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تسجيل

Enzyme immunoassays , Antibody-capture systems

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  p382-384

2026-11-04

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Enzyme immunoassays

Antibody-capture systems

Figure 1 is a schematic representation of an EIA based on a microplate antibody capture. The system uses anti-drug antibodies coated onto a microplate well by passive absorption. A microplate is a tray of 96 wells (each having a volume of approximately 400 μL) that are generally arranged as 12 X 8-well strips. The coating can be achieved by simply adding a solution of the antiserum or purified antibody in a bicarbonate buffer at pH 9 using high-precision pipettes. A suitable incubation period (e.g. overnight at room temperature) allows the anti-bodies to bind to the plastic microplate well. Following this incubation, the solution is washed from the solid phase and dried to leave an antibody-coated well. In some cases, a secondary coating of a non-relevant protein is performed to increase stability of the solid-phase antibody and to prevent nonspecific binding of assay components to the plastic well of the microplate. This process forms part of the reagent manufacturing process of commercial kits provided as a dry microplate with the antibody pre-coated. In running the assay, a sample (10–50 μL) is added to the microplate followed by a buffered solution (typically 100 μL) that contains a fixed amount of drug labelled with an enzyme such as horseradish peroxidase or alkaline phosphatase. The plate is then left for sufficient time to allow the horseradish peroxidase-labelled drug and any drug present in the sample to compete for binding to the solid-phase antibody. This is referred to as the incubation period. In the absence of drug, the maximum binding of the enzyme label occurs. Increased amounts of drug in the sample result in decreased amounts of antibody-bound enzyme label. After the incubation period, the microplate is washed with a suit able buffer (the separation step) to remove all traces of unbound enzyme label. Antibody bound enzyme conjugate (and antibody-bound drug) is left immobilised to the wall of the antibody-coated microplate. After the wash step, a substrate reagent is added to the microplate well and left to develop colour. The substrate of choice for horseradish peroxidase is dilute hydrogen peroxide with tetramethylbenzidine (TMB) as chromagen, which is available as a liquid-stable, ready-to-use product. TMB provides a blue-coloured signal that can be measured at 630 nm using a standard laboratory microplate reader. In routine use, the colour development is stopped, after it has developed over a 20–30 min incubation period, by the addition of 1 M dilute sulfuric acid. This causes a shift in absorbance that is read at 450 nm. The whole assay process described here can be fully automated or performed with the simplest of laboratory equipment.

The amount of enzyme-labelled drug bound to the solid-phase antibody, and therefore available for colour development, is inversely proportional to the amount of drug in the specimen. The colour produced at the final stage of the EIA is therefore inversely proportional to the amount of drug in the specimen, which gives a calibration curve similar to that found with RIA. The amount of enzyme-labelled drug bound to the solid-phase antibody, and therefore available for colour development, is inversely proportional to the amount of drug in the specimen. The colour produced at the final stage of the EIA is therefore inversely proportional to the amount of drug in the specimen, which gives a calibration curve similar to that found with RIA.

Figure 1 A heterogeneous EIA.