Enzyme multiplied immunoassay technique EMIT

Pankey et al.21 described a rapid, reliable, and specific enzyme multiplied immunoassay technique (EMIT ) for amitriptyline, nortriptyline, imipramine, and desipramine in sera. To overcome crossreactivity, solid phase extraction was included in sample pretreatment. Disposable 1 mL columns packed with covalently labeled silica gel were conditioned with HPLC-grade methanol (1 mL) and then with de-ionized or distilled water (1 mL). Serum (calibrator, control, or patient sample, 500 L) was applied onto the column, eluted to waste, washed with 900 /uL of wash solution containing acetonitrile (236.1 g/L) and ion-pairing reagent in acetate buffer, pH 4.2, washed with 500 fiL of mobile phase solution containing acetonitrile (393.5 g/L) in methanolic phosphate buffer, pH 7.0,... 

With enzyme-multiplied immunoassay technique (EMIT) assays, enzyme tags are used instead of radiolabels. The antibody binding alters the enzyme characteristics,... 

Enzyme Multiplied Immunoassay Technique (EMIT). This technique employs enzyme-labelled antibiotics which react analogously to the fluroimmunoassay in that a reduction of enzjrme activity is attributed to antibody binding. Higher concentrations of unlabelled drug in the sample result in less enzyme-labelled drug bound to the antibody. 

Figure 15.6 Comparison of the values measured by an enzyme-multiplied immunoassay technique (EMIT) assay and a molecularly imprinted sorbent assay (MIA) for determination of theophylhne in 32 patient semm samples. The correlation coefficient was 0.98. Reprinted from Vlatakis et al. (2003). Copyright 1993 Macmillan Ltd.

In studies performed by Arnold " and Sachs and Arnold, hair samples were treated with a solution of 1 M NaOH and boiled until the hair disintegrated. The hydrolyzed sample was neutralized with 1 M HCl. The same approach was employed by Kintz and Mangin. Hair digests were analyzed directly by the enzyme multiplied immunoassay technique (Emit , Syva Corporation, Palo Alto, CA) and fluorescence polarization immunoassay (FPIA, Abbott Laboratories, Abbott Park, IL). 

In the enzyme-multiplied immunoassay techniques (EMIT), the antigen or antibody is labeled with an enzyme (e.g., iysozyme, alkaline phosphatase, horseradish peroxidase, or glucose-6-phosphate dehydrogenase) instead of a radioisotope. For example, an alkaline phosphatase-labeled drug can be made to compete with an unlabeled drug for binding sites on... 

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). 

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). 

Types of enzyme-linked immunoassay include enzyme-linked immunosorbent assay (ELISA), enzyme multiplied immunoassay technique (EMIT), and CEDIA. 

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-... 

Enzyme-linked immunosorbent assays (ELIZA) and the enzyme-multiplied immunoassay technique (EMIT) are enzymatic assays in which the radioactive tag has been replaced with an enzyme. 

A comparison of fluorescent immunoassay and enzyme-multiplied immunoassay techniques (EMITs) concluded that they are similar in terms of accuracy, precision, and simplicity however, the sensitivity of fluorescent immunoassay is better by a factor of 8 than that of EMITs, but the assay time for fluorescent immunoassay is longer. The comparison was performed on the measurement of serum carbamazepine. 

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. 

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... 

Enzyme-Multiplied Immunoassay Technique (EMIT, Figure 4.29)... 

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