patient’s visit to the doctor’s office can significantly improve patient’s glycemic control by making immediate necessary adjustments of treatment or patient behavior or both. Therefore, a point-of-care device for the HbA1c test that can provide accurate and reliable HbA1c results at doctor’s office is highly desirable. Diazyme, recently, developed a point-of-care HbA1c test using a novel direct enzymatic method and a portable device (Smart 700). The direct enzymatic HbA1c assay method utilizes a specific enzyme (FVO) to quantify glycated hemoglobin amount without the need for a separate measurement of the total amount of hemoglobin, whereas the portable device utilizes an optical detection system (700 nm) with a two-reagent based cartridge and an RF card. The test consists of two simple steps as follows: taking twenty �L of blood sample from a fingerstick with a sample collector where the blood is immediately lysed in the collector; adding eighty �L of the lysed blood sample to the reagent cartridge which is inserted into the Smart 700. The test result is reported in 7 min after pressing the start key on the screen of the Smart 700. Other features of the Smart 700 HbA1c test include wide dynamic range of 4-18% HbA1c; high precision with CV < 3%; high accuracy comparable to large lab results; results traceable to NGSP values; no interference from Hb variants like HbC, HbE, and HbS , no calibration needed, and cost effective. The Smart 700 results correlate well with results obtained by the HPLC (Tosoh) and immunoassay (Roche) methods as shown in the figure. The device may also be used at home for HbA1c self-monitoring by patients.

Poster 44 A novel point‐of‐care assay measurement platform using imaging technology. Dylewski S, (Alverix, Inc., San Jose, Calif.)

Lateral flow assays are well established and have enabled a wide variety of point-of-care tests. Despite the benefits of faster results, adoption can be limited by:

• Complicated test procedures or sample preparation • Lack of automatic record keeping • Insufficient sensitivity • Difficult interpretation of quantized line strengths • Lack of sophisticated measurement tools for assay development

Alverix has developed a low-cost, flexible platform for assay measurement which helps to solve some of these problems. The system uses an image sensor backed by a microcontroller to quantify and report the assay line strengths. When using this assay measurement platform, there are many advantages, including automatic record keeping and quality control, increased sensitivity, and accurate quantization. Both reflective and fluorescent instruments are being made today. Without the ability to accurately measure assay performance, scientists spend excessive time developing assays, thus reducing their market competitiveness. The Alverix platform can be used during assay development to generate quantized performance results and quickly optimize a wide variety of lateral flow assays. With this integrated platform, algorithms are designed or modified quickly during assay development, and then encoded into a low-cost handheld instrument for commercial use. This poster will report results from label-only fluorescent dilutions, quantized measurements, and improved performance compared with existing measurement techniques.

Poster 45 NanoplexTM Biotags: nanoparticulate optical detection tags for homogeneous multiplexed assays of proteins, nucleic acids and cells. Xu H, Sha M, Brown B, Golightly B, Getman B, Doering B, Holland E, Walton I, Natan MJ, Cromer R. Oxonica Inc., Mountain View, Calif. Oxonica has developed a novel optical detection tag for use in point of care diagnostics, cellular imaging assays, and life science bioassays. Nanoplex biotags are silica coated gold nanoparticles that, by virtue of surface enhanced Raman scattering (SERS) active molecules adsorbed to the metal surface, can be used in numerous assay formats for analyte quantification. Multiple unique‐signal tags can be generated by varying the adsorbed Raman active molecule, thus allowing multiplexed detection to be performed. Importantly, because the tags are excited at near‐IR wavelengths, the optical output generated is unaffected by biological matrices offering robust measurement in tissue and whole blood samples. Combining the advantage of magnetic microspheres and Nanoplex biotags, we have developed a homogeneous, no‐wash, one–step, multiplexed assay platform that provides rapid, sensitive, and precise detection of multiple targets in a single tube. The sandwich‐type immunoassay employs a regular PCR tube containing magnetic particles and Nanoplex biotags respectively conjugated to capture and detection agents (antibody or nucleic acid). After addition of the biological sample, the mixture is incubated for a 30 minutes before concentration of the magnetic particles along with immunologically‐bound biotags in a specific location of the tube. The immuno‐complex thus formed is then interrogated for SERS activity and analyte quantified while the remaining sample is present, unwashed in the tube. The power of this platform is illustrated by the simultaneous detection of the cardiac biomarkers Troponin I, NT‐proBNP and CKMB. For the three analytes, the assay results in good analytical sensitivities (<100 picogram/ml), with the precision and accuracy typically expected of heterogeneous assays, but conducted in the presence of biological matrices (serum or whole blood). We have also developed a homogeneous assay for detection of multiple DNA targets. Enteric food‐borne bacterial pathogens such as E. coli O157:H7 and Salmonella spp. were detected with good performance. Good sensitivities (<10 femtomolar) were obtained for the rapid detection of specific gene sequences in the presence of genomic DNA and without the aid of amplification. Similarly, whole‐cell assays are compatible with this platform that permits detection and counting of rare cell events. Using SKBR3, a tumor cell line expressing Her2/NEU, as circulating tumor cell model for breast cancer, we were able to detect less than a hundred cells spiked into a milliliter of whole blood (<100 cells/ml) without requiring a separation or wash step. In conclusion, the use of magnetic microspheres and Nanoplex biotags offers fast solution‐phase kinetics in a simple assay format circumventing sample preparation. Combined with simple instrumentation, this assay platform delivers exquisite performance and illustrates the enormous promise SERS‐active nanoparticles can bring to diagnostic testing, both in the central laboratory and at point of care.

Correlation Between HbA1c (Diazyme) and HbA1c (Roche)

y = 0.9594x - 0.0637R2 = 0.9469

2.00

6.00

10.00

14.00

4.00 6.00 8.00 10.00 12.00 14.00 16.00

HbA

1c%

(Roc

he)

HbA1c% (Diazyme)


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Poster 46 High sensitivity detection of cardiac troponin I using fluorescence single‐molecule‐counting immunoassays. Qiu H2, Ferrell EP2, Tabrizi A1, Pawlak C1, Nolan N1, Nalefski EA2, Whitney DH2, Phelps BH1.( 1 Aviir, Palo Alto, Calif, 2US Genomics, Woburn, Mass.)

Background: Laser-induced excitation and fluorescence confocal microscopy can be exploited for the detection and counting of individual dye-labeled reporter molecules utilized in quantitative diagnostic assays. Previously we described the application of this methodology as a reporter technology in single-molecule immunoassays (SMIA) enabling quantification of analytes in the femtomolar range (Clin. Chem. 52: 2172). More recently we extended these studies by developing SMIAs for detection of several cytokines and chemokines at sub-ng/L levels (Clin. Chem. 53: 2010). In this study we developed and verified performance of a high sensitivity SMIA for cardiac troponin I (cTnI). Measurment of low cTnI concentrations (in the 1 to 10 ng/L range) may be useful for the detection of patients who have minor myocardial muscle damage (silent MFI) and are at short term risk for significant adverse cardiac events. Methods: cTnI was captured from 5.0 �L serum or plasma samples onto polystyrene microparticles covalently bound with monoclonal antibodies directed against cTnI and then labeled with fluorescently tagged monoclonal antibodies. The reporter antibodies were released into solution and counted on the US Genomics Trilogy® 2020 Single Molecule Analyzer, an instrument capable of detecting photon bursts corresponding to individual fluorescent molecules. Parameters of SMIA performance, including analytical and functional sensitivity, linearity of dilution, and potential for interference by commonly administered drugs and high concentrations of common serum constituents, were assessed using native material spiked into troponin-free serum as well as authentic clinical serum and plasma samples. Results: Assay sensitivity was measured according to CLSI standards (EP17-A, Vol. 24, No. 34). For the cTnI SMIA, the lower limit of the blank was 0.39 ng/L and the lower limit of detection was 1.8 ng/L. For detecting cTnI in 5.0 �L serum samples, the lower limit of quantification was 5.0 ng/L, and the upper limit of quantification was at least 96 ng/L. Linearity in cTnI quantification was maintained from 5.0 �L down to 0.31 �L sample. Cross-reactivity against related troponin isoforms, skeletal TnI and cardiac TnC, was low (<0.005% at 1000 �g/L). There was little or no interference from commonly adminstered drugs (streptokinase, acetaminophen and ibuprofen), high concentrations of common plasma constituents (cholesterol, bilirubin, triglycerides and hemoglobin), or high concentrations of rheumatoid factor on the cTnI SMIA. Conclusion: We have demonstrated the feasibility of measuring low levels of cTnI (≤5 ng/L) in human serum samples using SMIA reagents developed for use on the Trilogy 2020 system. We plan to conduct clinical vaidation studies to further support the clinical utility of monitoring changes in low level cTnI concentrations.

Poster 47 Withdrawn

Poster 48 A novel method for the analysis of HDL 2b% in human serum samples. Chang E1, Franz T2, Kincaid R1, Konigshofer Y1, Kratzmeier M2, McNulty M1, Mueller O1, Schneider J2,Tian X2, Yang D1and Van‐Cleve M1. (1Agilent Technologies Inc., Santa Clara, Calif., 2Agilent Technologies, Waldbronn, Germany.)

Pursuant to recent journal publications, clinical studies report that High Density Lipoprotein (HDL) sub-fractions are among the important predictors of cardiovascular risk. In particular, these studies reveal that HDL 2b, the largest of the HDL subclasses, has the strongest inverse correlation to cardiac events. A method was developed to separate, analyze, and report HDL 2b% in a more cost effective and easy-to-use manner than current methods of sub-fraction detection. The system uses lab-on-a-chip technology and a bench-top instrument for HDL 2b% analysis. Human serum samples are incubated with a lipophilic dye and applied to a microfluidic chip. HDL sub-fractions are then separated electrophoretically though a polymer matrix based on size and charge. Stained lipoproteins are detected via laser induced fluorescence and the resulting electrophoretic traces are deconvoluted using a proprietary algorithm. Nine serum samples, along with two quality control samples and one calibrator, can be analyzed on one chip within one hour. 10μL of serum sample is required per run.

The scientists evaluated the correlation-to-gradient gel electrophoresis (GGE), precision, inter-site correlation, dilution, and limits of detection of the HDL method through extensive sample testing. The system reported that HDL 2b% correlation to GGE is r=0.80 using thirty samples. A study using 148 samples was then conducted among three external unaffiliated sites resulting in HDL 2b% inter-site correlation of r=0.90, r=0.91 and r=0.94 with a bias < 0.5% units. A five-day internal precision study, indicated that within-run, between-run, between-day and total-precision had a SD < 2.5%. Sample dilution 1:2 using human delipidated serum did not alter sub-fraction distribution. The limit of detection was 11% HDL 2b%. Our system offers a robust, precise, and efficient way to measure HDL 2b% in serum samples.

Poster 49 Simultaneous quantitative analysis of multiple immunosuppressant drugs in transplant patient blood by liquid chromatography‐tandem mass spectrometry using a simple, robust protein precipitation method. Hammond GW, Calton LJ, Cooper DP and Holt CA. (Clinical Business Operations Group, Waters Corporation, Manchester, UK.)

Simple liquid chromatography-tandem mass spectrometry methods for therapeutic drug monitoring (TDM) of single immunosuppressant drugs have been widely adopted in clinical chemistry laboratories. A demand for a multi-analyte methodology with a simple and robust sample preparation procedure has arisen from the increasing use of combination therapy and the need to streamline laboratory workflow. This study investigates the use of advanced tandem mass spectrometry technology in combination with simple a chromatographic method to deliver a rapid multi-analyte immunosuppressant TDM assay. A Waters Acquity TQD tandem mass spectrometer, operating in positive ion electrospray mode, was optimised for the analytes (tacrolimus, sirolimus, everolimus and cyclosporin A) and their internal standards (ascomycin, 32-desmethoxyrapamycin and cyclosporin D). Whole blood was treated with 0.1M zinc sulphate and extracted with acetonitrile containing the internal standards. The supernatant was injected onto a Waters TDM column that was eluted using a water/methanol gradient containing ammonium acetate and formic acid, providing a total cycle time of 2.5 minutes. Seven multiple reaction monitoring (MRM) channels were monitored. Commercially available calibrators were used to construct calibration curves for each analyte. Whole blood samples from the Tacrolimus International Proficiency Testing (IPT) Scheme (Prof. David Holt, London, UK) were used to determine intra- and inter-day (n=5) precision and accuracy. Correlation coefficients, r>0.995, were obtained for the calibration curve for each analyte. The results of the intra- and inter-day imprecision of the low, medium and high QC for each analyte were acceptable. All the IPT samples produced results within the IPT scheme’s acceptance criteria. This data demonstrates a simple, robust protein precipitation method using liquid chromatography-tandem mass spectrometry for the determination of multiple immunosuppressants in a single experiment.

Poster 50 Microfluidic polymer monoliths for bacterial lysis and DNA isolation to detect urinary tract infections. Kulinski MD¹, Singh S², and Klapperich CM¹,³. (¹Department of Manufacturing Engineering, Boston University, Boston, Mass., ²Boston University Medical Center, ³Department of Biomedical Engineering, Boston University, Boston, Mass.)

Urinary tract infections result in millions of physician visits annually with estimated costs due to infection in the billions of dollars. Most infections are diagnosed using dipstick tests that detect the presence of leukocyte esterase and nitrite. The dipstick test does not give any information about the infecting organism. For this information, time-consuming urine culturing is required. As a result, initial treatments are given based on empiric symptoms, which may lead to the inappropriate use of antibiotics or to leaving a sick patient untreated. An integrated device with the capability of detecting specific strains of infecting organisms will enable clinicians to distinguish between those that are antibiotic resistant and those that are not. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic device designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis


14

followed by a solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped hematuric urine samples with E.coli at a concentration of 105 colony-forming units/ml (common definition of a UTI) to simulate real patient samples and successfully performed on-chip lysis and on-chip DNA extraction from these samples. The bacterial DNA was then amplified with real-time PCR establishing efficient lysis and isolation of intact bacterial DNA. Infection was confirmed by the presence of more than one gene, and the results were compared to commercial kits for lysis and solid phase extraction. These two steps are essential in the construction of a fully integrated device for the detection of bacterial infections in real patient samples at the point of care, and can be extended to sample preparation on-chip using other bodily fluids.

Poster 51 Gene expression analysis and genomic DNA extraction from biological samples in plastic micro‐solid phase extraction columns. Bhattacharyya, A², and Klapperich CM1,2. (¹Department of Manufacturing Engineering, Boston University, Boston, Mass., and 2Department of Biomedical Engineering, Boston University, Boston, Mass.) We have successfully isolated nucleic acids from microliter and submicroliter volumes of human blood, urine and stool using polymer/silica composite microscale solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here we quantify our ability to isolate and purify genomic DNA and mRNA via solid-phase extraction in this plastic microfluidic system. The microfluidic chips were fabricated in a cyclic polyolefin by hot-embossing with an electroformed master-mold. The solid-phase consisted of a photopolymerized microporous monolith embedded with functional microparticles (appropriate to each isolation application) and covalently attached to the channel walls via photoinitiated grafting. For DNA purification, the polymer monolith was impregnated with silica particles, and the extraction was achieved due to the preferential binding of nucleic acids to the silica particles in the presence of chaotropic salts. In order to assess the performance of the monoliths, extractions of human genomic DNA from whole blood samples were performed. The extraction efficiency of the system was approximately 60% and the nucleic acid binding capacity of the solid-phase was found to be approximately 3.5 ng. The solid-phase system was also used for the extraction of mRNA by embedding oligo(dT) beads in the porous monolith. The system was able to extract mRNAs of both abundant and rare genes from isolated total RNA as well as from whole cell lysates. The micro solid-phase extraction technology presented lays the groundwork for a high-throughput nucleic acid sample preparation platform that can be coupled with a downstream amplification/detection module to form an integrated molecular diagnostic device.

Figure: Plot of the amount of recovered DNA as a function of the concentration of the loaded sample. 60% yield is achievable in the 2 ng concentration range. The bottom right corner is an SEM of a representative solid phase extraction column.

Poster 52 Withdrawn

Poster 53 A Molecular‐based Biosensor Configured in a Manual/Rapid Format. Koss H1, Latorra D1, Weed D2, Schraufnagel J1, Klonoski J1, Torres R3, Jenison R1. (1Great Basin Scientific, Longmont, Colo., 2Denver Health Hospital, Denver Colo., 3Utah State University, Logan, Utah.) The incidence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically increased over the past decade. If not diagnosed and treated appropriately, these human pathogens can cause a range of life-threatening illnesses including septicemia and toxic shock syndrome. Early diagnosis and treatment of these infections has been associated with improved outcomes in patients. Clinical outcome studies have shown that reducing the time to diagnosis decreases the patient’s length of stay, leading to significant cost savings to the hospital, and reduces morbidity and mortality. Recently, two relatively rapid molecular-based tests for detecting MRSA have been introduced to the clinical setting. These tests rely upon using a real-time PCR platform requiring expensive instrumentation that may not be available in smaller hospitals or point of care settings. We describe here the development of a novel and inexpensive on-chip amplification/detection platform. The amplification is isothermal at 65 C, requiring only a simple heat block, and the signal is permanent and detectable by the naked eye on simply configured silicon chips. Analytical sensitivity of 10 copies of genomic DNA per sample has been demonstrated. From either a 10 uL blood culture aliquot or a nasal swab, organisms are extracted, amplified and detected within 60 minutes. The test may be done manually, with fewer than 12 total steps and 10 minutes active time, so it is configured appreciably like simple rapid tests used currently to detect primarily respiratory viruses. We recently validated the platform by testing 22 clinical blood culture samples. The described method showed 100% concordance for identification of MRSA with standard microbiological methods that take 24-72 hours to determine a result.

Poster 54 Withdrawn

Poster 55 Spontaneous amplification of surrogate markers: using bacteriophage for MRSA screening. Sportmann BP, Steinmark T, Dreiling B, Bush D, Rees J, Smith BC, Izzo M, Fiechtner M, Smith D. (MicroPhage, Inc., Longmont, Colo.) Background: The rapid increase in community- and hospital-acquired MRSA infections is driving a large increase in demand for screening potential MRSA carriers. Molecular technologies, especially PCR, have been developed to capitalize on this demand. Although potentially rapid and accurate, PCR requires complex and expensive instrumentation that is not accessible to many health care institutions. Bacteriophage growth is a form of exponential amplification that can attain high signal levels in a few hours with inexpensive reagents (broth) and minimal instrumentation (an incubator). Combined with low-cost dipstick-type detectors, bacteriophage amplification technology provides a means for simple, accurate detection of MRSA carriers. Methods: Clinical isolates were spiked on to nasal swabs using “challenge” conditions: low (500 cfu) levels of target MRSA strains, and high (50,000 cfu) levels of MSSA and coagulase-negative Staphylococcus non-target strains. The test panel consisted of 87 MRSA, 81 MSSA and 156 CNS strains. The swabs were incubated in two bacteriophage-containing broth mixtures, one with no antibiotic and one with cefoxitin. Bacteriophage amplification in the no-cefoxitin broth indicates the presence of S. aureus, and thus serves as an ID test. Amplification in the plus-cefoxitin broth additionally indicates methicillin-resistance. Results: Evaluation of test results after 10 hours incubation shows the bacteriophage amplification test to be highly accurate. Sensitivity and specificity for S. aureus identification were 96% and 99%, respectively. The methicillin-resistance test showed 91% sensitivity and 98% specificity. Incubation of the tests for longer periods resulted in increased sensitivity of MRSA detection, with only a small loss in specificity, mostly due to signal from methicillin-resistant S. epidermidis. These results were obtained using classical assay methods to detect bacteriophage amplification. A large subset of phage reactions were also tested in ELISA format using antibodies directed against the bacteriophage. Amplification was


15

readily detected by immunoassay with no false positives, yielding performance equivalent to classic methods. We have begun developing rapid-test immunoassay (dipstick-type) devices to further simplify the phage assay. Prototype devices show appropriate sensitivity with no evidence of positive or negative interference effects. Conclusions: We conclude that bacteriophage amplification technology has the intrinsic sensitivity and specificity required to create a highly accurate MRSA screening test. It also appears that low-cost rapid-test devices are adequate to serve as detectors for phage amplification. The combination of these two technologies should result in an MRSA surveillance and screening test that has high performance, low cost and minimal requirements for hands-on time and equipment.

Poster 56 MRI molecular imaging monitors downstream anti‐angiogenic effects of mTOR inhibition. Guimaraes AR1,2 , Ross R3,4, Figuereido JL1, Schultz, CP1,5 , Waterman P1,2, Weissleder R1,2.(1Center for Molecular Imaging Research, Dept. of Radiology, Massachusetts General Hospital, Charlestown, Mass., 2Center for Systems Biology, Massachusetts General Hospital, Boston, Mass., 3Lank Center for Genitourinary Oncology, Dana Farber Cancer Institute, Boston, Mass., 4Infinity Pharmaceuticals, Cambridge, Mass., 5Siemens Molecular Imaging, Siemens Medical Solutions, Chicago, Ill.) Introduction: Drugs inhibiting the VEGF and mammalian target of rapamycin (mTOR) pathways are approved for metastatic renal cell cancer (RCC). Although VEGF inhibition (e.g. sorafenib) has anti-angiogenic effects, less is understood about mTOR inhibition (e.g. rapamycin). Our group recently validated the use of magnetic resonance imaging (MRI) enhanced with magnetic nanoparticles (MNP) for the examination of tumor vascularity in vivo. Our aim was to evaluate in vivo, mTOR inhibition on the vascularity of a RCC mouse model using MNP enhanced MRI and to compare these effects to the established VEGF inhibitor, sorafenib. Method and Materials: One million CAK-1 RCC cells were subcutaneously implanted into each of 20 nude mice. When tumors reached ~750μl, four daily treatment arms began and continued for four weeks: (Rapamycin (mTOR inhibitor) 10mg/kg/day; Sorafenib (VEGF inhibitor) (high dose (80 mg/kg/day) and low dose 30 mg/kg/day); and saline control. Weekly MRI (4.7T Bruker Pharmascan) was performed before and after IV MION-48, a MNP. Vascular volume fraction (VVF), a surrogate marker of angiogenesis, was quantified as ∆R2 (from multi-contrast T2 sequences) and normalized to assumed muscle VVF of 3%. Linear regression compared VVF to histologic (CD-31 stained) microvascular density (MVD). Results: VVF correlated with MVD (R^2 0.95). Figure 1 demonstrates vascular volume fraction (VVF) parametric maps superimposed over T1 weighted images of mice visualizing in vivo VVF decreases in mice treated with rapamycin and sorafenib as compared to control (p<0.05). Quantitative VVF declined weekly with treatment and demonstrated dose-dependent changes with sorafenib; rapamycin was similar to high-dose sorafenib. Conclusion: This is the first in vivo demonstration of mTOR inhibitory, antiangiogenic effects. Work is ongoing to translate these animal protocols to human MRI scanners to evaluate for human clinical trials. The parallel development of in-vitro assays for biomarkers related to the tumor response could support such new imaging technologies during therapy. Funding: Dana Farber RCC SPORE Career Development Award

Poster 57 Detection of BCR‐ABL fusion proteins using a flow cytometry based BD™ Cytometric Bead Array assay system. Weerkamp F,1,2 Chen R,3 Ng P,1,2 Campbell K,3 Staal FJT,2 Nguyen B,3 Liu YP, 3 vanDongen JJM,2 1Dynomics, Rotterdam, NL, (2Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands, 3BD Biosciences, San José, Calif.) Objective: Detection of the BCR-ABL translocation t(9;22) is critical for diagnosis and selection of appropriate therapy for chronic myeloid leukemia (CML). The objective of this study was to develop an assay to detect the BCR-ABL fusion protein generated concurrently with the translocation using the BD™

Cytometric Bead Array (CBA) immunoassay system (San Jose, CA, USA). This system employs a suspension array of uniformly sized and spectrally discrete microspheres to capture and detect analytes using flow cytometry. The system is intrinsically equipped with multiplexing capability. Methods: The BCR-ABL fusion protein assay is a sandwich immunoassay composed of monoclonal capture antibodies (Abs) against the BCR portion of the fusion protein covalently coupled to CBA microspheres, and ABL-specific detection Abs labeled with phycoerythrin (PE). Cell lysates from cell lines either negative for the fusion protein or containing one of the three major forms (p190, p210, and p230) of the BCR-ABL fusion protein were used to determine assay specificity. Assay sensitivity was determined by spiking the reference cell line, K562 containing the BCR-ABL fusion protein, in a background of normal peripheral blood mononuclear cells (PBMCs) or white blood cells (WBCs). The median fluorescence intensity (MFI) of PE was measured with the BD FACSCanto™ II flow cytometer using BD FACSDiva™ software. The MFI is proportional to the concentration of BCR-ABL present in the cell lysate. Results: The flow cytometry based CBA BCR-ABL fusion protein assay showed high specificity with detection of 5 of 5 (100%) of BCR-ABL positive leukemia cell lines (TOM-1, LAMA-84, MEG-01, K562, and AR230) representing all the major fusion protein forms, and with negative results of 6 of 6 (100%) of BCR-ABL negative cell lines (KASUMI-1, 697, REH, RS4, NB4, and ME-1). Assay sensitivity was 0.1% K562 cells diluted in a background of PBMCs or 1.0% cells in WBC preparations. Intra-run and inter-run precision was <6.0%. An essential component of the fusion protein assay was the inclusion of a pretreatment reagent. Conclusion: The CBA immunoassay system offers a convenient, sensitive, and specific approach for the development of multiplexed assays for fusion proteins (eg, BCR-ABL and others), formed from reciprocal chromosomal translocations.

Poster 58 Towards personalized medicine: development of a multiplex gene expression panel for assessing therapeutic response in castration resistant metastatic prostate cancer. Rai AJ, Kamath R, Gerald W, Fleisher M, Scher HI. (Memorial Sloan Kettering Cancer Center, New York, N.Y.) Advancements in technology and refinements in procedures in recent years have led to great progress in gene expression profiling analyses. To date, numerous microarray studies have identified subsets of 5-25 gene targets which can separate two or more diagnostic groups. Progress to validate these biomarker signatures has been lacking, partially due to the paucity of technologies or established protocols to reproducibly demonstrate the classification power of the candidate genes of interest. We have designed a multiplex panel for the simultaneous detection of 17 genes whose expression is altered in castration resistant metastatic prostate cancer. These genes are involved in androgen receptor activation/signaling and steroid synthesis/metabolism. Gene selection was based on previous microarray analyses which identified aberrant pathways deregulated in castration resistant prostate cancer specimens and corresponding cell lines. We designed primer sets allowing amplification of fragments between 140-310 bp. After RT-PCR amplification, fragments are separated by capillary electrophoresis and visualized in an electropherogram (see Figure below). The area-under-the-curve (AUC) of each peak correlates with the abundance of a particular transcript. The detection limit is 3 ng RNA and intra-assay CV <14%. We demonstrate differences amongst expression profiles of tissues from castration-resistant metastatic prostate cancer patients, benign hyperplastic prostate tissue, non-prostate cancer patients, non-diseased human tissues, prostate cancer cell lines (LnCAP, PC3, and VCAP), and additional tumorigenic cell lines. This multiplex panel provides a tool for simultaneous detection of these 17 genes and this signature can serve as a validated biomarker to assess the effectiveness of new therapies targeting the androgen receptor and steroid metabolism pathways in castration resistant metastatic prostate cancer.

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17

The diagnostic platform is a low-cost, microfluidic biochip which enables simultaneous measurement of 6-8 biomarkers in a few drops of blood using a compact, high-sensitivity detection system. The chip is based on the Open Lateral Flow (OLF) platform developed by Åmic (Sweden). The polymer chip is injection-moulded and its key feature is a dense array of micro-pillars, which, by virtue of their spacing and surface chemistry, facilitate flow the blood sample along the chip by capillary action. An array of antibodies that selectively recognise the respective biomarkers is printed on the chip and advanced fluorescence techniques are used to quantify the binding of these biomarkers to the antibodies. We have developed a POC measurement system for CVD risk assessment based on a combination of the OLF technology and supercritical angle fluorescence (SAF). SAF is a technique in which the emitted fluorescence is only collected above the critical angle of the sample-substrate interface. This approach provides both high efficiency of signal collection and high surface selectivity, thereby discriminating against background signals. The resultant combination yields higher signal-to-noise ratios and lower limits of detection. In the first phase of this development, we have focused on a dual-analyte biochip comprising two fluoro-immunoassays for two of the markers on our CVD Risk Panel. We will present data from the platform that illustrates the high performance achievable in a system that is compatible with POC application using disposable polymer chips.

Poster 64 Label‐free epstein‐barr virus assay. Batliwala H, Vallejos PE, Gerner F, Regnier F, Lawrence G, Camarata C, and Harold N. (Quadraspec, Inc., West Lafayette, Ind.)

We developed a label-free, simultaneous multi-analyte interferometric immunoassays for the detection of Epstein-Barr Virus antibodies. The method can aid in the laboratory diagnosis of EBV-associated infectious mononucleosis. EBV, which infects >90% of the human adult population, is considered to be the classic example for immunesurveillance of persistent viral infections in humans. The assay results are compared with commercially available qualitative methods such as ELISA. We interrogated for antibodies using three antigens EBV viral capsid antigen, EBV early antigen, and EBV nuclear antigen. The interferometric method is called Spinning Disc Interferometry (SDI), and consists of an optical disc fabricated with hydrophobic barriers; these barriers form up to 272 incubation wells. The label free modality allows for ease of multiplexing and high throughput since secondary reagents are not needed. Titration curves are reported as well as results from a number of specimen samples.

Poster 65 The LiMA‐2 technology. A rapid method for bacterial detection. Van der Loos A, Wilson S, Stanley C. (ISEAO Technologies Ltd, , London, UK.)

We have reported earlier on the LiMA (Ligase Mediated ATP Assay) technology that detects the intracellular metabolite ATP using a molecular diagnostics (or ‘NAT’) readout (1). The original LiMA method uses an ATP-dependent DNA ligase to join two oligonucleotides in a nicked DNA substrate and create a primer that can start a DNA amplification reaction. The extent of DNA amplification is proportional to the amount of ATP present in the sample. LiMA was developed as a method for bacterial enumeration and viability determination on an existing molecular diagnostics detection platform, such as those available for PCR or TMA. The newly developed LiMA-2 technology described here is a variant of the Ligase Mediated Assay that uses the same molecular amplification platform as the original LiMA method but instead detects the bacterial enzyme, NAD-dependent ligase, rather than ATP (see Figure). NAD ligase is found exclusively in eubacteria (2) and has not been reported in mammals. Hence the LiMA-2 technology is ideal for use in the rapid and sensitive detection of bacteria in clinical samples where background from the host would otherwise be a problem (as is the case for the original LiMA method). An additional advantage of the LiMA-2 method is that there is a further amplification step in the system since NAD ligase generates many molecules of the DNA primer prior to NAT amplification. So LiMA-2 is a more sensitive approach than the original ATP-based method; currently the detection limit for E.coli and S. aureus is in the range 100 to 1000 cells in culture samples.

NAD ligase (bacterial source) plus NAD

PCRDetection by

1. The LiMA Technology: Measurement of ATP on a Nucleic Acid Testing Platform. Banin S, Wilson SM and Stanley CJ (2007). Clinical Chemistry 53, 2034-2036 2. Recent developments in ligase-mediated amplification and detection. Cao W (2004). Trends in Biotechnology 22(1), 38-44

Poster 66 A multiplexed microbead immunoassay for detection of ovarian cancer markers. Braun PJ, Shivraj L, Groelke JW, Harden AT, Russell DP, Hall LB, Swan RJ Jr, Venetta TM, Baker JP, Cheek RL, Gallagher P, Felton IS, Taylor AJ, Malinowski D, Fischer TJ. (BD Diagnostics/TriPath, Durham N.C.) Objective: Improvements are sought for diagnosis and treatment of ovarian cancer via development of improved assays for early detection of ovarian cancer, differentiation between benign and malignant tumors, therapeutic monitoring, and detection of recurrent cancer. Recent studies suggest that the sensitivity and specificity for these diagnoses could be improved through use of multiple biomarkers. The objective of this study was to investigate preliminary development of a multiplexed immunoassay for ovarian cancer biomarkers in an efficient, automatable test format. Methods: Three serum proteins were selected as candidate markers, based on previous testing using conventional immunoassay methodology (ELISA). Markers included cancer antigen 125 (CA-125), human epididymus protein 4 (HE 4) and glycodelin. Suitable antibody pairs were identified based on ELISA screening, and antibodies were conjugated to MagPlex ™ magnetic microbeads and phycoerythrin fluorescent tag. Testing was performed on the Luminex 200 platform. Assays were initially tested in single-plex mode using antigens in buffered media, then combined into tri-plex format for assay of serum samples. Assay format consisted of incubation of test samples with microbeads, followed by washing, incubation with fluorescent Ab-conjugate(s), final wash, resuspension and data acquisition. Preliminary assessment of analytical performance included testing of precision, range and sensitivity, dilutional linearity, and recovery of spiked antigen, tested according to CLSI guidelines where applicable. Preliminary assessment of clinical performance included testing of normal post-menopausal sera (n = 38), specimens from patients with benign masses (n= 19), ovarian cancer stage 1-4 (n=36) and other cancers (n=3) using the triplex immunoassay as well as reference ELISA methods. Results: All markers could be measured at medically relevant levels, with sensitivity as low as 100 pg/ml. Linear recovery of all markers was estimated at 70-120% based on antigen spiked into normal human serum. Serum specimens showed linear dilutional recovery for dilutions of 5-fold and greater. Total imprecision for controls with elevated antigen was estimated at 6-20% CV. Within-run imprecision was estimated at 2-8% CV. Testing of clinical specimens indicated that the three individual markers were elevated in the majority of ovarian cancer sera, and not elevated in the majority of normal sera or patients with benign masses. Correlation of multiplex results with reference ELISA methods showed linear correlation coefficients (r^2) of 0.71-0.98. Concordance with reference methods using a diagnostic cutoff of mean of normals + 2SD was 88-91%. Conclusions: Results indicate that multiplexed immunoassay for ovarian cancer markers including CA125, HE4 and glycodelin can be adapted to a microbead assay format and that elevated values are confirmed in sera from patients with ovarian cancer. This system can support further testing to determine the utility of multiple biomarkers for detection and monitoring of ovarian cancer.


18

Poster 67 On‐board pneumatic valves and reservoirs for multiplexed, contained assays. Levine LM, (McDowell J. ALine, Inc., Redondo Beach, Calif.) Complex assays that require multiple steps that include dispensing of reagents and washing, require multiple fluid streams. For compact, low dead volume applications, this in turn requires reagents to be integrated with the device, and for dispensing between different fluid streams to be performed using valves integrated into the device. We have developed a flexible pneumatic valve and method of dispensing from on-board reservoirs that addresses this need for a four-plex analysis platform. Our focus is on the development of scalable, repeatable manufacturing processes that allow this functionality to be used for the development and commercialization of self-contained, disposable devices. Using our precision laser cut and bonded laminate fabrication process, we demonstrate the performance of a device suitable for multiplexed assays.

Poster 68 & 69 Withdrawn

Poster 70 Detection of multiple cystic fibrosis transmembrane conductance regulator (CFTR) mutations using high resolution thermal melt analysis on a microfluidic chip. Mason JA1, Boles DJ1, Pryor RJ2, Poulson MD2, Wittwer CT2, Inoue H1, Knight IT1. (1Canon U.S. Life Sciences, Inc., Rockville, MD and 2The University of Utah, Salt Lake City, Utah.) Cystic Fibrosis is an autosomal recessive genetic disease that is caused by mutations in the CFTR gene. The current method of detection of this disease is via a positive sweat chloride test followed by sequencing of the 23 mutations recommended by the American College of Medical Genetics which is very costly and time consuming. We have utilized an unlabeled probe assay followed by high resolution thermal melt analysis to distinguish genotypes by differences in melting temperature using a saturating double stranded DNA binding dye (LC Green Plus). This method utilizes PCR primers and a 3’ phosphate-modified probe that covers the mutation of interest. As the DNA is denatured, the intercalating dye is released from the helix and becomes non-fluorescent in a

sequence dependent manner. Differences in the probe melting temperatures indicate the presence or absence of a mutation. We have developed a microfluidic lab-on-a-chip system capable of differentiating homozygous, heterozygous and compound heterozygous mutants of all 23 CFTR ACMG mutations.

Figure 1: A 93 bp PCR product was generated in the presence of LC Green Plus from samples representing wild type, heterozygous and double heterozygous CFTR human DNA. The DNA samples were loaded on a microfluidic chip mounted in a modified Caliper LabChip 3000S instrument. A 15 nL volume of PCR reagents and DNA samples were first loaded onto the microfluidic chip where they were mixed on chip via lateral flow and amplified while continuously flowing through the microchip. Fluorescence was monitored in the microfluidic channel during thermal denaturation and the melting temperature was extrapolated using a third party software system. The results above display four melting profiles of wild type and mutations in Exon 11 that were accurately genotyped on our microfluidic chip. The thermal melt profiles for the unlabeled probe that binds to the 93bp product are shown above and represent 7 different human genomic DNA samples.

Poster 71 Rapid and sensitive detection of RNA in live cells using molecular beacons. Bao G, Santangelo P. (Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Ga.) The ability to detect RNAs, including mRNAs and viral RNAs in living cells with high sensitivity offers tremendous opportunities for biological and disease studies. Here we describe a novel method to directly detect the viral genome of a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells using molecular beacons, which are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other. They are designed to fluoresce only when hybridized to a complementary target. By imaging the fluorescence signal of molecular beacons, the spreading of bRSV was monitored for 7 days with a signal-to-noise ratio of > 50 (Fig. 1), and the measured time-course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2x103.6 TCID50/ml diluted a thousand-fold, demonstrating high detection sensitivity. Low background in uninfected cells, and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. We also observed the dynamics of assembled viral filaments, and characterized viral RNA dynamics by imaging the 3D distribution of cellular vRNA. Taken together, this new live-cell-based method provides a powerful tool for rapid viral infection detection, basic virology studies of RNA viruses, and the design of new antiviral drugs.


19

Figure 1. Live-cell fluorescence imaging of bRSV viral genome following the progression of infection for 7 days post-infection (PI). Molecular beacons were designed to target repeated sequences that allowed for the binding of 3-9 molecular beacons to each viral genome, providing signal amplification and significantly increasing the signal-to-noise ratio.

Poster 72 Chemically‐heated non‐instrumented nucleic‐acid amplification assay platform. Weigl BH1, Domingo GJ1, Gerlach J1, Wilmoth J1, Tang D1, Harvey D1, Talwar N1, Fichtenholz A1, van Lew B1, LaBarre P1. (1PATH, Seattle, Wash.)

Several pathogens that impact global health are best diagnosed through pathogen-specific nucleic acid (NA) detection. Making nucleic acid amplification tests (NAATs) accessible to underserved populations in low resource settings would have great benefits. This can be done either by enhancing centralized laboratory testing or by facilitating NAATs at the point of care (POC) or primary health care facilities. We have developed components of a diagnostic disposable platform that provides both molecular diagnostics at POC as well as a stabilized specimen for transport to a centralized surveillance system. Minimally trained health workers can operate the disposable with no additional instrumentation. The system will be almost as rapid and simple as a lateral flow strip test, while offering the sensitivity and specificity of a NAAT. The low-cost integrated device has three components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing NAs, (2) an isothermal NA amplification subunit based on the LAMP methodology, and (3) a visually read amplicon detection subunit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal NA amplification. The aim of this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used, but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics. Patients would be tested with the disposable in the field, and a NA sample would be preserved within the spent disposable, which could then be sent to a central laboratory facility for further analysis if needed.

Figure 1: Prototype (left) and schematic (right) of a manually operated, chemically heated nucleic acid amplification disposable diagnostic platform.

Poster 73

Rapid isothermal DNA amplification and visual detection of herpes simplex virus for point‐of‐care applications. Ferguson TM1, Tan E1, Erwin B1, Dames S2, Voelkerding K2,3, Niemz A1. (1Keck Graduate Institute, Claremont, Calif., 2ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, 3University of Utah, Department of Pathology, Salt Lake City, Utah.) Novel nucleic acid technologies are needed for the rapid and specific diagnosis of human pathogens in a simple, inexpensive, and user-friendly format. Rapid detection of Herpes simplex virus (HSV) infections is important for HIV positive or immuno-compromised individuals, pregnant women, and newborns. Isothermal DNA amplification methods facilitate DNA detection with less complex instrumentation than PCR. Rapid isothermal amplification can be accomplished through the Exponential Amplification Reaction (EXPAR), and related methods. In the herein reported assay, a short, specific trigger oligonucleotide is generated from target genomic DNA, is exponentially amplified, and converted to a reporter oligonucleotide. The reporter can aggregate DNA functionalized gold nanospheres in solution resulting in a visually detectable color change. Using this approach, we recently reported detection of 1 pM (6 million copies in a 10 μl reaction volume) of a recombinant vector model system containing an HSV-1 insert in less than 10 minutes total assay time. In the current study, we have further optimized the assay and are able to detect as low as 10 aM (60 copies in a 10 μl reaction volume) of our HSV-1 vector model system. This improved performance has enabled us to implement the assay starting from HSV-1 genomic DNA (104 copies in a 10 μl reaction volume), and from DNA isolates of HSV-1 positive clinical swab samples in less than 10 minutes with minimal equipment needs. We also since implemented the assay using nucleic acid lateral flow as an alternate detection scheme, with similar sensitivity. Therefore, EXPAR coupled with visual detection offers an appealing approach for rapid pathogen detection in point of care based clinical diagnostic applications.

Poster 74 Exploiting the non‐linear properties of magnetic beads enables robust and versatile diagnostic bioassays. Nikitin M, Legastelois S, Lenglet L. Magnisense SA, Paris, France. We introduce an innovative rapid test platform, which offers high sensitivity in a simple field/POC format. Our approach utilizes the non-linear response of nanometric sized magnetic beads in a fluctuating magnetic field to effectively isolate signal from noise. Our goal was the development of a more sensitive and robust method of counting magnetic beads which can accommodate a variety of affordable immunoassay formats, and is compatible with immunoconcentration and magnetic enrichment of analytes. The presence of magnetic beads in a sample can be detected by different sensors thanks to their magnetization response. However , approaches which detect a linear magnetization response encounter several limitations which have prevented them from being adopted at the POC. These approaches (e.g., GMR, Hall sensors, coils) intrinsically exhibit a poor sensitivity as the undesirable contribution of water, sample holders, migration or filtration media to the measured signal may equal or even be higher than the specific signal of small quantities of target magnetic material. Attempts to overcome this limitation have resulted in complicated compensation systems requiring moving parts (coil sensors) or microfluidics (GMR sensors). Additionally, because of this large background magnetic noise, these approaches have not capitalized on the unique quality of magnetic fields to pass through sample matrix, plastics, water, etc. Thus no magnetic technology has yet enabled a true volumetric detection. Magnetic beads (MBs) are exposed to an alternating magnetic field at two frequencies f1 and f2. The amplitude A1 of the field component with the lower frequency f2 is chosen high enough to magnetize the beads to saturation (Fig.1). The component periodically “switches” on and off the capability of MBs to further magnetize. When MBs can be further magnetized, the higher frequency component f1 will contribute to the resulting induction signal. As a result, the response signal U(t) will be modulated by both frequencies (Fig.1).We measure the response at combinatorial frequencies fi = m·f1+n·f2. The values of m and n may be varied in order to achieve the best signal-to-noise ratio (SNR). For many MBs, it is not necessary to reach complete saturation by A1, and small non-linearity of the beads provides sufficiently high SNR ratio, for example, at f = f1


20

± 2·f2. The maximal SNR was realized for such amplitude A1 when the MBs were magnetically saturated for about half of time. For biosensing, SNR of f is much higher for two ac frequencies, which allowed us to measure a relative change of magnetic susceptibility at the level of up to 10-8 and use this principle for MB counting. The response at the combinatorial f is proportional to the amount of non-linear magnetic material (or the number of MBs) provided the beads have small size variance.

Poster 75 The ultrasensitive NADiA® total PSA (tPSA) assay reliably predicts prostate cancer recurrence after radical prostatectomy (RP). McDermed J1, Klem R2, Saunders R2, Sanders R2, Adams T2, and Diamandis E3. (1Iris Diagnostics, Chatsworth, Calif., 2Iris Molecular Diagnostics, Carlsbad, Calif., 3Mount Sinai Hospital, University Health Network and Toronto Medical Laboratories, Toronto, ON, Canada.) Background: Clinical studies show that biochemical relapse (BCR) can be detected earlier by monitoring changes in serum tPSA using ultrasensitive tPSA assays. However, the functional sensitivity of such assays is limited and cannot reliably report tPSA levels <0.01 ng/mL (10 pg/mL). The NADiA tPSA assay’s functional sensitivity is 0.5pg/mL (0.0005ng/mL, Clin Chem 53(6)Suppl., 2007, #C-15) and may be able to provide more reliable detection of early BCR. Objectives: To measure serial serum tPSA levels in post-RP patients and compare results against those from an ultrasensitive immunofluorometric (IFM) assay, and to determine concordance with their clinical outcome. Methods: Serum samples (N=435) leftover from a previously published study (J Urol 157:913-8, 1997) were used in this study. Samples were collected in 1993 and 1994, stored frozen at -40°C and had results reported using the IFM tPSA assay, the performance of which was described previously (Clin Chem 39:2108–14, 1993). The 85 patients studied had baseline tPSA <100pg/mL post-RP (Abbott IMx) and >3 post-RP serial samples (mean 5.0, median 5, range 3-6). Median (range) age was 63 years (51-73), pre-RP tPSA was 7.1ng/mL (0.1*-49.0), Gleason score was 7 (5-9) and % tumor involvement was 25% (1-90%). Clinical stages were T1a-c (16), T2a-b (34) and unknown (33). 14 patients received pre-RP therapy (hormones=12; radiotherapy=2). The median (range) nadir or first tPSA value post-RP was 4.1pg/mL (0.2-147pg/mL). BCR was defined as >2 successive IFM tPSA increases reaching >100pg/mL, with relapse backdated to the first tPSA increase. tPSA values for stored specimens were re-analyzed by the IFM method and showed no significant differences compared to original values. Results: Eighty-four (98.8%) patients were evaluable for biochemical evaluation and 60-70% of them were evaluable clinicopathologically. Median (range) age was 63 years (51-73), pre-RP tPSA was 7.1ng/mL (0.1*-49.0), Gleason score was 7 (5-9) and % tumor involvement was 25% (1-90%). Clinical stages were T1a-c (16), T2a-b (34) and unknown (33). Fourteen patients received pre-RP therapy (hormones=12; radiotherapy=2). The median (range) nadir or first tPSA value post-RP was 4.1pg/mL (0.2-147pg/mL). Comparing stable vs. BCR patients, univariate analysis revealed significant differences in Gleason score (p=0.0276), % tumor involvement (p=0.0008), margin involvement (p=0.0006), peri-prostatic tissue invasion (p=0.0006), extracapsular extension (p=0.0181), nadir or first tPSA value post-RP (p=0.0001), and PSA doubling time (p=0.0001). Multivariate analysis revealed nadir or first tPSA and PSA doubling-time to be independent predictors of PSAR. The correlation between NADiA and IMF tPSA methods was excellent (R2=0.92 [95% CI: 0.88-0.96], Slope=1.29, y=-0.8). Conclusions: The ultrasensitive NADIA tPSA assay can reliably measure tPSA concentration as low as 0.5pg/mL providing precise PSA nadir results and PSADT calculations. On average, NADIA detected a rising tPSA 34 months before the tPSA value reached 100pg/mL (0.1ng/mL).

Poster 76 Comparison Between Label Free Internal Reflection Ellipsometry and Commercial ELISA Diagnostic Kits for Autoimmune Disease. Dultz S. Maven Biotechnologies, Pasadena, Calif. Label free internal reflection ellipsometry (LFIRE) in a multiplexed format was used in a feasibility study for comparison with existing commercial ELISA kits to

measure individual biomarkers of autoimmune disease. The detection of autoantibodies associated with occurrence of autoimmune disease is important for diagnosis and lack of single marker disease specificity often requires measurement of multiple autoimmune markers for accurate diagnosis. LFIRE is a new immunoassay detection platform characterized by the rapid time-resolved detection of polarization changes of reflected monochromatic light due to interactions with biomolecules near a glass-liquid interface, permitting simultaneous detection of multiple ligand binding interactions in-situ. For a Lupus (SLE) panel, microarray spots of purified RNP (calf thymus), SSB, SCL70, SSA and Smith antigen were spotted on our high sensitivity slides and a flow cell configuration was used (Fig. 1A) to measure both end-point binding and real-time kinetics. Thirty patient sera were diluted in buffer and assayed using commercially available multi-step ELISA kits and LFIRE single-step detection for multi-plexed auto-antibodies. All results were compared to the reference cutoff used by the ELISA manufacturer and a single normalization factor was used to compare with LFIRE signals Figure 1B depicts a subset of data for one of the five Lupus antigens (RNP) where the commercial ELISA positive cutoff was 25 units. 100% concordance was obtained for the 15 samples tested in the determination of positives and negatives. Statistical analysis showed the signals to be highly rank correlated as well (Spearman r = 0.01, p < .01). For 150 total results, concordance between ELISA and LFIRE was > 99%. Our results demonstrate the utility of LFIRE for rapid screening of autoantibodies in disease, with significant advantages in reduced assay time and cost.

Poster 77 Withdrawn

40th

Annual Oak RRidge Confereence – April 17–18, 2008 Pooster Abstractts

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Poster 83 Third generation POC technology based on highly sensitive fluorescent assay system. Richard Egan1, Stan Vukajlovich1, Randy Madsen1, Alexander Belenky1, Barry Bluestein1, David Booker1, Wendy Benson2, Graham Lidgard1. 1.(Nanogen, Inc, San Diego, CA: 2, HX Diagnostics, Emeryville, Calif.) Nanogen independently and with development partner HX Diagnostics and with support from CDC has developed a novel highly sensitive and simple to use Point Of Care (POC) assay system suitable for use with a wide variety of analytes. This system has been designed to detect multiple analytes at high sensitivity from a single sample. The assay was designed around low cost lateral flow technology using nitrocellulose as the fluid transport medium. The assay system is in advanced stages of development and is composed of a low-cost, icon-driven fluorescent reader that has been designed to easily interface with the Test Device (TD), read, analyze and report the result by analyte. The TD contains a wicking pad to quickly adsorb the extracted specimen and assay reagents, a wash buffer packet, and nitrocellulose striped with multiple detection reagents. A sampling device containing extraction reagent designed to use with swabs, is described as an example. The Sample Collection Device is a fully integrated device containing both sample extraction reagents and assay chemistry. A unique feature of the system is the use of synthetic nucleotide polymers (non-DNA and non-RNA), termed pRNA, that have been designed to bind at high affinity with its homologous binding partner. The assay can use one or many such pRNA pairs where one member of a pair is striped onto the nitrocellulose and the second member of the pair is conjugated to a capture reagent. For a protein analyte, detection is facilitated using a sandwich approach where the analyte protein bridges between the detection and capture MAb leading to an accumulation of fluorescent microbeads at the specific analyte test line. The capture and detection reagents are separately lyophilized into small assay reagents pellets. Two separate assay systems were developed and demonstrate the capability of the assay format and detection system. One assay system was designed to maximize sensitivity while the second assay was designed to detect at high sensitivity multiple analytes of Influenza (Type A, Type B, H1/H3 and H5 specific antigens). Studies to date have demonstrated the high sensitivity of the system by showing an LOD of 0.36 pg/mL of NT-proBNP after 15 minute incubation with a limit of quantitation of about 1pg/mL. The multianalyte assay for influenza, in advanced stages of development, is showing a sensitivity for Influenza Type A and Type B, for example, that is 2 logs more sensitive than a currently marketed lateral flow assay. In summary, Nanogen with support from the CDC and HX Diagnostics, has developed a third generation low cost highly sensitive, multianalyte POC system with a supporting low cost Reader yielding final results in 20 minutes or less. We acknowledge significant support from the U.S. Department of Health and Human Services (HHS), the Centers for Disease Control (CDC), and the Office of Assistant Secretary for Preparedness and Response (ASPR) for advanced product development under Contract No. HHS200-2007-19345.

Poster 84 Rapid antimicrobial resistance detection using DNA chips. Leinberger DM1,2, Grimm V2, Rubtsova M3, Henn A2, Egorov A3, Schröppel K4, Wichelhaus TA5, Knabbe C3, Schmid RD2, Bachmann TT. (1Division of Pathway Medicine, University of Edinburgh Medical School, Edinburgh, UK, 2Institute of Technical Biochemistry, University of Stuttgart, Germany, 3National Research Center for Antibiotics, Moscow, Russia, 4Department of Clin. Chem. and Laboratory Medicine, Robert Bosch Hospital, Stuttgart, Germany, 5Institute of Medical Microbiology and Hygiene, J.W. Goethe‐Universität, Frankfurt am Main, Germany.) Conventional, culture based antimicrobial susceptibility testing methods are too slow to enable prospective therapy and sometimes error-prone. Molecular methods such as DNA chips open the way for rapid testing. Microbial resistance often stems from the occurrence of Extended Spectrum Beta-Lactamases (ESBL). The majority of ESBL variants derive from the genes blaTEM-1 or blaSHV-1 by mutations that alter the amino acid configuration around the active site or from the acquisition and mutation of genes from the CTX-M gene family. In the past

decade OXA lactamases and plasmid-mediated AmpC lactamases have emerged in the clinics as a new severe problem as they are likely to hydrolyze modern cephalosporins. Here we describe the development and application of fluorescence based diagnostic oligonucleotide microarrays for the genotyping of TEM, SHV, and CTX-M beta-lactamase variants based on allele specific hybridization. To increase the coverage of detectable lactamase species, we also developed new chip modules to genotype plasmid-mediated AmpC and OXA-type beta lactamases [2]. To present a single all-in-one beta-lactam resistance chip for the clinic, we integrated individual chip modules (TEM, SHV, CTX-M) to a single ESBL chip platform [3]. The chip was validated using target DNA originating from 60 clinical samples of different gram negative organisms, which were phenotypically characterized as ESBL. The detected variants included TEM-1, SHV-1, SHV-5, SHV-12, CTX-M3, CTX-M9, CTX-M14 and CTX-M15. Analytically difficult samples were correctly identified such as mixed resistances of various genotypes. All ESBL phenotypes could be ascribed to the presence of a CTX-M variant (78%) or SHV variant (22%), whereas no ESBL TEM variant was found. As the total assay could be performed in less than 4 hours it offers a highly competitive alternative to conventional identification of beta lactam resistance within the clinical routine diagnostics. Future routes of further assay integration (e.g. sample prep) and commercialisation will be discussed. [1] Grimm V, Ezaki S, Susa M, Knabbe M, Schmid RD, Bachmann TT; Rapid resistance genotyping of TEM beta-lactamases using DNA-microarrays, J Clin Microbiol, 2004, 42: 3766-3774 [2 - 3] Leinberger D et al. unpublished

Poster 85 Discovery of SLE autoantibody biomarkers utilizing protein microarray technology. Mattoon DR1, Smith M1, Brodey M1, Chen G1, Alcorta D2, Patel D3, Falk RJ4, Schweitzer B1. (1Protein Microarray Center, Invitrogen Corporation, Branford, Conn., 2University of North Carolina, Division of Nephrology and Hypertension, Department of Medicine, Chapel Hill, N.C., 3Novartis, 4University of North Carolina, Division of Pathology and Laboratory Medicine, Chapel Hill, N.C.) The presence of autoimmune disease-associated autoantibodies prior to the presentation of clinical symptoms has been well-documented, and can be exploited for the development of multiplex assays offering improved sensitivity, selectivity, and reproducibility over currently available immunodiagnostics. This study utilized high-content protein microarrays comprised of more than 5,000 human proteins, including 25 known autoantigens, to evaluate immunological profiles across panels of serum samples derived from healthy donors and Systemic Lupus Erythemasosus (SLE) patients. Three statistical approaches were applied in parallel, utilizing M-statistics, volcano analysis, and fold change calculations applied to quantile normalized or unnormalized signal intensity data, to identify candidate biomarkers. Independent expression and purification of these putative autoantigens was carried out in order to develop both custom protein microarrays and Luminex bead sets for validation studies. A validation rate of approximately 70% was observed across both technology platforms when the same set of disease and normal serum samples were used as probes. Improved discrimination between the two populations was observed when Principle Component Analysis was applied to data derived from novel, protein microarray-defined proteins relative to autoantigens with annotated associated with SLE. Leave-one-out cross-validation analysis using support vector machine learning calculated a classification error rate of 3.3% for the array-defined candidate biomarkers, relative to an error rate of 13.3% calculated for the annotated SLE biomarkers. Taken together, this study provides the experimental and statistical framework to support the adoption of protein microarray technology as a tool for immunological profiling for disease biomarker discovery. The protein microarray technology has been validated for use in developing immunological profiles for a number of biological fluids in addition to serum, including plasma, urine, saliva, tears, and aqueous humor. Data supporting each of these applications will also be presented.


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Poster 86 A highly parallel flow cell enabling multi‐channel sensing in diagnostic applications. Eddings MA1, 2, Miles A1, Liu JP2, Myszka DG2, Shumaker‐Parry J2, Eckman JW1, Sams G1, Gale BK1, 2. (1Wasatch Microfluidics, Salt Lake City, Utah, and 2University of Utah, Salt Lake City, Utah.) A highly parallel microfluidic flow cell array has been demonstrated for a range of diagnostic applications. The flow cell array can be used with a variety of sensing platforms and can quickly convert a sensing technology into a high throughput diagnostic tool by enabling the analysis of multiple samples in parallel. The flow cell array is based on a microfluidic platform for manufacturing protein, lipid, and cell microarrays, called the Continuous Flow Microspotter (CFM), which has been shown to produce spots with less than a 3% CV between spots, increasing the accuracy of microarray based assays and expanding their use in diagnostic applications. Assay sensitivity for protein-protein reactions has been shown to improve by a factor of up to 20,000, as shown in Figure 1a. Assay performance times have been improved up to 100 fold. In addition, the flow cell array has shown the ability to selectively print proteins onto sensor surfaces directly from crude samples, circumventing the need for costly sample preparation steps while simultaneously providing high sensitivity protein microarrays for diagnostic assays. The flow cell array also enables an “array of arrays” concept wherein multiple pre-printed subarrays of binding ligands can be addressed by a network of flow cells. By passing different patient samples through each flow cell, a multiplex screen of 48 patient samples can be performed in parallel on a label free platform, rivaling the throughput of current fluorescent multiplex diagnostic instruments while removing the need for expensive labeling reagents. Figure 1b presents real-time SPR binding curves which illustrate the performance of multiple label-free binding events in a parallel fashion. Similar results have been obtained for a variety of assays including biomarker measurement from serum samples. This technology enables the high-throughput label free screening of rare proteins, such as biomarkers, engineered antibodies, or protein-based drugs from serum. Overall, the flow cell array enables greater multiplexing and higher throughput performance of diagnostic assays, while improving sensitivity and decreasing analysis times.

Figure 1. Representative data from CFM and flow cell array (a) Comparison of printing methods for an anti-protein assay showing a 20,000 fold increase in sensitivity using CFM printing compared to an optimized pin printing protocol. (b) Protein binding assay completed in 12 channels simultaneously on an SPR instrument. Poster 87 Evaluation of a new multiplex PCR‐based device for rapid pathogen detection in post‐surgical trauma and burn patients with suspected septicemia. Tran NK1, Galante JM2, Wisner D2, Battistella F2, Louie RF1 ,Greenhalgh D3, Albertson TE4, Cohen S5, Kost GJ1.1UC Davis,( Lawrence Livermore National Laboratory Center for Point‐of‐Care Technologies, Divisions of 2Trauma and Emergency Surgery, 3Burn Surgery, 4Pulmonary and Critical Care Medicine, and 5Infectious Disease, Livermore, Calif.) Introduction. Sepsis is defined as two or more manifestations of the systemic inflammatory response syndrome (SIRS) associated with a suspected or identified source of infection. SIRS includes: (a) hypothermia (<36°C) or hyperthermia (>38°C), (b) tachycardia (>90 beats/min), (c) tachypnea (>20 breaths/min) or a PaCO2 <32mmHg, and (d) leukopenia (<4,000 cells/μL), leukocytosis (>10,000 cells/μL) or >10% band forms. Inadequate antimicrobial therapy during the first 24-hours of hospital stay significantly contributes to the high mortality (25-50%) in septic patients. Traditional blood culture methods are too slow. BC pathogen speciation times may range from 13.3 to 376.1 hours (mean ± SD, median = 53.4 ± 36.1, 46.3). Rapid multiplex polymerase chain reaction (PCR)-based technology can achieve faster turnaround times (5-6 hours) to facilitate early evidence-based treatment. Methods. We are evaluating a multiplexed PCR-based instrument for early detection and differentiation of 25 bacterial/fungal pathogens frequently associated with bloodstream infections in post-surgical trauma and burn patients. Key pathogens on our assay include: Acinetobacter baumanii, coagulase negative Staphylococcus (CoNS), Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella oxytoca/pneumoniae, E. coli, and Candida albicans. Serial whole blood samples (1.5mL) from 50 high-risk patients are collected with BC and compared to paired BC results. DNA primers specific for bacterial and fungal internal transcribed spacer regions are targets for amplification via hot-start PCR. Fluorescent resonance energy transfer (FRET) coupled to DNA melting point analysis is used for pathogen speciation. Melting point cut-off values are evaluated to identify the resolution between contaminating CoNS versus real CoNS infections. The significance of FRET signal overlap for poorly resolved Klebsiella oxytoca and Klebsiella pneumoniae melting curves, a common pathogen in burn patients, is


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also being determined. Following testing of fresh samples, all positive PCR samples are archived and frozen (-25°C) for 30 days. Frozen samples are retested to determine the recoverability of pathogen DNA. PCR inhibitory effects due to white-blood cell (WBC) counts <1,000 cells/μL or >30,000 cells/μL (manufacturer specifications) are evaluated using samples from patients experiencing leukopenia and leukocytosis. Results. Preliminary results show PCR cut-off values correctly identifying false positive results. PCR inhibition did not occur when testing a sample with a WBC count of 1,800 cells/μL. All but one PCR result agreed with BC where one PCR sample set was positive and BC was negative for a single pathogen. Pathogen DNA was recovered from the positive frozen PCR samples. Conclusions. Pre-determined melting point cut-off values appear adequate for screening out CoNS contamination. Low WBC counts did not inhibit the PCR reaction, detection of pathogen DNA or PCR controls. Overall agreement between PCR and BC for fresh and frozen samples were acceptable. Results from this study will properly focus multiplexed PCR-based pathogen detection technology on targeted surgical patient populations. Poster 88 Localized surface plasmon resonance (LSPR) as a novel detection method for IVD tests. Huber M, Gerion D.( LamdaGen Corporation, Menlo Park, Calif.) Introduction: LamdaGen Corporation has developed a process to manufacture biosensors, which exhibit strong shifts in wavelengths in response to changes at their surface due to localized surface plasmon resonance (LSPR). The surfaces are robust and stable and can be grown on a variety of substrates. Biomolecules can be directly adsorbed onto the sensor surfaces or preferably the sensors are coated with a layer, which minimizes non-specific binding, while providing functional groups for ligand immobilization. A binding event at the sensor surface leads to a wavelength shift of the plasmon’s absorption maximum. The shifts can be quantitatively observed in real-time using a spectrophotometer and appropriate software. Or, with amplification, wavelength shifts can be visually monitored with the naked eye. Hence, traditional ELISAs can be performed on the LSPR biosensors with results visually detected to obtain qualitative or semi-quantitative data. Alternatively, quantitative measurements can be made with the use of an instrument (CCD camera, spectrophotometer or other optical means). Results: We have demonstrated the ability to perform assays covering a number of different applications. Here, we focus on the detection of C-reactive protein (CRP) in assay buffer and whole blood. The CRP level in whole blood samples (EDTA anti-coagulant) was determined against a calibration curve of known CRP concentrations in assay buffer (see table). Values measured in whole blood were compared to results obtained from samples diluted with assay buffer to test whether whole blood causes signal suppression. CRP concentrations in two samples were independent of assay medium, whereas one sample showed signal suppression as is often observed in ELISAs. Summary: We have developed a CRP ELISA on our specialized LSPR active surface which exhibits the ability to detect CRP concentrations < 1.0 mg/L (“hsCRP”) in whole blood. We evidence the system’s use as a platform for development of rapid, highly sensitive and visual POC diagnostic tests. CRP concentrations [mg/L] Dilution Sample

None (whole blood)

3x 4x 5x 10x

1 0.12 ± 0.02 nd nd nd 0.14 ± 0.06 2 0.54 ± 0.13 0.51 ± 0.09 nd 0.35 ± 0.03 nd 3 0.09 ± 0.02 0.17 ± 0.03 0.19 ± 0.02 0.21 ± 0.03 0.39 ± 0.09

POSTER ABSTRACTS 40th Annual Oak Ridge …clinchem.aaccjnls.org/content/suppl/2008/10/09/54.11...40th Annual Oak Ridge Conference Breakthrough Technologies for Clinical Diagnostics

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