Enzyme-antibody conjugate immunoassay

O Sullivan, M., Gnemmi, E., Morris, D., Chieregatti, G., Simmonds, A., Simmons, M., Bridges, J., and Marks, V. (1979) Comparison of two methods of preparing enzyme—antibody conjugates Application of these conjugates for enzyme immunoassay. Anal. Biochem. 100, 100-108. 

The response of a surface antigen-antibody reaction can also be mediated by an electron transfer reagent. This has been demonstrated by Robinson et al. in their studies on electrochemical immunoassays for hCG and thyroxine. In the analysis of hCG, a two-site amperometric immunoassay was developed in which monoclonal capture antibodies were immobilised on the surface of a glassy carbon electrode. A second antibody against hCG was labelled with glucose oxidase. The electrode was used both to separate free from bound enzyme-antibody conjugate and to assay the enzyme activity electrochemically by use of dimethylaminomethyl ferrocene as an electron transfer mediator. This method was found to correlate well with an immunoradiometric assay. In the analysis of thyroxine, another ferrocene derivative, namely ferrocenemonocarboxylic acid, was used as the electron transfer mediator. ... 

Pool the fractions containing antibody and immediately mix with an amount of maleimide-activated enzyme to obtain the desired molar ratio of antibody-to-enzyme in the conjugate. Use of a 4 1 (enzyme antibody) to 15 1 molar ratio in the conjugation reaction usually results in high-activity conjugates suitable for use in many enzyme-linked immunoassay procedures. 

Jeanson, A., Cloes, J.M., Bouchet, M., and Rentier, B. (1988) Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent. Anal. Biochem. 172, 392. 

Tussen, R, and Kurstak, E. (1984) Highly efficient and simple methods for the preparation of peroxidase and active peroxidase—Antibody conjugates for enzyme immunoassays. Anal. Biochem. 136, 451-457. 

Chemiluminescence offers yet another sensitive detection system, which is easily implemented with simple instrumentation, but suffers to some extent from background interference in complex matrices. A recent example of an enzyme immunoassay with chemiluminescence as the detection system is the assay for 8-oxoguanine in DNA, which uses a secondary antibody conjugated with peroxidase-anti-peroxidase complex and a substrate solution containing hydrogen peroxide, luminol and p-iodophenol. 

In plant hormone enzyme-linked immunoassays (ELISA), the tracer is an enzyme-hapten conjugate, usually constructed in the same way as the immunogen. The separation of free from bound tracer is achieved by initially immobilising the antibody by adsorption... 

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