AIMS: To determine the antibody concentration by antibody captures ELISA. It involves the following experiments:
> Coating the wells with Antigen > Blocking & Incubation with Primary and Secondary Antibody > Detection
Principle: Enzyme-linked immunosorbent assay, commonly known as ELISA is a popular format of analytic biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. In this technique, an enzyme conjugated with an antibody reacts with a colourless substrate to generate a coloured product. Such a substrate is called a chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline phosphatase, (AP) horseradish peroxidase (HRP) etc.
BioBharati Antibody Capture ELISA kit is HRP based. The antigen is first coated in the plate followed by incubation with primary and HRP tagged secondary antibody. The HRP tagged secondary antibody is detected using hydrogen peroxide as a substrate and 3, 3’, 5, 5 Tetramethyl benzidine (TMB) as a chromogen. TMB acts as a hydrogen donor for the reduction of hydrogen peroxide to water by horseradish peroxidase. The TMB oxide is deposited wherever enzyme is present and appears as bluish colour chromogen. The intensity of the colour is measured using a spectrophotometer at 450 nm. The developed colour is directly proportional to the amount of antibody present in sample.
In this kit, antigen, primary antibody and their respective diluents buffers will be provided. Students will dilute the antigen and the primary antibody with their respective diluents as mentioned in the procedure just prior to initiate the experiment. Additionally, Horse Radish Peroxidase (HRP) conjugated secondary antibody and developing reagent (consists of three components) along with the detachable ELISA plate and blockerare provided in this kit.
# BB-ITK060 is designed to perform 4 set of reactions.