Immunoassay coupled enzyme system

Coupled enzyme systems have been used in enzymology to significantly increase the sensitivity of detection of enzyme-substrate reactions. The addition of coupled enzyme systems to an enzyme immunoassay should lead to several orders of magnitude increase in the sensitivity (48, 67). 

The EMMIA system was developed by Ngo and Lenhoff (N3, N4). In this assay, enzyme activity is modulated by an enzyme modulator which is coupled to antigen (free form) but not by the complex of enzyme modulator-antigen and antibody (bound form). As shown in Fig. 2 and Table 6, in an enzyme inhibitor immunoassay, an enzyme inhibitor is used as a negative modulator. For example, the reaction mixture for measuring thyroxine consists of acetylcholine inhibitor-thyroxine conjugate [I-Ag], acetylcholinesterase [E], unlabeled thyroxine [Ag], and antithyroxine antibody [Ab]. When the amount of unlabeled thyroxine, which binds to antibody [Ab Ag], is increased, the free form of acetylcholine inhibitor-thyroxine conjugate [I-Ag] increases, and the enzyme activity decreases. Therefore, the enzyme activity is inversely proportional to the concentration of unlabeled thyroxine. A tetrazyme kit (Abbott) is now available for measuring thyroxine. 

The general method utilized to prepare E5-Ab solutions obviates the need for stocking large numbers of reagents which would be necessary if different activation methods were used for each antibody. A number of specific antibodies immobilized by this process have shown response similar to that of the same antibodies when adsorbed as immune complexes in the Stratus system. In addition, the dendrimer-coupled antibodies have shown dramatic improvements in sensitivity, flexibility and precision for the enzyme immunoassay system. Feasibility demonstration of an assay for DNA probes is a prelude to what can possibly be achieved with these dendrimer-based reagents. 

An enzyme immunoassay using adenosine deaminase as the enzyme label has been described (318). Potentiometric rate measurements were made with an ammonia gas-sensing electrode. The immunoassay system employed is based on competition between a model haptenic group, dinitrophenyl (DNP) covalently coupled to adenosine deaminase, and free DNP hapten for the available binding sites on the anti-DNP antibody molecules. The detection limit is 50 ng antibody. 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

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