Immunoassay enzyme-multiplied

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,... 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Figure 7.15 Enzyme-multiplied immunoassay (EMIT). The three reactants, test (or standard) antigen, enzyme-labelled antigen and a limited amount of antibody are allowed to react and reach an equilibrium position. The unbound labelled antigen which remains is the only source of enzyme activity, the bound enzyme being inactivated. This free enzyme can be quantitated using a direct kinetic assay method and is proportional to the amount of unlabelled antigen originally present.

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. 

EMIT enzyme-multiplied immunoassay test epi epinephrine EPS extrapyramidal symptoms (tardive dyskinesia, tremors and rigidity, restlessness [akathisia]. 

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.

An ingenious method of decreasing detection limits in biosensors that is frequently employed is enzyme amplification. An example is enzyme multiplied immunoassay technology (emit). 

Yamamoto, Y., Yamamoto, K., Comparison of Enzyme Multiplied Immunoassay Technique and High Performance Liquid Chromatography in Determination of Amphetamine in Urine, Nippon Floigaku Zasshi, 34,158,1980. 

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). 

Quite recently, Schafroth et al. (1994) determined the major urinary compounds of eight common benzodiazepines (flunitrazepam, diazepam, midazolam, clonazepam, bromazepam, temazepam, oxazepam, and lorazepam). The used MEKC with 75 mM SDS in a phosphate-borate buffer (pH 9.3). After enzymatic hydrolysis and extraction with commercial double-mechanism cartridges, the sensitivity was reportedly better than that obtained with the common immunoassay EMIT (enzyme-multiplied immunoassay technique). 

Hosotsubo, H. et al. Analytic validation of the enzyme multiplied immunoassay technique for the determination of mycophenolic acid in plasma from renal transplant recipients compared with a high performance liquid chromatographic assay. Then Drug Monit. 2001, 23, 669-674. 

Gulbis B, Van der Heijden J, van As H, Thiry P. Whole blood cyclosporin monitoring in liver and heart transplant patients Evaluation of the specificity of a fluorescence polarization immunoassay and an enzyme-multiplied immunoassay technique. J Pharm Biomed Anal 1997 15 957-63. 

McDonagh, J.E., Nathan, V.V., Bonavia, I.C., Moyle, G.R., Tanner, A.R. Caffeine clearance by enzyme multiplied immunoassay technique a simply inexpensive, and useful indicator of liver function. Gut 1991 32 681-684... 

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... 

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. 

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). 

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