Use of solid-phase immunoenzyme analysis for the serodiagnosis of pseudotuberculosis

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.     

The results of using direct solid-phase immunoenzyme analysis for assessing the specific activity of rabies vaccines

The effectiveness of the solid-phase enzyme immunoassay (EIA) in the determination of the specific activity of rabies vaccines is evaluated in comparison with that of the protective test in mice. Inactivated tissue-culture, concentrated tissue-culture and purified cerebral tissue vaccines for human use were studied. The methods for performing two EIA variants and evaluating the results are described. The average level of correlation between the results of EIA and the protective test for vaccines of different groups was revealed (0.546), the highest correlation index being obtained for tissue-culture vaccines: 0.753. On the basis of the data obtained in this study the expediency of using EIA for the determination of the specific activity of rabies vaccines has been substantiated.     

The standardization of conjugates for the indirect solid-phase immunoenzyme analysis reaction

The comparative study of several batches of conjugates has revealed that enzyme immunoassay techniques can be used for the standardization of conjugates by their affinity level. The study has shown that this can be done only if the concentration of specific antibodies in the conjugate is known and the amount of the conjugate is in excess to that of the antigen adsorbed on the plate.     

Use of solid-phase immunoenzyme analysis for determining allergen-specific IgE antibodies

An ELISA system for the detection of allergen-specific IgE antibodies to ragweed allergen has been developed. The system is highly sensitive and specific. Ragweed pollen allergen has been obtained by the dialysis of water-soluble extract through a kidney membrane. The high molecular fraction of ragweed allergen, showing the whole of the allergenic activity detected by skin tests in untreated patients, has been used for coating polystyrene assay plates. To detect IgE antibodies to ragweed allergen, the conjugate of sheep anti-IgE antibodies with horse-radish peroxidase has been used. The level of allergen-specific IgE antibodies has been determined on the basis of the data on the optical density of the samples in comparison with that of the normal sera. The correlation factor of the results obtained in the assay of specific IgE antibodies with the newly developed assay system and with the commercial kit Phadezyme RAST manufactured by Pharmacia AB (Sweden) has proved to be 0.82 at n = 39, p less than 0.01, while the variation factor in the reproduction of the assay results has proved to be 12% at n = 40.     

Use of direct solid-phase immunoenzyme analysis for assessing the immunological activity of a vaccine against tick-borne encephalitis

A high correlation between the current index of the effectiveness of tick-borne encephalitis vaccine, its protective activity in mice, and the results of the direct solid-phase enzyme-immunoassay has been established, which permits the use of this assay as an auxiliary method for the immunological evaluation of newly prepared commercial purified tick-borne-encephalitis vaccine.     

Study of the affinity characteristics of monoclonal antibodies by solid-phase immunoenzyme analysis

An approach is proposed for measuring the binding constant (Kb) for monoclonal antibodies (MA) interacting with an immobilized antigen in indirect ELISA. This approach allows the measurement of optical density (A405) in the peroxidase reaction initiated by the conjugate at different concentrations (C0) of antibodies. Using the Scatchard plots, the dependence of A405/C0 = f (A405) for the whole range of MA concentrations was examined, and the tangential of the slope (tg alpha = Kb) of the linear portion of the antigen molecule was calculated. Analysis of MA affinity parameters by using this approach may find wide use in immunodiagnostic studies aimed at measuring antigen and antibody concentrations in biological fluids as well as for estimating the efficiency of vector drugs in which the diagnostic or therapeutic component is conjugated with the vector (MA or F(ab) fragment) responsible for the drug transport to target cells. The method proposed was used for testing mouse (BALB/C) monoclonal antibodies (IgG1) to pig insulin produced by various hybridomas as well as for estimating the effect on MA of pH, temperature and hydrophobization. The minimal detectable concentration (method sensitivity) was found to depend on Kb.     

The diagnosis of antileptospiral antibodies in human subjects by solid-phase immunoenzyme analysis

The possibility of using the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of leptospirosis has been shown. This method has proved to be more simple and sensitive than the leptospiral microagglutination and lysis test. The data on obtaining genus-specific leptospiral antigens are presented. As revealed in this study, the antigens obtained by the complex treatment of microbial cells with ultrasound and detergents show the maximum activity in ELISA. The optimum parameters of the ELISA system for the diagnosis of leptospirosis have been established.     

Rapid serological screening of human sera for the presence of measles virus antibodies in solid-phase immunoenzyme analysis

A single dilution technique has been used for the determination of antimeasles antibody titer. The method involved the plotting of the calibration curve and the characterization of the serum by arbitrary "evaluation units" in comparison with the specially selected positive serum whose titer was taken to be equal to 100 "evaluation units". By means of this method 57 sera obtained from children immunized against measles and 118 sera from non-vaccinated adults aged 18-22 years were examined. The values of the calculated titers were similar to those determined experimentally. This recommends this method for seroepidemiological investigations aimed at determining the level of herd immunity to measles.     

Use of immunoenzyme analysis for controlling the activity of antisera to insulin

Antisera to insulin were obtained from guinea pigs hyperimmunized with the use of Freund's adjuvant. To control the specific activity of antisera, the highly specific modern method of ELISA was used. Insulin was allowed to adsorb on the surface of the wells of polystyrene microplates, then consecutive dilutions of the tested antisera to insulin were placed into the wells. The insulin-antibody complexes thus obtained were detected by means of immunoperoxidase conjugate. Antibody titers in antisera obtained from different animals varied from 5 X 10(-3) to 10(-5). A good correlation between the results obtained by ELISA and by radioimmunoassay was observed.     

Choice of the suitability criteria of polystyrene-based solid-phase carriers for performing immunoenzyme analyses

The authors discuss a tentative approach to the choice of criteria indicating the optimal suitability of different solid-phase carriers made of polystyrene for use in the enzyme immunoassay (EIA), viz. the dependence of specificity, sensitivity, reproducibility and reliability of EIA results on the adsorption properties, transparency expressed in percent and transparency variations of the plates under test. The evaluation of the carriers by four parameters is proposed with the use of assay plates manufactured by Nunc A/S (Denmark) for control. To ensure the objective evaluation of the suitability of polystyrene plates for use in EIA, the choice of uniform criteria is necessary.     

The use of immunoenzyme analysis in the laboratory diagnosis of plague]

The possibility of using the enzyme immunoassay (EIA) for the early diagnosis of pneumonic plague was studied in experiments on monkeys. EIA was shown to be more effective than the passive hemagglutination test. The diagnostic value of blood serum samples was found to be higher than that of nasopharyngeal mucus samples taken from the sick animals. The conclusion on the suitability of EIA for the early laboratory diagnosis of this disease was made.     

Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.     

Problems of selecting the reagent concentration in the solid-phase immunoenzyme method for determining antibody concentrations

The problems of selecting the concentration of reagents with the aim of increasing the accuracy and sensitivity of reactions, as well as decreasing the consumption of reagents, are shown as exemplified by the antigen-antibody reaction of the first order. A new approach to the ELISA reaction is proposed, which makes it possible to present the totality of consecutive interactions of the reagents as block diagrams described by known mathematical expressions. The limitations of the linear dependence of the results of the reaction are shown, and the methodological recommendations for overcoming these limitations are given.     

The indirect solid-phase method of immunoenzyme analysis, its accuracy and sources of error

The study of the accuracy of the enzyme-linked immunosorbent assay has revealed that the accuracy of this assay is low. The influence of instruments (dispensers, photometers, plates) on the accuracy of the assay has been studied. As shown in this study, the main sources are titration procedures and differences in the adsorption and optical properties of available plates, those manufactured in the USSR giving greater errors.     

Application of competitive immunoenzyme assay for analysis of group B fumonisins]

A varriant of competitive ELISA, making it possible to detect fumonisin B1 (up to 0.4 ng/well) was developed, based on the use of specific polyclonal antibodies obtained by immunization of rabbits with a horseradish peroxidase conjugate of fumonisin B1. The minimum concentration of fumonisin B in water-acetonitrile extracts of corn grain, dry corn products, and feeds, detected by the ELISA version developed is 0.2 mg/kg.     

Use of an immunoenzyme method on a solid-phase carrier for determining the tularemia antigen

The method of the highly sensitive (up to 10 ng/ml) and specific determination of soluble tularemia antigen, based on the use of "sandwich" type ELISA techniques, has been developed. The dependence of the specificity and sensitivity of the method on the degree of purification of antibodies and their peroxidase conjugates used in the assay has been studied. The study has revealed that the best results can be obtained with the use of purified IgG and its conjugate free of unbound peroxidase. Both foreign peroxidase preparations and type A enzyme manufactured in the USSR can be equally used as enzymatic labels.     

The use of immunoenzyme analysis and immunoblotting for the serological characterization of mycobacterial antigens]

The enzyme immunoassay and immunoblotting were used for the study of the serological activity of different mycobacterial antigens and the spectrum of antibodies to them in patients with different forms of tuberculosis and healthy persons. Antibodies in patients' sera were shown to bind antigens with different molecular weight. The level and spectrum of antibodies to purified protein fraction I made it possible to differentiate between patients with various forms of tuberculosis and healthy persons.