Mechanism of Antibody Based Diagnostic Tests

1. PHM142 Fall 2012 Instructor: Dr. Jeffrey Henderson Mechanism of Antibody Based Diagnostic Tests Presented By: Colette Papagiannis, Boya(Connie) Xiong and Tianna Costa

2. Overview • What is an antibody? • Antibody Techniques • ELISA • Indirect • Sandwich • Competitive • Methods of Detection • ELISA in Diagnosis • Cancer Diagnosis

3. What is an Antibody? • Antibody: binds to bacteria, viruses or large molecules identified as foreign and targets them for destruction • Antibodies recognize a specific part of the foreign target (antigen), called the epitope

4. Antibody Techniques • Many ways to use antibodies: • ELISA • Immunoflorescence • Western Blot • Immunodiffusion • Immunoelectrophoresis • Magnetic Immunoassay

5. ELISA • ELISA: Enzyme Linked Immunosorbent Assay • Most common and widely used methods for both detecting and quantifying the amount of antibody or antigen present • 3 main methods are used: • Indirect • Sandwich • Competitive

6. Indirect ELISA • Bind the antigen to a solid support • Add the antibody directed against the target antigen • Add a second antibody that binds to the first antibody • Note: If the first antibody is a human antibody the second antibody must be non-human • Second antibody is linked to an enzyme • Substrate is added and a colourimetric or luminescent signal is measured • Colour intensity/signal is proportional to amount of antibody in the sample

7. Sandwich ELISA • Antibody bound to microtitre well • Sample added and allowed to react with the immobilized antibody • Target antigen binds • Well is washed and second antibody added – second Ab binds different epitope • Well is washed to remove any remaining second antibody • Substrate is added and colour reaction is measured • Colour intensity/signal is proportional to amount of antigen in the sample

8. Competitive ELISA • Primary antibody is incubated with sample solution • Target antigen binds to Antibody • Antigen-Antibody mixture added to antigen-coated well Higher antigen concentration in the sample results in less free antibody to bind with antigen coated in the well • Well is washed and secondary antibody specific for the isotype of the primary antibody is added to the well • Well is washed and substrate added • Colour intensity/signal is inversely proportional to antigen concentration in sample

9. Detection Colour Reaction: • Detection Ab has a biotin molecule attached • Strepavidin-Horseradish peroxidase (HRP) complex is added • Strepavidin binds to biotin (high affinity) • Tetramethylbenzidine (TMB) substrate reacts with HRP to generate a blue-coloured product • Measure at 450 nm

10. Detection Continued Bioluminescence: • Detection Ab linked to Luciferase • Add luciferin and Ca2+ to microtitre well • Luciferin oxidized to oxiluciferin and light Fluorescence: • Detection Ab is linked to a fluorescent molecule • Fluorescence is quenched by an attached dinitrophenyl (DNP) substituent • Enzyme cleaves DNP resulting in fluorescence • Measure Fluorescence • Luciferin + O2 Oxiluciferin + Light • Luciferase

11. ELISA in Disease Diagnosis Indirect ELISA: • Diagnosis of HIV, and Tuberculosis Sandwich ELISA: Cancer diagnosis Cancer cell: marked with Altered protein expression that lead to • Uncontrolled division • Altered cell morphology • Non-functional • Formation of tumor (metastatic)

12. Cancer Diagnosis • Biopsy—although the only definitive method, it is invasive and only performed when other tests suggest presence of cancer • Blood test —Altered protein expression: biomarker • The presence • The level of expression ex: alpha-fetoprotein for liver cancer

13. Target biomarker in serum sample as the antigen Signal intensity mark the level of expression Help manage the progress of treatment and detection of recurrence as well ELISA in Cancer Diagnosis Patient serum Biomarker

15. Summary • Antibody: recognize a specific part of the foreign target (antigen), called the epitope • ELISA: Enzyme Linked Immunosorbent Assay • Most common and widely used methods for both detecting and quantifying the amount of antibody or anitgen present • 3 main methods of ELISA used: • Indirect: Colour intensity/signal is proportional to amount of target molecule in sample • Sandwich: Colour intensity/signal is proportional to amount of target molecule in sample • Competitive: : Colour intensity/signal is inversely proportional to amount of target molecule in sample • Detection Methods: Colour reaction, Fluorescence and Bioluminescence • Application in cancer diagnosis: cancer cells have altered protein expression, and these proteins are called biomarkers • Sandwich ELISA can measure the level of biomarkers in serum

16. References Brennan, DJ., O’Connor, DP., Rexhepaj, E., Ponten, F. and Gallagher, WM. (2010) Antibody-based proteomics: fast-tracking molecular diagnostics in oncology. Nature Reviews, 10, 605-617. Ferrier, CM., de Witte, HH., Straatman, H., van Tienoven, DH., van Geloof, WL., Rietveld, FJR., Sweep, CGJ., Ruiter, DJ. and van Muijen, GNP. (1999) Comparison of immmunohistochemistry with immunoassay (ELISA) for the dectection of components of the plasminogen activation system in human tumor issue. British Journal of Cancer. 79(9/10), 1534-1541 Goldsby, R.A., Kindt, T.J., Osborne, B.A. and Kuby, J. (2003) Immunolgy 5th Edition. W.H. Freeman and Company, New York. Hoehn, Katja and Marieb, Elaine N. (2010). Human Anatomy & Physiology 8th Ed. Pearson Education Inc., California. Kim, K., Visitin, I., Alvero, AB. and Mor, G. (2009) Development and validation of a protein based signature for the dectection of ovarian cancer. Clin Lab Med. 29(1)47-55. Nelson, David L. and Cox, Micheal M. (2008). Lehninger Principles of Biochemisty 5th Ed. W.H. Freeman and Company, New York.