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An enzyme immunoassay technique for the detection of sulfamethoxypyridazine in honey, developed using rabbit polyclonal antibodies raised against N-sulfonyl-4-aminobutyric acid, which contains a structural group characteristic of sulfonamides, is proposed. Under the optimized conditions, the sulfamethoxypyridazine detection limit was 0.05 ng/ml, with the entire analysis procedure taking 2 h. In total, 24 honey samples were tested using the protocol based on tenfold dilutions of samples without their preliminary treatment.

The approaches for high sensitive and specific determination of nonylphenol (NP) with the help of immune enzymatic (ELISA) method have been developed. The process of preparation of conjugates of NP with proteins, antiserum obtaining, purification of immunoglobulin (Ig) fractions and study of specificity of the obtained antibodies were described in detail. It was shown that the antiserum and total Ig fraction do not differ in respect of specificity and binding abilities. The developed algorithm of ELISA method fulfillment is able to provide NP revealing with the sensitivity in the range of 20-50 ng/ml and working controlled concentrations--from 20-50 to 1000 ng/ml.

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3-63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 microg/l) by several orders of magnitude.

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,2,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3-7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Equilibrium and kinetic constants were determined for interactions between alpha-amylase from Bacillus subtilis and polyclonal antibodies (with immobilization of either reagent). The effects of desorption of immobilized molecules and intermolecular complexes on the immunochemical reaction were studied. Models of sandwich enzyme immunoassay, whereby complexes of immobilized antibody-determined antigen-labeled antibody were formed, were developed from these results. The results obtained from the model and experiments were compared. The desorption was shown to cause the hook-effect, that is, a decrease in the label binding at high concentrations of the antigen.

Competitive solid-phase enzyme immunoassays for the detection of 2,4-dichlorophenoxyacetic (2,4-D) and 2,4,5-trichlorophenoxyacetic (2,4,5-T) acids have been developed and optimized. The sensitivity of the assay is 3 ng/ml for 2,4-D and 5 ng/ml for 2,4,5-T. The time of the assay is 1.5 h. The sensitivity of the assays increases after immobilization of antibodies on staphylococcus protein A, in the case of using monovalent derivatives of antibodies, and as a result of chemical modification of hapten. The detection limit for the pesticides is 0.1 ng/ml in this case, which is close to the sensitivity of the assay for the haptens conjugated with proteins.

A complex of immunoglobulin A (IgA) with peroxidase was obtained as a result of interaction of IgA with the enzyme, modified with sodium periodate. The molar ratio of IgA/peroxidase was equal to 1 in the complex, as shown by gelfiltration and sedimentation analysis. Peroxidase maintained 75% of the initial activity in the complex. The antigenic and enzymatic properties of the complex were retained completely within 5 months, if the preparation was kept at 4 degrees in phosphate buffer, pH 7.4. The complex isolated was used for quantitative estimation of IgA in human blood serum. The method is based on the reaction of competitive binding between free and labelled with peroxidase IgA molecules and antibodies, immobilized on inorganic porous carrier--sylochome. The method enables to estimate quantitatively IgA concentration within the limits of 102=105 ng/ml.

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.

A technique for quantitative determining horseradish peroxidase was developed using an amperometric sensor that makes it possible to register up to 50 pM of the enzyme in the reaction of the peroxidase oxidation of iodide ions by hydrogen peroxide. The sensor, composed of combined carbon and Ag/AgCl electrodes, is intended for enzyme immunoassay and may be used for registering the results in the very wells of the polystyrene plates widely used in clinics and laboratories. The dependences of the initial rate of the enzymatic reaction on the substrate concentration and pH of the medium were determined, on the basis of which the conditions for the optimal operation of the sensor were found. The amperometric and spectrophotometric methods of registration on a model system for determining antibodies against X-virus of potato were compared in order to show a possibility of the sensor applying to enzyme immunoassay.

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.