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The Basics of ELISA Immunoassays

Understanding the basic principles of immunoassays is easy. The essential components of antibody-based immunoassay systems are threefold: an antigen to detect and perhaps quantitative; a specific antibody to this antigen; and a system to measure the amount of antigen in a given sample.

Although the system looks very simple, in many cases a number of other test materials are required to enable fast and convenient measurement. The bndf ELISA is also a widely used application for detecting and quantifying proteins and antigens from various samples.

The immunometric test, also known as a sandwich ELISA (Enzyme-Linked Immunosorbent Assay), uses two antigen-specific antibodies to capture or detect the antigen in the detection well.

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Immunometric analysis shows a direct relationship between antigen concentration and substrate response. Immunometric tests typically use plate-coated "catch" antibodies to bind to the desired antigen.

During the second incubation, the antigen binds to a second "detection" antibody that is also specific for the antigen. The detecting antibody can be bound by secondary antibody-enzyme conjugation, or the detector antibody itself can be conjugated.

When a chromogenic substrate is added to the assay for color, a sample with a high antigen concentration will produce more signals than a sample with a low antigen concentration, resulting in a signal that is directly proportional to the number of antigens in the sample.

This correlation can then be used to extrapolate the antigen concentration from the standard curve to an unknown sample. In enzyme-linked immunosorbent assays (EIA), the antigens in the sample compete for a limited antibody binding site against the antigen conjugated with the reporting enzyme. This results in an inverse relationship between antigen concentration and substrate turnover.