Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen. An ELISA is a five-step procedure:
coat the microtiter plate wells with antigen;
block all unbound sites to prevent false positive results;
add antibody to the wells;
add anti-mouse IgG conjugated to an enzyme;
reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction.
There are many different types of ELISAs. One of the most common types of ELISA is "sandwich ELISA."
This assay is used to determine the antigen concentration in unknown samples. This ELISA is fast and accurate, and if a purified antigen standard is available, the assay can determine the absolute amounts of antigen in unknown samples. The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies.
To utilize this assay, one antibody (the "capture" antibody) is purified and bound to a solid phase. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the "detection" antibody) is allowed to bind to the antigen, thus completing the "sandwich". The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. A major advantage of this technique is that the antigen does not need to be purified prior to use, also that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified at "matched pairs", meaning that they can recognize separate epitopes on the antigen.
Unlike Western blots, which use preciptating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those which convert a colorless substrate to a colored product, e.g. p-nitrophenylphosphate (pNPP) which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2?-azo-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3?,5?tetramethylbenzidine base (TMB), which yield green orange and blue colors, respectively. A table of commonly-used enzyme-substrate combinations is included in Appendix A.
The sensitivity of the Sandwich ELISA is dependent on four factors:
The number of molecules of the first antibody that are bound to the solid phase.
The avidity of the first antibody for the antigen.
The avidity of the second antibody for the antigen.
The specific activity of the second antibody.
The amount of the capture antibody that is bound to the solid phase can be adjusted easily by dilution or concentration of the antibody solution. The avidity of the antibodies for the antigen can only be altered by substitution with other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains. Antibodies can be labeled conveniently with iodine, enzymes, or biotin.
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