Homogeneous enzyme immunoassay EMIT

Inhibition of the activity of an enzyme molecule labeled with a hapten (e.g., morphine) as a result of combination with a specific antibody is the basis of homogeneous enzyme immunoassay (EMIT, Syva Corp., Palo Alto, Calif.). See Chapter 9 for further details. 

S., Kubo, S., and Iwai, H. Comparison of Homogeneous Enzyme Immunoassay (EMIT) and Gas-Liquid Chromatographic Measurements of Plasma Phenobarbital, Diphenyl-hydantoin and Primidone Concentrations in Epileptic Patients Kawasaki Med. J. 3(l) 59-66 (1977) CA 87 126838u... 

Fig. 1. Schematic representation of the principle of homogeneous enzyme immunoassay developed by Rubenstein (emit). [Cited and modified from Fig. IV-4, Miyai, K., in Ishikawa et al., eds. (15).]...

Homogeneous enzyme immunoassays have also been developed for serum T4 determination. These procedures are rapid and simple to use and have also been applied to several major automated instruments.For example, the enzyme-multiplied immunoassay technique (EMIT) for T4 measurement uses glucose-6-phosphate dehydrogenase covalently hnked to T4 as the enzyme label.Binding of T4 specific antibody to this label reduces enzyme activity, perhaps as a result of steric or allosteric inhibition As the concentration of unlabeled T4 increases, less enzyme-labeled hormone is bound by the antibody. As a result, the catalytic activity of the unbound enzyme conjugate increases in direct proportion to the amount of T4 in the specimen. The indicator reaction involves oxidation of glucose-6-phosphate with simultaneous reduction of nicotinamide-adenine dinu-... 

W Roos, et al. A homogeneous enzyme immunoassay for benzodiazepine alkaloids (EMIT). Pharmazie 42 213, 1987. 

Homogenous radioimmunoassay (RIA) e.g.. Scintillation proximity assay (SPA) Homogenous enzyme immunoassay (EIA) e.g.. Enzyme monitored immunotest (EMIT)... 

Reagents for the analysis of a number of drugs and hormones have recently become available commercially from the Syva Company (Palo Alto, Ca) as EMIT homogenous enzyme Immunoassays. Table III lists the clinical tests currently available from Syva which do not require prior separative steps. 

Both competitive and noncompetitive methods have been incorporated into homogeneous enzyme-labeled immunoassay kits that ultimately relate enzyme activity to analyte concentration.22 The competitive-binding assays are called enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescein immunoassay (SLFIA), apoenzyme reactivation immunoassay (ARIS), and cloned enzyme donor immunoassay (CEDIA), while a noncompetitive method is called enzyme inhibitory homogeneous immunoassay (EIHIA). 

Some enzyme immunoassays for uptake do not require separation of the reactants. In the homogenous EMIT assay, a serum sample is incubated with a known amount of exogenous T4, which binds to available sites on TBPs ... 

Although unlike RIA, enzyme immunoassays can be carried out in homogeneous systems without a separation step (based on the change in enzyme activity during the immune reaction), in practice, heterogeneous (enzyme-linked immunosorbent assay) methods are much more frequently used. The antibody, or in the case of the double-antibody method the second antibody, is immobilized, either covalently or by coating enzyme multiplied immunoassay technique (EMIT). This can be done on the walls of microtiter plates. After the immunogenic reaction, the enzyme activity, which is the equivalent of radioactivity in RIA systems, can be measured by suitable photometric methods on the microtiter plates themselves. 

In the homogeneous immunoassays there is no separation step because they are based on a changing signal by formation of the immune complex. The first assay of this type was the EMIT method (enzyme-modulated immunoassay technology) described by Engvall et al. (1970). The main disadvantages of homogeneous assays are low sensitivity and a more pronounced susceptibility to interferences. 

Homogeneous immunoassays rely on a change in the intensity of a label signal that occurs when labeled antigen binds with antibody. When the label is an enzyme, a reduction in the rate of enzyme catalysis forms the basis for the assay. This technique is commonly known as an EMIT (enzyme multiplied immunoassay technique) assay. This enables free, labeled antigen to be distinguished from bound antigen with no separation step necessary. 

Enzyme-Multiplied Immunoassay Technique. EMIT is a homogeneous method for the quantitation of haptens, especially hormones, therapeutic drugs, and drugs of abuse. This method is a competitive assay, in which hapten and enzyme-labeled hapten compete for a fixed, insufficient quantity of antibody (Eq. 6.16). 

Figure 9-15 CEDIA and EMIT homogeneous immunoassays. A, Enzyme acceptor ED, enzyme donor.

Enzyme-multiplied immunoassay technique Perhaps the best known homogeneous assay format is the enzyme-multiplied immunoassay technique (EMIT), in which the analyte is covalently attached to an enzyme, and the formation of an analyte-antibody complex blocks the active site and inhibits enzyme activity. When this blocked enzyme is mixed with the experimental sample, there is competition between the enzyme-linked analyte and the sample analyte for occupation of the antibody s antigen-binding site. The more of the analyte present in the sample, the more of the enzyme is released from inhibition, and the level of enzyme activity can thus be used to determine the quantity of the analyte. 

These four methods have been combined to develop a number of EIAs for quantifying antigens, antibodies, and haptens. For example, the enzyme multiplied immunoassay technique (EMIT ) is a commercially available homogeneous and competitive EIA for the determination of a variety of drugs. In addition, double antibody methods that utilize a heterogeneous and competitive EIA system are available. In this case, enzyme-labeled antigen and sample antigen are mixed... 

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