1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

3. ELISA Dr V.Mazaheri

4. Introduction Enzyme Linked Immunosorbent Assay Immunologic test Enzyme as a reporter label Qualify &Quantify suited to diagnosis at the screening level, surveillance where larger scale sample research Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

5. Introduction Many of the accepted standard assays in many scientific fields are ELISA-based In conjunction with molecular method for identifying and characterizing disease agents and to assess disease prevalence 2 Research groups in Sweden and Netherlands Literature search from 1970s Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

6. Introduction Why ELISA? -Passive attachment to solid phase -Easy separation of bound &unbound reactants - Simple -Microtiter plate -Kit feasible Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

7. Introduction Sensitive Quantifiable - color, read by eye -Multichannel spectrophotometer -Data can be stored -Data can be analyzed statistically Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

8. Brief definition of terms Solid phase Usually a microtiter plate well Adsorption The process of adding an antigen or antibody, diluted in buffer Washing with a buffered solution to separate bound (reacted) from unbound (unreacted) reagents Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

9. Brief definition of terms Antigens A protein or carbohydrate Antibodies Produced in response to antigenic stimuli Antispecies antibodies E.g. guinea pig serum injected into a rabbit elicits the production of rabbit anti-guinea pig antibodies. Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

10. Brief definition of terms Enzyme A substance that can react at low concentration as a catalyst to promote a specific reaction Enzyme conjugate An enzyme that is attached irreversibly to a protein, usually an antibody Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

11. Brief definition of terms Substrate A chemical compound with an enzyme reacts specifically (a color reaction) Stopping The process of stopping the action of an enzyme on a substrate Reading Measurement of color produced Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

12. ELISA Methods Direct ELISA Indirect ELISA Sandwich ELISA Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

13. Direct ELISA Summer School Influenza Unit, Pasteur Institute of Iran summer 2012 AgWashAb specific for Ag (conjugate) Wash Substrate

14. Indirect ELISA Summer School Influenza Unit, Pasteur Institute of Iran summer 2012 Ag Ab Ab conjugated (antispecies) Wash Substrate

15. Sandwich ELISA -Measures the amount of antigen between two layers of antibodies - The antigens must be contain at least two antigenic sites - Valuable when the concentration of antigens is low Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

16. Direct Sandwich ELISA Summer School Influenza Unit, Pasteur Institute of Iran summer 2012 Ab Ag Ab conj (same on s.p or different species) Substrate

17. Indirect Sandwich ELISA Summer School Influenza Unit, Pasteur Institute of Iran summer 2012 AbAg Ab (different species) Anti-species conj Substrate

18. General information Solid Phase -Microtiter 96-well plate - Polyvinyl chloride(flexible) -Polystyrene (inflexible) Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

19. General information Coating - Diffusion coefficient of the attaching molecule - Ratio of the surface area being coated to the volume of the coating solution - Concentration of the substance being adsorbed - Temperature - Time of adsorption - Coating buffer Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

20. General information Washing -Separate bound and unbound -Washing buffer - At least 3 times for every well - Washing methods Dilution buffer Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

21. General information Blocking Conditions and Nonspecific Reactions - To prevent nonspecific adsorption of proteins to wells from samples added after the coating of the solid phase before, during, or as a combination of both times -High background color - Blocking agents: BSA, Nonfat dried milk, Gelatin,Detergents,… Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

22. General information Enzyme Conjugates -Usually antibodies are conjugated to enzymes -The species in which the antiserum is produced can be important - Different sources and batches of conjugates from the same manufacturer may vary - The linkage of an enzyme to an Ag or Ab affect the specificity of an assay Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

23. General information Enzyme Conjugates -Horseradish Peroxidase Plus Hydrogen Peroxide Substrate such as OPD, TMB,ABTS,… -AlkalinePhosphatase Plus p-Nitrophenylphosphate Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

24. General information Substrate -Substrate concentrations must be optimized -Certain solutions can be made and stored e.g.OPD Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

25. General information Stopping Reactions -Molar concentrations of strong acids or strong bases stop enzymic activity by quickly denaturing enzymes -Wavelength & Color -TMB(Blue) Yellow,450 nm -OPD(Green/Orange) Orange/Brown,492 nm Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

26. General information Reading -Spectrophotometric Reading -Optic Density Summer School Influenza Unit, Pasteur Institute of Iran summer 2012

27. Thanks