ELISA - Enzyme-linked Immunosorbent Assays

ELISA combines the specificity of antibodies with the sensitivity of simple enzyme assaysm by using antibodies or antigens coupled to an easily-assayed enzyme that possesses a high turnover number. ELISA can provide a useful measurement of antigen or antibody concentration.

Then the reaction is detected by colour change or flourometric reaction that can be measured quantitatively.

There are two types of ELISA developed for detection of mycotoxins.
Direct competitive ELISA and Indirect competitive ELISA.

Direct competitive ELISA involve incubation of a toxin-enzyme conjugated and the unknown sample in the presence of the antibody, typically coated in wells of a microtiter plate or other solid support. Toxin in the sample and the toxin-enzyme conjugate compete for antibody binding sites during incubation and then the solution is washed away. An appropriate substate solution is added to react with any enzyme that is bound by the antibody. The color produced by this reaction can be measure visually by comparison with standards or with a spectrophotometer to acheive quantitation.

Indirect competitive ELISA which uses a mycotoxin-protein conjugate coated on the microplate is incubated with the particular antibody and the test sample. The toxin in the sample competes with the toxin-protein conjugate on the support for antibody binding sites present in the solution. The amount of antibody bound to the plate is determined by reaction with an enzyme complex and subsequently with a substrate.