Enzyme-linked Immunosorbent assay (ELISA) can provide a useful measurement of antigen or antibody concentration. A standard ELISA is a five-step procedure:
- Coat a microtiter plate wells with antigen.
- Block all unbound sites to prevent false positive results.
- Add antibody to the wells.
- For indirect methods: add secondary antibody conjugated to an enzyme.
- Reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. ELISA may be run in a qualitative or quantitative format.
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A prime advantage of ELISA applications is that the results are quantifiable. Performing an ELISA involves at least one antibody to detect a particular antigen or the presence of another antibody. The sample with an unknown amount of antigen is immobilized on a solid support. The "detection antibody is then added, forming a complex with the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Instead of using radioactive materials, which have been utilized on a large scale in the beginning of molecular biology, to determine the results of a test, the ELISA uses enzymes which react with antibodies to form colored products. The development of color in an ELISA kits test indicates a positive result. There are two main variations on this method: It can either be used to detect the presence of antigens that are recognized by an antibody (direct method) or it can be used to test for antibodies that recognize an antigen (indirect method).
As summarized above, in a direct ELISA the labeling occurs with the antibody itself. Microwell plates are coated with a sample containing the target antigen. The binding of labeled antibody can be quantified. Advantageous is, that the direct ELISAs are relatively quick, due to just one antibody being applied. It also avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample. However, the direct ELISA requires the labeling of every primary antibody, which can be time-consuming and more expensive than in indirect methods. Additionally, certain antibodies may be unsuitable for direct labeling. Another downside of the assay is that direct methods do not allow for signal amplification in contrast to methods that use a secondary antibody.
The indirect ELISA is a two-step method using labeled secondary antibody for detection. First, a primary antibody is incubated with the antigen. Then a labeled secondary antibody that recognizes the primary antibody is utilized. A disadvantage of the indirect ELISA is that cross-reactivities occur, potentially leading to strong ba ckground signals. On the positive side the signal-amplification due to application of secondary antibodies improves the signal strength. Most importantly, the versatility is greatly improved. The same primary antibody can be used with differently labeled secondary antibodies.
The sandwich ELISA measures the amount of antigen between two layers of antibodies. Sandwich assays are restricted because the antigens to be measured must contain at least two antigenic sites, since at least two antibodies act in the sandwich. Sandwich ELISAs are especially useful if the concentration of antigens is low or they are contained in a mix of high concentrations of contaminating protein.
To utilize this assay, one antibody (the capture antibody)is bound to a microtiter plate well. Antigen is then added and bound to the antibody. Unbound products are then removed, and a labeled secondary antibody (the detection antibody) is added, thus completing the sandwich. The assay is then quantified by measuring the amount of labeled secondary antibody (the 3rd antibody) through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, due to its high specificity. Disadvantageous is that not all antibodies can be used.
An unlabeled primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and samples with unknown protein content. After this reaction is allowed to go into equilibrium, conjugated antigen or enzyme-linked antibody is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled antigen. Thus, the more unlabeled antigens in the sample or standard, the lower the amount of conjugated antigen bound. A substrate and color change is then measured.
The advantage to the competitive ELISA is that non-purified primary antibodies may be used. In competitive ELISA, there is an inverse relationship between the signal obtained and the concentration of the target protein in the sample, i.e. the more target protein the lower the signal.
Quantification of the signal
After binding of the antibody or antibodies a usually colorless reagent is added. The antibody-conjugated enzyme cleaves the reagent and a color reaction occurs. Even small amount of bound enzyme would cleave the reagent completely if given enough time, hence the reaction needs to be terminated, usually by adding a weak acid. Otherwise all samples would yield the same optical density and would be have been rendered indistinguishable. After stopping the reaction at when an optimal contrast has been reached photometric measurement will yield quantifiable results.
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