Indirect ELISA is a two-step process which involves binding of primary antibody and labeled secondary antibody. Primary antibody is incubated with antigen followed by incubation with secondary antibody. However, this may lead to nonspecific signals because of cross-reaction that secondary antibody may cause.
In this enzyme assay test, sample antibody is sandwiched between antigen coated on plate and an enzyme-labeled, anti-species globulin conjugate. Addition of an enzyme substrate chromogenic reagent causes color to develop.
This color is directly proportional to amount of bound sample antibody. More the antibody present in sample, stronger the color development in test wells. This is suitable for determining total antibody level in samples.
Antibody can be detected or quantitatively determined by indirect ELISA. In this technique, antigen is coated on the microtiter well. Serum or some other sample containing primary antibody is added to microtiter well and allowed to react with coated antigen.
Detection of Antibody
Any free primary antibody is washed away and antibody bound to antigen is detected by adding an enzyme conjugated secondary antibody that binds to the primary antibody. Unbound secondary antibody is then washed away and a specific substrate for enzyme is added. Enzyme hydrolyzes the substrate to form colored products. The amount of colored end product is measured by spectrophotometric plate readers that can measure the absorbance of all the wells of 96-well plate.
Quantitative Estimation of Antibody
Indirect Enzyme assay has been used for quantitative estimation of antibodies in serum and other body fluids. In this method, specimens are added to microtiter plate wells coated with antigen towards which specific antibodies are to be detected.
After a period of incubation, wells are washed. If antibody was present in sample, antigen–antibody complex would have been formed and will not get washed away. On the other hand, if specific antibody was not present in specimen, there would not be any complex formation.
Then, an anti-isotype antibody conjugated with an enzyme is added and incubated. After another washing step, a substrate for the enzyme is added.
If there was complex formation in the initial step, secondary anti-isotype antibody would have bound to primary antibody, and there would be a chromogenic reaction between enzyme and substrate. By measuring optical density values of wells, after a stop solution has been added to arrest chromogenic reaction, one can determine the amount of antigen–antibody complex formed.
- Different visualization markers can be used with the same primary antibody.
- Versatile as many primary antibodies can be made in one species and same labeled secondary antibody can be used for detection.
- Wide variety of labeled secondary antibodies are available commercially.
- Increased sensitivity, since more than one labeled antibody is bound per primary antibody.
- Maximum immunoreactivity of primary antibody is retained because it is not labeled.
- Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody.
- Cost savings, since fewer labeled antibodies are required.
- Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.
- An extra incubation step is required in procedure.
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