ELISA assay can be done with various modifications based on the basic procedure – direct, indirect, sandwich ELISA test.
Direct ELISA – This is the simplest form of ELISA assay, in which the antigen is absorbed into a multi-well plate and then bound to a labeled antibody for detection. The antigen-coated plate is detected by the antibody, which binds directly to the enzyme. Because only one antibody is used in direct ELISA, they are less accurate.
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Indirect ELISA – The indirect ELISA technique uses a two-step detection process. This assay involves two binding processes for the primary antibody and the labeled secondary antibody. First, the unlabeled primary antibody binds to the specific antigen.
The enzyme then binds to a secondary antibody directed against the host's primary antibody. This method is very sensitive because more than one labeled antibody binds to the primary antibody. Indirect ELISA is a less expensive method than direct ELISA test. However, this test carries a potential risk of cross-reactivity by secondary antibodies.
Sandwich ELISA – Sandwich ELISA or sandwich immunoassay is one of the most widely used test formats and is designed to measure antigens between 2 antibody layers (antibodies to capture and detect).
This assay requires a combination of antibodies to two different epitopes on the target protein for greater specificity. The antibodies were immobilized on the surface of the multi-well plate and incubated first with the target protein and then with the specific antibody labeled with the enzyme.
This makes antigen detection easier. Sandwich ELISA analysis is very sensitive to direct and indirect ELISA assays. It gives very specific results because it uses two antibodies to detect the antigen.