Use of immunoenzyme analysis for determining antibacterial antibodies in diphtheria infection

A diagnostic EIA system for the detection of antibacterial antibodies in diphtheria infection has been developed. As antigen, homogeneous membrane protein (mol. wt. 64 KD) obtained from Corynebacterium diphtheriae cell walls has been used. This protein antigen has been prepared with the use of nonionic detergent NP-40.     

Use of cooperative monoclonal antibodies in immunoenzyme analysis for determining human tumor necrosis factor

A cooperative sandwich enzyme immunoassay (EIA) based on the newly produced pair of cooperative monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was developed and characterized. It was found that, when used simultaneously, cooperative mAbs was capable to bind TNF from its preformed complexes with soluble TNF receptors (sTNF-R), thus providing the effective TNF detection in ex vivo samples by the respective one-step cooperative EIA. While demonstrating typical analytical characteristics regarding variability, dynamic range and specificity, a cooperative EIA offers an advantageous combination of high sensitivity (< 2 pg/ml) and short-time TNF capture protocol (1 hour). Application of cooperative EIA for TNF detection in clinical samples has demonstrated an increased serum TNF levels in patients with the mixed connective disease and infectious endocarditis that positively correlated with severity of systemic inflammatory reactions. Production and EIA application of cooperative mAbs would be promising in development of standardized and clinically applicable immunoassays for cytokines.     

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.     

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.     

A rapid enhanced chemiluminescent immunoassay for allergen-specific IgE antibodies

An enhanced chemiluminescent immunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed.     

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.     

Use of monoclonal antibodies in immunoenzyme analysis for demonstrating antigenic monovalency

To prove the monovalence of the antigen a method has been developed consisting of ELISA with the use of monoclonal antibodies in combination with the antibody neutralization test. Yersinia pestis capsular antigen was disintegrated by heating at 100 degrees C for a short time and subsequently passed through a column packed with Sephadex G-50. The portions of the eluate, showing high activity in the antibody neutralization test and low activity in ELISA (the double antibody sandwich scheme), contained mainly the monovalent antigen. This antigen was replaced by the polyvalent antigen from the antibody complex, but if such complex had been previously fixed by treatment with glutaraldehyde, no replacement of the monovalent antigen by the polyvalent one occurred.     

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.     

Use of immunological adsorption in heterogeneous immunoenzyme analysis in determining antibodies to the protective determinants of Bacillus anthracis

In a heterogeneous enzyme immunoassay system involving the use of polystyrene assay plates, the method of immunological adsorption has been used for studying the spectrum of specific antibodies to individual chromatographically pure fractions of B. anthracis toxin. The relationship between the characteristics of acquired stability and the level of serum antibodies to individual biologically active and biologically inactive toxin antigens in guinea pigs, immunized with live vaccines in a single injection, has been studied. As revealed in this study, the level of serum antibodies to chromatographically pure toxin fractions does not reflect acquired immunity to anthrax.     

Use of an artificial antigen simulating the Salmonella O-determinant 4 for determining antibodies to Salmonella group B O-antigen in immunoenzyme analysis

The use of the artificial antigen abequosylmannoside copolymer with acrylamide in the enzyme immunoassay for the determination of antibodies in the sera of salmonellosis patients has enhanced the specificity of the serological diagnosis of group B salmonellosis in comparison with the use of the natural antiren, S. typhimurium lipopolysaccharide.     

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.     

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 specific Bordetella pertussis antibodies in immunoenzyme analysis for detecting the pertussis antigen

The competitive EIA technique with the use of peroxidase-labeled B. pertussis antigen has been developed. The data obtained in our investigations suggest the possibility of using this technique for the detection of B. pertussis antigen in faucial smears obtained from patients.     

Use of commercially available monoclonal antibodies for immunoenzyme double staining

Summary An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.     

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.     

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.     

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.     

An immunoenzyme system for determining antibodies to Streptococcus pneumoniae polysaccharides in biological fluids

The enzyme immunoassay (EIA) system for the determination of antibodies to capsular polysaccharides of pneumococci, serotypes 1, 3, 6B, 8, 9N, 15F, 23F, and C-polysaccharide has been developed on the basis of poly-L-lysin-modified antigens. The use of isotype-specific conjugates in this system permits the detection of IgG and IgA antibodies in different biological fluids: blood serum, pleural fluid, saliva, milk. Samples obtained from children with pneumococcal infection and from nursing mothers have been studied. As shown in this study, the EIA system can be used for the evaluation of the dynamics of pneumococcal infection in children.     

The use of the microdot immunoenzyme method for determining antibodies to Mycobacterium tuberculosis antigens

The microdot enzyme immunoassay (EIA) has been used for the determination of antibodies to M. tuberculosis protein fractions, crude antigenic preparations, PPD and old tuberculin in tuberculosis patients and healthy persons. Purified protein fractions have been found to possess the highest sensitivity and specificity in microdot EIA. The determination of antibodies to these fractions has permitted the differentiation of persons infected with M. tuberculosis from healthy ones. The use of M. tuberculosis protein fractions permits the determination of IgA and IgC in the sera of tuberculosis patients.