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Aptamers are single-stranded nucleic acids, typically 20-80 nucleobases (nb) in length, which can bind different compounds with high affinity and selectively. Their ligand-binding properties can be attenuated by adding short complementary strands. These interactions open new opportunities for aptamer-based assays. Strong dependence between the length and electrophoretic mobility of short nucleic acids makes polyacrylamide gel electrophoresis a powerful tool for studying their complexes. The interactions between the 36 nb DNA G-quadruplex aptamer (5’-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3’) specific to ochratoxin A and 9 complementary single-stranded DNA (ssDNA) were studied. The length of ssDNA varied from 5 to 9 nb. To maintain ligand-binding conformation of the aptamer, a high ionic strength buffer was used. The best resolution between the aptamer and its complex was provided for the gel with 15% monomer and a monomer/cross-linker ratio of 15:1. Bands of free aptamer and ssDNA were observed for all studied variants. If the ssDNA length was less than 9 nb, the position of the aptamer’s band remained unchanged, independent of the aptamer/ssDNA ratios, and additional bands did not appear. The longest ssDNA (5’-CGC-CAC-CCA-3’) did not lead to the appearance of a new band, but it slowed the aptamer’s migration depending on the ssDNA concentration. Under a 27-fold excess of the given ssDNA, the relative mobility of the aptamer band changed from 0.566 to 0.468. Thus, electrophoresis visualizes aptamer-ssDNA interactions and can be used in the development of aptamer-based analytical systems.

As revealed by double immunodiffusion and the competitive immunoenzyme assay, the immunization of animals with ribosomes isolated from group A streptococci leads to the formation of antibodies to M-protein and does not lead to the formation of antibodies to A-polysaccharide and lipoteichoic acid, which is seemingly caused by the presence of M-protein in ribosomes. Antiribosomal serum has permitted the detection of more antigenic determinants in M-protein than commercial antiserum to homologous M-protein. The immunoenzyme assay for the quantitative determination of ribosomes has been developed.

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.

To control the effectiveness of vaccination against influenza, the optimum conditions for making the enzyme-linked immunosorbent assay (ELISA) with a view to determine the level of anti-influenza antibodies in human blood sera have been established. The kinetics of influenza virus adsorption in the wells of ELISA polystyrene plates and the kinetics of the interaction between the immobilized antigen and species-specific peroxidase-labeled antibodies have been studied. The method has been shown to be more sensitive than the hemagglutination inhibition test in the determination of seroconversion in persons immunized with influenza vaccine.

Studies aimed at the development of a variant of ELISA for the determination of hepatitis B virus surface antigen (HBsAg) showed the assay to be most effective when a modified periodate method of conjugation of highly purified horseradish peroxidase with the IgG-fraction of antiserum to HBsAg was used. With the resulting conjugates, the "sandwich"-test on polystyrene solid phase could detect HBsAg in concentrations up to 5 ng/ml which is several thousand-fold higher in sensitivity than counter immunoelectrophoresis, one order of magnitude more sensitive than the passive hemagglutination test, and comparable with the radioimmunoassay (RIA). The specificity of the method was confirmed by positive results of the neutralization test. By this method, HBsAg in the sera of hepatitis B patients was detected as frequently as by the RIA.

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 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.