Biochemical Techniques and Instrumentation

3.2.3. Components of ELISA

1. Antigen

Any substance that stimulates an immune response is known as antigen. It is the target protein in the sample that binds to the antibody.

2. Antibody

An antibody is a protein made in response to an antigen. Each antibody binds in the epitope region of its antigen. Two types of antibodies viz. monoclonal or polyclonal can be produced and used as the capture and detection antibodies. The monoclonal antibodies are used as detection antibody as they have monospecificity toward a single epitope and allows fine detection and quantitation of small differences in antigen.

3. Enzyme conjugate

An enzyme conjugate is an antibody joined with an enzyme. The joining of the enzyme to antibody is called conjugation. The detection enzyme can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP).

4. Substrate

A substrate is a compound that undergoes change. The substrate binds to active sites on the surface of enzyme and gets converted by the change of color. The choice of substrate depends upon the assay sensitivity and instrumentation available for signal-detection such as spectrophotometer, fluorometer or luminometer. The common ELISA substrates are:

1. PNPP (p-Nitrophenyl phosphate, Disodium Salt) is used to detect alkaline phosphatase. It produces a yellow water-soluble reaction product that absorbs light at 405 nm.
2. ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is used to detect horse radish peroxidase (HRP). It produces a water-soluble green end reaction product which has two major absorbance peaks, 410 nm and 650 nm. ABTS is less sensitive than OPD and TMB
3. OPD (o-phenylenediamine dihydrochloride) is used to detect horse radish peroxidase and yields a water soluble yellow-orange reaction with an absorbance maximum of 492 nm.
4. TMB (3,3',5,5'-tetramethylbenzidine) is used to detect horse radish peroxidase. It yields a blue color with absorbance maxima at 370 nm and 652 nm. It is very sensitive.