ELISA| Enzyme-linked Immunosorbent

 It is the enzyme-linked immunosorbent assay, is a plate-based immunoassay used to detect and quantify biomolecules such as antibodies, proteins, hormones or peptides, as well as for the characterization of protein-protein and protein-nucleic acid interactions. In an enzyme-linked immunosorbent assay the target of interest, typically an antigen, is immobilized onto a solid surface and subsequently probed with an antibody conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Quantification is achieved by incubation with a substrate, which is catalyzed by the enzyme, resulting in a measurable byproduct.

Overview of ELISA (enzyme-linked immunosorbent assay)

ELISA assays are traditionally performed in more than 90-well polystyrene microtiter plates. These plates are designed to more passively bind and immobilize antigens, typically through direct adsorption to the surface of the microtiter plate or indrectly via pre-coated "capture" antibodies. If the Once immobilized, than primary antibodies are introduced to the sample, forming an immune complex with the antigens. Primary antibodies can be either covalently labeled to an enzyme, such as HRP, or can itself be indirectly detected using the enzyme-labeled secondary antibodies or streptavidin conjugates if the primary antibody is labeled with biotin. Detection is achieved by evalutating the conjugated enzyme activity via incubation with the appropriate substrate, which produces a measurable byproduct. Substrates vary in sensitivity and compatability with imaging equipment, and should be considered carefully when designing an ELISA.

Direct vs. Indirect Detection

Direct ELISA assay

In a direct ELISA, the antigen is bound to the bottom of the microplate well, then it is bound by an antibody specific to it and also conjugated to an enzyme or other molecule that allows detection.

Indirect ELISA

In an indirect ELISA, the antigen is bound to the bottom of the microplate well, then an antibody specific to the antigen is added. A secondary antibody, conjugated to an enzyme or other detection molecule is then linked to the first antibody.

Competitive ELISA

In a competitive ELISA, a reference antigen is bound to the bottom of the microplate wells. Sample plus antibody are added to the wells, and if any antigen is present in the sample, it competes with the reference antigen to bind to the antibody. The unbound substance is removed. The greater the amount of antigen in the sample, the lower the amount of antibody bound by the reference antigen to the bottom of the wells, and the weaker the signal.

Sandwich-type ELISA assay

For the sandwich type ELISA, two antibodies specific to two different epitopes on the target antigen are used. The capture antibody is bound to the bottom of the microplate well and binds to an epitope of the antigen. The detection antibody binds to the antigen at a different epitope and is conjugated to an enzyme that allows detection. (If the detection antibody is not conjugated, then a detection antibody conjugated to a secondary enzyme is required).

Steps to Run a Sandwich-Type ELISA Assay

Most sandwich-type ELISAs are run in microplates, with the bottom of the microplate wells serving as a solid surface to which antibodies and other reagents attach. A microplate washer is used to remove non-specific substances from the wells, and an absorbance ELISA microplate reader detects the color change produced when the target antigen is present. Microplate reader software is used to plot the standard curves and calculate the results.

Enzymes-linked Immunosorbent Sorbent  and applications

 The enzyme immunosorbent assay is a commonly used analytical technique in many research and biotech laboratories. Below is a collection of application notes, research and technology for significant ELISA assays and applications.