ELISA Kits

Enzyme-Linked Immunosorbent Assays (ELISA) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISAs can provide a useful measurement of antigen or antibody concentration.

We offer a variety of analytically validated, preconfigured ELISA assays, reagents and accessories for the study of metabolic disease markers and other vital research targets.

Methodology

ELISAs are used to determine the concentration of specific molecules in a sample. This method is typically highly sensitive while usually simple to perform. This solid-phase assay is possible due to the fact that proteins (antibodies) can be positively attached to plastics (96-well microtiter plate).).

Sandwich ELISA Assays

One of the most useful of the immunoassays is the two antibody ‘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 amount of antigen in an unknown sample. 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.

Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

Unlike Western blots, which use precipitating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., pnitrophenylphosphate (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 3,3’5,5’-tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively.

The sensitivity of 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.

Competitive ELISA Assays

When two “matched pair” antibodies are not available for a target, another option is the competitive ELISA. Another advantage to the competitive ELISA is that non-purified primary antibodies may be used. Although there are several different configurations for competitive ELISAs, below is an example for one such configuration. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody.

Note: Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color change.

Factors that Influence Assay Performance

The greatest impact of the behavior of the assay is typically based on the antibody pair itself. If the pair is a “good” match for the desired analyte, other unwanted factors are greatly minimized. If the pair is a “poor” match with lower affinity for the desired analyte, it may take a lot of time and creativity to achieve acceptable parameters.
Non-specific binding can cause high background values and poor recovery and linearity, which can often be controlled with IgG antibodies or serum.
False positive values can occur on Non-specific binding of samples to one or both of the antibodies present which is often detected when performing spiked recovery and linearity testing. In the ELISA format, these samples can typically be controlled by the use of IgG antibodies and/or heterophilic blockers such as Ultra HBR.
Hook Effect may give incorrect sample values for highly concentrated samples and can usually be detected during linearity studies.

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