Slides on ELISA - Definition, Procedure etc.Immunology.Heart of various diagnostic procedures

1. Enzyme-Linked ImmunoSorbant Assay … By Dr. Deshkar DW

2. 1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner) 1890 - Demonstration of antibody activity against diphtheria and tetanus toxins. 1890 - Beginning of humoral theory of immunity. (Emil von Behring & Shibasaburo Kitasato) 1900 - Antibody formation theory (Paul Ehrlich) 1938 - Antigen-Antibody binding hypothesis (John Marrack) 1948 - antibody production in plasma B cells 1959 -1962 - Discovery of antibody structure E nzyme L inked I mmuno S orbent A ssay {ELISA} History

9. Four major advantages of ELISA Data analysed statistically ELISA Enzyme Linked Immunosorbent Assay Why ELISA? Versatile Simple Sensitive Quantifiable Many systems using different combinations of reagents are possible Uses micro-titre plates and allied equipment Enzyme catalyst amplification Colour Passive attachment to solid phase High Capacity Ideal range of sensitivity for diagnosis Read by eye Easy separation of bound and unbound reactants by washing step Rapid Cheap Kits feasible Multi-channel spectrophotometers Data can be stored

12. A Microtiter plate (96-well) used for ELISA

13. General Procedure Of ELISA

14. Reading plates • Select proper wave-length on machine. • Carefully wipe bottom of plate to remove excess moisture. Plates that are wet may diffuse your light source, giving inaccurate readings. • Mix the plate. This will distribute color evenly. Some machines have a ‘mix’ setting. General Procedure Of ELISA

15. HOMOGENOUS ELISA There is no need for phase separation. It is less sensitive than heterogenous ELISA. Uses: It is suitable for determination of low molecular weight substances. e.g drug monitoring. PRINCIPAL: Enzyme labeled Ag & unknown Ag compete together for Ab-binding sites . The enzyme labeled Ag that remain free is enzymatically active & can be determined in the presence of the bound form.

19. DIRECT ELISA

20. DIRECT ELISA

21. DIRECT ELISA

24. INDIRECT ELISA

25. INDIRECT ELISA

29. SANDWICH ELISA

31. SANDWICH DIRECT

32. SANDWICH DIRECT

33. SANDWICH INDIRECT

34. SANDWICH INDIRECT

36. COMPETITIVE ELISA

39. PRECAUTIONS Negative control with strong signal The excessive background signal can be caused by inadequate rinsing of plates, reagents not sufficiently diluted, inadequate blocking of plates or non-specific binding of enzyme conjugate. The appearance of color in negative control wells may also indicate cross-reactivity of secondary antibody with components in the antigen sample.

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