Presentation of elisa test

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1. ELISA (Enzyme linked ImmunoSorbant Assay) DEPARTMENT OF MICROBIOLOGY GOVERNMENT COLLEGE UNIVERSITY FAISALABAD Iqbal Danish m.Phi l 2 nd semester

2. Introduction ELISA, are quantitative immunological procedure in which Ag- Ab reaction is monitored by enzyme measurements. It is used to detect the antibodies in the blood. The ELISA test was the first screening test commonly for HIV. It is highly sensitive.

3. Principle Use an enzyme to detect the binding of Ag –Ab. The enzyme converts a colorless substrate to colored product. That indicate the presence of Ag. It can be used to detect the presence of both Ag and Ab.

4. Antigen (Ag) Any molecule that induces production of antibodies when introduced in the body is called antigen. OR Any thing that is foreign to immune system. E.g Bacteria, Virus, etc.

5. Antibody ( Ab ) These are proteins that produced by the immune system which help defend against antigens.

6. Antigen –Antibody Interaction

7. Material Required Test Sample Micro titeration plate ELISA Reader Washing Buffer Stop Solution Substrate Incubator

8. Material Required Test Sample Micro titeration plate ELISA Reader Washing Buffer Stop Solution Substrate Incubator

9. Procedure Take 96 wells plates coated with antigen. Use blocking agend . Take serum and then add into the wells. Add Ab with enzyme . Add substrate to make color. Add stop solution. Use spectrophotometer to read color.

10. Types Frequently there are 3 types of ELISA on the basis of binding structure between the Antibody and Antigen. Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

11. Specimen Samples for ELISA Serum Cerebrospinal fluid Sputum Urine Supernatant of Culture etc

12. Direct ELISA  Is one where there is only one set of antigens and one set of antibodies to react. In this ELISA method, antigens from the patient sample fixed to the Elisa plates are made to react with an antibodies sample which is tagged to a marker enzyme I.e. directly to the antigen in the test an enzyme-linked antibody is added to produce a color reaction with externally added substrate i.e. Elisa reagent. Ag or Ab + Ab or Ag- (e)  −−−−−→ Reaction color.

13. Procedure Antigen is immobilized onto the wells of a 96-well polystyrene plate via passive adsorption. An enzyme-labeled primary antibody specific for the target antigen is added to the wells and directly binds to the antigen. A respective enzyme substrate is added, which upon reaction with the enzyme, produces a visible colorimetric output that can be measured by a spectrophotometer or absorbance microplate reader.

14. Direct ELISA

15. Advantages It is used to detect antibodies. It is simple test. It is quick and easy than other. It is used to detect antigen .

16. Indirect ELISA This has a difference to the Direct Elisa in that one more additional antibody is added in the reaction. To the antigen (fixed to Elisa plate) an antibody is added. Again secondary antigen is added which is enzyme-linked. This requirement is due to the reason that sometimes in patients antigen of the disease-causing agent may not be present but a corresponding antibody is available in the patient sample. which can be traced. These are of two types like normal Indirect Elisa and other is sandwich, Elisa. Ag or Ab + Ab or Ag +Ag or Ab - (e)   −−−−−→Reaction color.

17. Procedure Coat the micro titer plate wells with antigen. Block all unbound sites to prevent false positive results. Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and incubate the plate at 37°c. Wash the plate, so that unbound antibody is removed. Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).

18. Wash the plate, so that unbound enzyme-linked antibodies are removed. Add substrate which is converted by the enzyme to produce a colored product. Reaction of a substrate with the enzyme to produce a colored product.

19. Advantages A wide variety of labeled secondary antibodies are available commercially. Cost Effective. Different visualization markers can be used with the same primary antibody. Disadvantages Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal. An extra incubation step is required in the procedure.

20. Sandwich ELISA Antigen can be detected by sandwich ELISA Antibody is coated on the microtiter well. Sample containing antigen is added to the well and allowed to react with the antibody attached to the well, forming antigen-antibody complex. Washing can be done.

21. Enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. Washing can be done. Substrate is added to the plate which is hydrolyzed by enzyme to form colored products.

22. Advantages High specificity, since two antibodies are used the antigen is specifically captured and detected. Suitable for complex samples, since the antigen does not require purification prior to measurement. Flexibility and sensitivity, since both direct and indirect detection methods can be used.

23. Competitive ELISA. This test is used to measure the concentration of an antigen in a sample. Antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to the microtitre well which is coated with antigen.

24. The more the antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Washed wells. enzyme conjugated secondary antibody specific for iso -type of the primary antibody is added to determine the amount of primary antibody bound to the well. The higher the concentration of antigen in the sample, the lower the absorbance.

25. Advantages. High specificity, since two antibodies are used. High sensitivity, since both direct and indirect detection methods can be used. Suitable for complex samples, since the antigen does not require purification prior to measurement.

26. Application of ELISA. Presence of antigen or the presence of antibody in a sample can be evaluated. Determination of serum antibody concentrations in a virus test. Used in food industry when detecting potential food allergens. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc.

27. Qualitative and Quantitative Test Qualitative: Determine the present of antigen and antibody . Posive or negative result. Quantitative: Determine the quantity of antibody. No of positive and negative result.

28. Conclusion It is a good techniques and being popular today because of its simple and not involve radiation . It is easy than other test . It is most useful than other test because of their sensitivity. It is not harmful.