US/Ireland Emerging Technologies, Univ. of Massachusetts, Lowell, Oct 1 9 -2 0 th . , 2009 .

1. 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.

2. 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

3. Core Expertise Mutagenesis: Random and site-specific mutagenesis for protein improvement Display and Selection: Phage, yeast and ribosomal display of proteins Cloning: Cloning, expression and purification of antigens and biomakers Antibody Production: Monoclonal, polyclonal and recombinant (human, chicken, mouse and rabbit) Automated Screening: Custom written software for high throughput screening Fermentation: Large scale protein expression Biosensor Assays: Incorporation of biorecognition elements into biosensor platforms Protein Kinetics: Determination of interaction rate constants and thermodynamic profiles Lateral Flow Assays: Point of care tests for environmental and clinical applications Liposomes: Antibody labelling and dye/contrast agent encapsulation Biorecognition molecule: Antibodies, fragments, peptides, protein scaffolds and DNA

4. Research Interests Marine Toxins Sialic Acids Prostate Cancer Markers Cardiac Markers Food Contaminants: Multiple Myeloma Aflatoxins Drugs of Abuse

5. They didn’t mention this in my contract

6. CH1 VH VH CL VL VL CH1 CH1 VH VH VH VH CL CL VL VL VL VL CH2 Fab VL VL VH VH Recombinant antibodies F(ab')2 IgG scFv Dimeric scFv Dimeric bifunctional scFv

7. Characteristics Polyclonal Monoclonal Recombinant Ease of production ++++ +++ ++ Low cost ++++ +++ ++ Stability* +++ ++ ++ Availability ++++ +++ + () Ease of immobilization ++++ ++++ ++++++ Sensitivity* ++++ ++++ +++++ Capacity for improvement* - - +++++ Antibody Types

8. Avian Antibodies: IgY • Phylogenetically distant from humans • Single primer sets • Stable and long half-life • Do not react with RF, HAMA or surface Fc receptors Picture obtained from www.beckman.com

9. Derivatization of Target Target-Carrier Conjugate Design and Synthesis Target Linker Immunize Mouse Immunize Rabbit Polyclonal antibodies Recombinant antibodies Monoclonal antibodies Kinetic and Thermodynamic Characterisation Development of Target-specific tests using a range of antibody labels Biosensor-based Immunoassay Response (RU) RU Target Conc. Time Antibody Production, Characterication and Applications

10. Pimp My Antibody Nature446, 964-966 (26 April 2007)

11. Antibodies for Use – Key Issues

12. Extract RNA Genetic source of antibody fragments - Non-immunised/Immunised Mouse - Hybridomas Reverse Transcription VL SOE-PCR anneals the VH and VL regions together VH Transform into E.coli Chloramphenicol Phage Display scFv antibodies Ligation of amplified DNA into plasmid F1 PRODUCTION AND CHARACTERISATION OF MURINE SINGLE CHAIN FV ANTIBODIES SfiI digest of SOE product

13. pIII phage coat protein Vector containing VH andVL DNA sequences Recombinant scFv antibody fragment Phage displaying scFv antibody fragment Diagram of filamentous phage expressing scFv on the surface of the phage as a fusion with the pIII phage coat protein. Light and heavy chain genes are present in the vector contained within the phage and the phage is ready for infection into E.coli.

14. 1. Phage antibody library 8. Prepare phage particles 7. Amplify bacteria Phage selection cycle 2. Incubate with antigen on immunotube 6. Re-infect selected phages into E. coli 3. Wash and remove non-binders 5. Analyse eluted phage by phage-ELISA 4. Elute specific phage

15. Robotic Screening • Screening capacity: 4000 clones per day • Automated screening (custom designed)

16. A100 – rapid kinetic characterisation • High throughput hardware and software • High quality, high content data • Parallel analysis array format system

17. A100 Screening approach

18. Stability early Stability late Stability late X 100 %left = Stability early Complex Stability of 95 clones

19. Listeria monocytogenes • Gram-positive bacterium, motile • Ubiquitous in the environment • ‘Listeriosis’ - manifested as • food poisoning (‘Influenza-like’) • spontaneous abortion (2nd/3rd trimester) • meningitis/encephalitis • >20% mortality rate

20. Food recalls • In 2002, 27.4 million pounds of turkey produce were recalled due to suspected contamination with L. monocytogenes • largest meat recall in the history of the United States • resulted in several fatalities and a subsequent nationwide class-action lawsuit Limits allowed: 1 micro-organism per 50 g

21. Internalin B (InlB) ~65 kDa p60 (60kDa), Hpt (cell growth/division) Actin Tail Filaments (ActA) 90 kDa (Intracellular motility) Listeriolysin (60 kDa), PI-PLC, PC-PLC (escape from vacuole) Internalin A (InlA) 80 kDa P66 (kDa) Aminopeptidase Virulence proteins Listeria monocytogenes

22. Antibodies

23. mAb2b3 cross - reactivity • Direct capture ELISA ( Anti-Inl A antibody) • mAb2b3 only bound to L. monocytogenes cells tested

24. Listeria monocytogenes determination

25. Detection of Listeria 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).

26. 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

27. Anti-aflatoxin scFv preincubated with aflatoxin Aflatoxin Anti-aflatoxin scFv Biacore assay development Antibody and free aflatoxin injected over aflatoxin-immobilised CM5 chip surface

28. 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 Conversion of scFv to Fab format

29. Improving Affinity Q E X X X Y S M D NNK NNK NNK Heavy chain CDR3 • in vitro (directed) evolution strategies

30. Comparison of Antibodies:Detection of aflatoxin B1

31. Summary of Lecture • Aim: offer perspective on selection and use • of antibodies • Specificity • Sensitivity • Sources • Selection • Screening • Sensing Six-s = success

32. Acknowledgements • Lab group • Biacore • FIRM • SFI • Fusion • EI