Methods of Enzyme-Linked Immunosorbent Assay (ELISA) Tests
There are two methods of ELISA;
1. Direct method
Used for the detection of Ag (Antigen).
2. Indirect method
Used for the detection of Ab (Antibody).
1. Direct Method
(Double Antibody sandwich ELISA)
Useful in detection of HepBAg, toxin, hormones.
It is used for the detection of antigen and known as sandwich ELISA as the antigen occurs between two Antibody.
1. Wells of micro-ELISA plate are coated with antibodies and specimen may contain antigen.
2. The solution (with Ag) to be tested for antigen is placed on Ab coated well.
The antigen if spefic to Antibody, wiii be forming Ag-Ab complex.
3. Now enzyme linked antibody (second antibody) is added.
4. Antigen is thus sandwiched between the first antibody (that coated to well) and enzyme linked second antibody.
5. Now specific substrate is added, if the antigen is present, then coloured reaction is produced.
Colour intensity is measured by spectrophotometer.
2. Indirect method
To detect antibody.
It is used for the detection of antibody.
1. In this case antigen is coated on plate.
2. The test serum (Ab) is added to antigen coated wells.The antigen if specific to antibody, bind and forming antigen- antibody complex.
3. Enzyme linked second antibody is added to wells.
4. Substrate is added. The development of colours indicates presence of antibodies in serum.
5. The intensity of colour is measured by spectrophotometer.
3. Competitive ELISA
It is very significant as it detect either antibody or antigen.
If the antigen interest is present, this bind to enzyme labeled Antibody and if absent, color indicates positive reaction.
Antigen binding to specific amount of labeled antibody.
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