AAI IMMIUNOLOGY 2019 Technological Innovations I Program 130.35 / Poster P1261 Identifying Predictive Cytokine Biomarkers in Asthma Using the Invitrogen ProQuantum High-Sensitivity Immunoassay Platform David M Bourdon 1 , Helena Ekoff 2 , Anders Sjölander 2 , Takao Fujisawa 3 , Mizuho Nagao 3 , and Magnus Molin 2 1 Thermo Fisher Scientific, USA; 2 Thermo Fisher Scientific, Sweden; 3 Institute for Clinical Research, Mie National Hospital, Japan Introduction Asthma and chronic obstructive pulmonary disease (COPD) affect hundreds of millions of people worldwide and are a significant cause of morbidity and mortality. These heterogenous and complex inflammatory diseases have few good biomarkers for disease etiology, diagnosis, and monitoring of treatment efficacy. Characterization has previously been performed at genomic and proteomic levels, as well as airway histology, lung function, and quality of life metrics. Since inflammation typically involves one or more cytokine cascades, or pathways responsible for triggering and Key Benefits of the ProQuantum High-Sensitivity Immunoassay Platform Sensitivity and broad dynamic range of qPCR A simple, yet powerful platform for target-specific protein quantitation • High-sensitivity – detect low levels of proteins with greater sensitivity than traditional methods like ELISA • Small sample consumption – 2 μL vs 75 μL for triplicate wells with other methods) • Fast, easy workflow – no wash steps, 2 hours from sample to answer • Broad dynamic range – 5 logs minimizes sample dilutions to ensure falling within the range • No proprietary instrument to purchase – runs on any real-time PCR instrument • Includes intuitive cloud-based software – for robust data analysis and statistical group wise comparison Specificity of matched antibody pairs + (c) rob3000 www.fotosearch.se The goal of the following study was to determine whether the Invitrogen ProQuantum High-Sensitivity Immunoassay platform (www.thermofisher.com/proquantum) could prove useful in uncovering biomarker profiles in asthmatic populations. Additional benefits included running the proteomic assay on ubiquitous qPCR instrumentation and the small 2 µL sample requirement. In this study, 168 human serum samples were analyzed (IL-5, IL-6, IL-8, IL-13, IFN-γ and eotaxin-1) across wheezing and asthmatic phenotypes, as well as healthy age- matched controls among both cohorts. Data presented include pairwise analyses that indicate that the ProQuantum high-sensitivity immunoassay has helped uncover predictive biomarkers in samples from wheezing and asthmatic children, that we not previously easily distinguishable. Source: Edwards et al. (2017) Addressing unmet needs in understanding asthma mechanisms, Eur Resp J.49:1602448 maintaining such inflammation, cytokine profiles are critical to advancing our understanding of the disease. However, a powerful immunoassay platform with a simplified workflow that is capable of early cytokine detection and minimum sample consumption is lacking in the asthma / COPD research space. Complementary splint oligo and robust DNA ligase generate qPCR template Antibodies and analyte in a homogeneous mixture Antibodies come into close proximity upon epitope binding Following DNA template ‘melting’ at 95˚C, all proteins (analyte, antibodies, etc.) are denatured and no longer a part of the assay C= cycle number to reach threshold Polymerization Strand displacement Cleavage Merging Powerful and Trusted Genomic and Proteomic Technologies Only need to program qPCR instrument once to create a template for repeated use * * Printable lab instructions (e-notebooks) Standard curve wizard Robust Analytics with Intuitive Cloud-based and Desktop Applications Additional feature: ProQuantum cloud and desktop applications can create GraphPad Prism files for direct integration and convenient creation of publication quality figures Plate setup tool 4PL/5PL analysis with outlier detection Group wise comparison featuring M-stats ProQuantum immunoassays are compatible with any qPCR instrument, and Applied Biosystems instrument users will experience enhanced utility of .EDS files (rapid data import, amplification plots, etc.) Assay Characteristics and Analytics ü Serum spike-and-recovery: 80% - 120% ü Plasma spike-and-recovery: 70% - 130% ü Parallelism and linearity of dilution: >0.9 ü Inter- and intra-assay reproducibility: <15% CV of calculated concentrations (in linear range of assay) ü Analyte cross-reactivity: <10% ü Benchmarking to ELISA ü Protein standard calibration to leading ELISA or NIBSC The ProQuantum Immunoassay Setup Experience in More Detail Prepare reagents Prepare setup plate (on 4˚C cold block) This is a typical example of a setup plate to run the ProQuantum Immunoassay on an entire 96-well plate using 5-µL sample volumes, and a general serial dilution diagram for a standard curve. Reconstitute protein standard Run assay The ProQuantum Immunoassay Workflow Is Rapid, Robust, and Flexible Streamlined Workflow and Intuitive Cloud Application Delivers Rapid Sample-To-Answer Efficiency While Preserving Precious Samples Step 1: antibody-antigen binding • 2* μL 10% serum + 2* μL antibody-conjugates • 1 hr incubation at room temperature Step 2: ligation and qPCR readout • Add 16* μL ligase and qPCR master mix • In-instrument ligation and 40 cycles of qPCR ProQuantum assays are scalable 5 μL volume: one 96-well plate (5+5+40) 2 μL volume: two 96-well plates (2+2+16) 1 μL volume: one 384-well plate (1+1+8) Step 1: Analyte Binding Step 2: Ligation / qPCR 1 2 3 4 Sample binding to plate – 2 hrs W Detection antibody binding – 1.5 hrs W Time (hours) Typical ELISA ProQuantum assays Analyze Results For Additional Information on the ProQuantum High-Sensitivity Immunoassay Platform visit thermofisher.com/ProQuantum For Research Use Only. Not for use in diagnostic procedures. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. The content provided herein may relate to products that have not been officially released and is subject to change without notice. Results Materials: Six ProQuantum Immunoassays To Detect Circulating Cytokines 16 18 20 22 24 26 28 30 32 34 Concentration, pg/mL C t human IL-5 1,000 100 1 10 0.1 0.01 0.001 0 10,000 16 18 20 22 24 26 28 30 32 Concentration, pg/mL C t human IL-13 1,000 100 1 10 0.1 0.01 0.001 0 10,000 18 20 22 24 26 28 30 32 Concentration, pg/mL C t human IFNγ 1,000 100 1 10 0.1 0.01 0 10,000 18 20 22 24 26 28 30 32 34 Concentration, pg/mL C t human IL-6 1,000 100 1 10 0.1 0.01 0 10,000 16 18 20 22 24 26 28 30 32 34 Concentration, pg/mL C t human IL-8 1,000 100 1 10 0.1 0.01 0.001 0 18 20 22 24 26 28 30 32 Concentration, pg/mL C t human Eotaxin 1,000 100 1 10 0.1 0.01 0 10,000 Example Standard Curves Indicating Assay Range 0 10 20 30 40 0 10 20 30 40 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IL-5 R= 0.8937 0 100 200 300 0 100 200 300 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IL-13 R= 0.8585 0 200 400 600 0 250 500 750 1000 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IFN-γ R= 0.8392 0 50 100 150 200 250 0 50 100 150 200 250 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IL-8 R= 0.9738 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 Expected concentration, pg/mL ProQuantum immunoassay, pg/mL human IL-6 R= 0.9265 0 20 40 60 80 100 0 20 40 60 80 100 ELISA, pg/mL ProQuantum immunoassay, pg/mL human Eotaxin R= 0.9087 Example Concordance Plots: Same Human Sera Samples Run on ProQuantum Immunoassays and ELISAs ProQuantum Interferon- g Serum conc. (pg/mL) IFN-g wheezing IFN-g healthy 0.01 0.1 1 10 100 1,000 10,000 ProQuantum IL-13 Serum conc. (pg/mL) IL-13 wheezing IL-13 healthy 0.01 0.1 1 10 ProQuantum IL-8 Serum conc. (pg/mL) IL-8 wheezing IL-8 healthy 0.01 0.1 1 10 100 1,000 10,000 ProQuantum Eotaxin Serum conc. (pg/mL) Eotaxin wheezing Eotaxin healthy 1 10 100 ProQuantum IL-5 Serum conc. (pg/mL) IL-5 wheezing IL-5 healthy 0.01 0.1 1 10 100 ProQuantum IL-6 Serum conc. (pg/mL) IL-6 wheezing IL-6 healthy 0.01 0.1 1 10 100 1,000 10,000 • Previously, sensitivity limitations of other immunoassay platforms made it difficult to accurately stratify samples based upon analyte concentration. • In this study 168 serum samples (healthy, wheezing, and asthmatic children) were analyzed across six targets: eotaxin, IL-5, IL-6, IL-8, IL-13, and IFN-!. • ProQuantum High-Sensitivity Immunoassays uncovered marked differences in circulating cytokines levels in human serum samples from both wheezing and asthmatic subjects, versus healthy age-matched controls. ProQuantum IL-5 Serum conc. (pg/mL) IL-5 asthma IL-5 healthy 0.01 0.1 1 10 ProQuantum IL-13 Serum conc. (pg/mL) IL-13 asthma IL-13 healthy 0.001 0.01 0.1 1 Serum conc. (pg/mL) Eotaxin asthma Eotaxin healthy 0.1 1 10 100 ProQuantum Eotaxin Conclusions and Future Directions • The Invitrogen ProQuantum High-Sensitivity Immunoassay platform has been used to detect serum cytokine levels from children afflicted with respiratory disease. • Utilizing the streamlined workflow, rapid sample-to-answer, and without consuming appreciable sample, marked differences have been identified between healthy controls and disease-specific samples. • Future directions include longitudinal analyses on independent sample cohorts to examine early detection profiles, as well as additional analyte targets for study of disease stratification (e.g. eosinophilic asthma versus healthy). Watch the How-To Video