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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 that possesses a high turnover number. ELISAs can provide a useful measurement of antigen or antibody concentration.
Sandwich ELISA Assays
One of the most useful of the immunoassays is the two antibody �andwich? 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.
To utilize this assay, one antibody (the ?b>capture?antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. 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 ?b>detection? antibody) is allowed to bind to the antigen, thus completing the �andwich? The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, 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, 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 �atched pairs? meaning that they can recognize separate epitopes on the antigen so they do not hinder each other� 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? tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively. A table of commonly used enzyme-substrate combinations is included in Appendix D.
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.
General Protocol for the Sandwich ELISA method:
- Before the assay, both antibody preparations should be purified and one must be labeled.
- For most applications, a polyvinylchloride (PVC) microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.
- Bind the unlabeled antibody to the bottom of each well by adding approximately 50 � of antibody solution to each well (20 �/mL in PBS). PVC will bind approximately 100ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 �/well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be s