ELISA (Enzyme Linked Immunosorbent Assay).pptx

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1. Techniques in Biotechnology… M.PHOOL BADSHAH

2. ELISA ( Enzyme Linked Immunosorbant Assay) M.PHOOL BADSHAH Lecture-11

3. Introduction It is a qualitative or quantitative immunological procedures in which the Ag-Ab reaction is monitored by enzyme measurements. Engvall & Perlma first explore the term ELISA in 1971. The E lisa test was the first screening test commonly employed for HIV. It has a high sensitivity.

4. Principle : Its principle is similar to RIA but depends on an enzyme rather than radioactive label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate. Some enzyme have been employed for ELISA including alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

5. Equipments: Microwell plate: it is flat bottom polystyrene plate containing 8 x 12 wells holding 350 micro-litter each.

6. Multipipettes: It is an 8 channel 100 microliter pipette which is a good help for even small scale work. Easily allowing for a single device to fill multiple wells at a single time. This enables the user to quickly and easily fill multi-well plates used in tissue culture, drug screening, or enzyme assays.

7. Washing Device: It is manually operated. Elisa Washer is a medical device specially designed to clean the microplate, and generally used in conjunction with the microplate reader.

8. Procedure: Wells are coated with antibodies. Sample is added which may contain antigen. The antigen-antibody interaction took place. Removal of unbound antigens by washing. Addition of another antibody which is linked with enzyme. Interaction of enzyme with particular substrate. This interaction produces colour through which we can observe a particular antigen (disease).

9. Types of ELISA: There are four types of ELISA: Direct ELISA Indirect ELISA Competitive ELISA Sandwich ELISA

10. 1- Direct ELISA: It involves attaching a labelled (enzyme-conjugated) antibody directly to the target antigen. Although simplicity and quickness are benefits, sensitivity could be less than that of other ELISA types.

11. 2- Indirect ELISA: It makes use of a primary antibody that attaches itself to the target antigen, which is then recognized by an enzyme-conjugated secondary antibody. increases the signal's amplification, which raises the sensitivity.

12. 3- Competitive ELISA: It makes use of two antigens, one labelled and the other unlabeled from the sample that compete to bind to a finite number of immobilized antibodies. The antigen concentration in the sample has an inverse relationship with the signal.

13. 3- Sandwich ELISA: It uses two antibodies, one labelled with an enzyme and the other immobilized on a solid surface. The two antibodies trap the antigen, creating a "sandwich." High sensitivity and specificity; frequently used in antigen detection.

14. Advantages: Cheaper reagent and have long life. It is easy to perform and quick procedure. Equipments are inexpensive and widely available. ELISA can be used to detect variety of infections. No radiation hazards occur during labeling or disposal of waste. It is highly specific and sensitive.

15. Disadvantages: Measurements of enzyme can be more complex. Enzyme activity may be affected by plasma constituents. Kits are commercially available but not cheap. False positive /negative possible, especially with mutated/ altered antigen. Very specific to a particular antigen, would not recognize any other antigen.