US/Ireland Emerging Technologies, Univ. of Massachusetts, Lowell, Oct 19-20 th ., 2009. 'Developing Immunoassays for Bioprocess Analysis and Diagnostics' Richard O’Kennedy, Professor of Biological Sciences/Vice- President, School of Biotechnology, Biomedical Diagnostics Institute and National Centre for Sensors Research, Dublin City University, Dublin 9, Ireland
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US/Ireland Emerging Technologies, Univ. of Massachusetts, Lowell, Oct 1 9 -2 0 th . , 2009 .
US/Ireland Emerging Technologies, Univ. of Massachusetts, Lowell, Oct 1 9 -2 0 th . , 2009 . 'Developing Immunoassays for Bioprocess Analysis and Diagnostics' Richard O’Kennedy, Professor of Biological Sciences/Vice-President, - PowerPoint PPT Presentation
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US/Ireland Emerging Technologies,
Univ. of Massachusetts, Lowell, Oct 19-20th., 2009.
'Developing Immunoassays for
Bioprocess Analysis and Diagnostics'
Richard O’Kennedy,
Professor of Biological Sciences/Vice-President,
School of Biotechnology, Biomedical Diagnostics Institute and National Centre for Sensors Research,
Dublin City University, Dublin 9, Ireland.
SUMMARY:
'Developing Immunoassays for Bioprocess Analysis and Diagnostics'
• Background
• Antibodies: ideal reagents for assay development
• Key Characteristics: specificity, sensitivity,
structural format, stability and immobilization.
• Generation
• Genetic engineering facilitates optimisation
• Potential applications
Core Expertise
Biorecognition molecule:Antibodies, fragments, peptides, protein scaffolds and DNA
Antibody Production:Monoclonal, polyclonal and recombinant (human, chicken, mouse and rabbit)
Fermentation:Large scale protein expression
Cloning:Cloning, expression and purification of antigens and biomakers
Protein Kinetics:Determination of interaction rate constants and thermodynamic profiles
Liposomes:Antibody labelling and dye/contrast agent encapsulation
Automated Screening:Custom written software for high throughput screening
Mutagenesis:Random and site-specific mutagenesis for protein improvement
Biosensor Assays:Incorporation of biorecognition elements into biosensor platforms
Display and Selection:Phage, yeast and ribosomal display of proteins
Lateral Flow Assays:Point of care tests for environmental and clinical applications
mAb2b3 cross - reactivity• Direct capture ELISA ( Anti-Inl A antibody)Direct capture ELISA ( Anti-Inl A antibody)
– mAb2b3 only bound to mAb2b3 only bound to L. monocytogenesL. monocytogenes cells tested cells tested
Listeria monocytogenes determination
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10
Cell Concentration (Cells/ml)
A/A
0..
Anti-InlA antibody-linked red light emitting quantum dots (605nm)
The development of rapid fluorescence-based immunoassays, using quantum dot-labelled antibodies for the detection of Listeria monocytogenes cell surface proteins - Int. J. Biol. Macromol., 39(1-3), 127-134. Tully, E., Hearty, S., Leonard, P. and O’Kennedy, R. (2006).
Detection of Listeria
AFB1
Aflatoxin B1
• Potent naturally occurring carcinogen
• Ability to induce specific mutations in specific mammalian genes
• Reported 1 million cases of liver cancer per year due to aflatoxins • Linked to human hepatocellular carcinoma
• Listed as group 1 carcinogens by the IARC
Anti-aflatoxin scFv preincubated with aflatoxin
Anti-aflatoxin scFv Aflatoxin
Biacore assay development
Antibody and free aflatoxin injected over aflatoxin-
immobilised CM5 chip surface
Conversion of scFv to Fab format
• Anti-AFB1 murine scFv was converted to a chimeric Fab format by the addition of human constant regions using a number of overlap extension polymerase chain reactions (PCRs)
• Potential benefits of converting
an scFv to Fab format in terms of
stability, sensitivity and specificity
were evaluated
• Influence of additional constant regions
and presence of the interchain disulphide
bonds in the Fab fragment was assessed
scFv
Fab
scFv
Fab
Heavy chain CDR3 Q E X X X Y S M D
NNK NNK NNK
Improving Affinity• in vitro (directed) evolution strategies
Antibody Fragment ELISA Limit of Detection
Biacore Limit of detection
Monomeric scFv 12ppb 0.390ppb
Dimeric scFv 3ppb 0.19ppb
Bifunctional dimeric scFv
3ppb n/a
D11 Fab 4.2ppb 0.29ppb
G6 Fab (mutant) 1.3ppb 0.12ppb
Comparison of Antibodies:Detection of aflatoxin B1