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The authors propose a method for determination of proteolytic activity, based on the hydrolysis of peroxidase-labeled molecules of bovine serum albumin immobilized on the surface of polystyrene microassay plates with the subsequent determination of peroxidase activity on the carrier or in the solution. The optimum conditions for the sorption of the labeled substrate have been established. The method permits the determination of bacillary alkaline protease at a concentration of 01. microgram/ml within 45 minutes. The determination of four proteases has demonstrated that this method shows good correlation with the routine one (r = 0.98), but is more sensitive and less time- and labor-consuming.

The solid-phase enzyme immunoassay for testosterone (TS), permitting the determination of this hormone at concentrations of up to 0.5 ng/ml, has been developed. The method comprises the adsorption of TS conjugated with soya trypsin inhibitor in the wells of a standard polystyrene assay plate, competition between adsorbed TS and TS under test for the binding sites of specific antibodies, and the detection of antibodies bound to the carrier by means of peroxidase-labeled antispecific antibodies. Antisera to TS have been obtained by the immunization of rabbits with TS conjugated with bovine serum albumin of a known composition. These antisera are specific to TS and do not interact with estrogens and progesterone. The study of their cross reactions with eleven TS derivatives has demonstrated that antibodies reveal the presence of structural changes in ring D of the molecule of TS and are insensitive to variations in ring A. The determinant comprising the 17-OH-group essentially contributes to the binding of antibodies.

The complexes of human immunoglobulin G (IgG) with peroxidase produced by a two-step synthesis using glutaraldehyde retain high enzymatic and immunochemical activities. The effectiveness of the complex formation depends on the concentration of glutaraldehyde used for enzyme modification. Optimal molar ratios of the constituent components during the synthesis of the complex and localization of the enzyme on the IgG molecule are discussed. Preliminary heating of IgG or the overall complex at 50 degrees C results in an increase of the complex stability (20-fold) under storage.

The diagnostic test system based on the solid-phase enzyme immunoassay (EIA) for the detection of antibodies to Yersinia pseudotuberculosis in the sera of patients with the use of Soviet-made preparations and reagents has been developed. The test has been performed in microchambers for immunological reactions, thus making it possible to decrease the consumption of reagents 10-20 times in comparison with the traditional technique with the use of plates. The results of the titration of 42 sera in EIA and in the passive hemagglutination test (PHAT) are indicative of the presence of positive correlation (r = 0.78; p less than 0.05) between antibody titers in EIA and PHAT. A fourfold or greater increase in antibody titers has been determined by means of EIA in 80% of cases and with the use of PHAT in 55% of cases. The minimum diagnostic titer yielded by EIA has been determined: 1:256.