Homogeneous Competitive Immunoassays

In recent years, routine laboratory screening of drugs of abuse in urine has mainly been carried out by homogeneous competitive immunoassays. The most widely used homogeneous drug-testing immunoassay technologies include enzyme-multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA), kinetic interaction of microparticles in solution (KIMS), and cloned enzyme donor immunoassay (CEDIA). The major assay labels and the technologies are implied in the respective immunoassay nomenclature.

The assay principle of EMIT is based on the modulation of enzyme activities by the binding of specific antibodies to the enzyme-labeled drug derivatives (4-6). Currently, EMIT-based DAT immunoassays can be purchased from several companies, and a common enzyme of choice is the genetically modified glucose-6-phosphate dehydrogenase (rG6PDH). The oxidation of enzyme substrate G6P to form glucuronolactone-6-phosphate is coupled with the reduction of the cofactor nicotina-mide adenine dinucleotide (NAD) to NADH. In the absence of drugs in the sample, the antibodies bind to the enzyme-labeled drugs and inhibit the enzymatic activity. Free drugs in the specimen compete for antibody binding, so fewer antibodies are available for binding to the drug-enzyme conjugates and enzymatic activity is less inhibited. The rate of NADH production, as reflected by the change in absorbance at 340 nm, is directly related to the G6PDH enzyme activity. Therefore, the change of absorbance can be plotted vs the corresponding calibrator concentration to construct a calibration curve for running a semi-quantitative assay. The assay can also be run qualitatively by comparing the sample rate to the calibrated cutoff rate.

The measurement of FPIA relies on detecting the degree of polarization of the emitted fluorescent light when the fluorophore label is excited with plane-polarized light (7,8). FPIA requires a specific FP photometer (9,10). A polarization filter (rotational) and an emission filter (stationary) enables the photomultiplier tube to read emitted parallel and perpendicular polarized light. The degree of polarization is dependent on the rate of rotation of the drug-fluorophore conjugate (tracer) in solution. Small molecules such as tracers can rotate rapidly before light emission occurs, resulting in depolarization of the emitted light. When bound to the antibody, the tracer rotates more slowly and the level of fluorescence polarization is higher. An optimized amount of the tracer competes with free drugs in the sample for binding to a limited amount of antibodies. Hence the drug concentration is inversely related to the degree of polarization. Calibrators containing known amounts of drugs interact with the tracers and antibodies to produce a calibration curve relating drug concentrations to arbitrary "milliPolarization" units (mP). The interactions of the drugs in the specimen, the tracers, and the antibodies under the same condition controlled by the analyzer yield mP units that can be correlated with the drug level in the specimen by making a comparison with the calibration curve.

The principle of microparticle agglutination-inhibition tests has been applied to various drug screening assay formats (11-15). One KIMS DAT format is based on the competition of microparticle-labeled drug derivatives and the free drugs in the specimen for binding to a limited amount of free antibodies in solution (14,15). The drug conjugates are labeled with microparticles through covalent coupling. These drug conjugates react with free antibodies and form particle aggregates that scatter transmitted light. The KIMS-II format contains soluble polymer drug derivative conjugates and microparticle-labeled antibodies (16). The binding of the conjugates to the antibodies promotes the aggregation and leads to subsequent particle lattice formation. In both cases, the aggregation reaction in solution results in a kinetic increase in absorbance values. Free drugs in the sample compete for antibody binding and inhibit the particle aggregation. The absorbance difference between a defined initial reading and final reading decreases with increasing drug concentration, and the signal generated can be monitored spectrophotometrically. The assay can be run qualitatively in comparison with the cutoff calibrator. The assay can also be run semi-quantitatively using four or five levels of calibrators to construct a calibration curve via a logit/log fitting function.

The measurement of CEDIA is based on the antibody modulation of the complementation of two inactive polypeptide fragments to associate in solution to form an active enzyme. The fragments of the recombinant microbial P-galactosidase are called the the enzyme donor (ED) and enzyme acceptor (EA). The binding of antibodies to the drug-ED conjugates can inhibit the spontaneous assembly of active enzymes (17,18). The CEDIA reagent composition includes the lyophilized EA and ED reagents and their respective reconstitution buffer solutions. The antibody binding to drug-ED conjugates in the analyzer reaction cuvet prevents the formation of active enzymes in the cuvet. Conversely, free drugs in the specimen compete for antibody binding and allow the drug-ED conjugates to reassociate with the EA fragments. Therefore, the drug concentration is proportional to the amount of active enzyme formed. The enzyme catalyzes the hydrolysis of selected substrate such as chlorophenol red-P-d-galactopyranoside, and the resulting absorbance rate change is measured as a function of time (mA/min). CEDIA assays can be run either qualitatively or semi-quantitatively based on an appropriate calibration curve.

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