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Homogeneous immunoassays

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  386-387

2026-11-05

186

Homogeneous immunoassays

Homogeneous immunoassays do not require the separation of bound and free fractions, and as a result have been automated successfully in a number of different systems. The most widely used and most cost-effective immunoassay used to screen large numbers of urine samples when there is no need for high sensitivity is the homogeneous EIA marketed by Syva (Dade-Behring) for many years as EMIT (enzyme multiplied immunoassay technique). This method, for use for the analysis of morphine, was first published in 1972 (Rubenstein et al. 1972). The EMIT technique is based on the enzyme activity of a drug-labelled enzyme, glucose 6-phosphate dehydrogenase (PDH), modulated by antibodies raised against the drug. Enzyme activity (in the presence of glucose-6 phosphate) results in the conversion of nicotinamide–adenine dinucleotide (NAD) to the reduced form NADH, and the subsequent increase in absorbance at 340 nm is monitored spectrophotometrically. Addition of the anti-drug antibody results in binding to the drug-labelled enzyme, which effectively reduces the enzyme activity (Fig. 1). Any drug in the sample competes with the drug-labelled enzyme in binding to the antibody, which allows unbound enzyme to become active and thereby increases absorbance at 340 nm. Increasing amounts of drug in the sample produce increased enzyme activity and hence an increase in the rate of change of absorbance at 340 nm. The use of bacterial PDH, which uses NAD as coenzyme, avoids interference from endogenous PDH, which uses nicotinamide–adenine dinucleotide phosphate (NADP). Earlier examples of the technique used lysozyme (Rubenstein et al. 1972) or malate dehydrogenase (Ullman and Maggio 1980; Kabakoff and Gr eenwood 1981).

Figure 1 A homogeneous enzyme multiplied immunoassay technique (EMIT).