The diagnostic effectiveness of an immunoenzyme test system for determining diphtheria toxin antigens in the blood serum in a clinical laboratory trial

The data on the approbation of the diagnostic value of the enzyme immunoassay (EIA) system for the determination of diphtheria toxin in the blood sera of diphtheria patients and persons suspected for diphtheria are presented. The EIA system was prepared on the basis of F(ab)2 fractions of purified antidiphtheria antibodies. 240 serum samples from diphtheria and tonsillitis patients and from healthy persons were studied. Diphtheria toxin was determined in all patients with the toxic form of diphtheria and in 41.3% of patients with its localized forms. Blood was taken mainly of the first week of the disease. In healthy persons the results of EIA were negative. Thus, the trial of the assay system in a clinical laboratory showed its good diagnostic effectiveness. The use of this EIA system in medical practice is believed to be quite promising.     

The coagglutination reaction for detecting diphtheria toxin

High-titer antidiphtheria antitoxic rabbit serum has been obtained, and on the basis of this serum a coagglutinating diagnosticum has been developed. The sensitivity of the test has been found to depend on the content of antitoxic antibodies in the serum and on its purity. Diagnostica prepared from native serum containing 500 I. U./ml (a titer of 1:51, 200 in the passive hemagglutination test) permit the detection of 0.02-0.03 Lf/ml of diphtheria toxin. A decrease in antibody titer to 5-25 I. U./ml leads to a drop in sensitivity to 0.2-2 Lf/ml. The use of LgG fraction and pure antibodies increases the sensitivity of the test to 0.002-0.003 Lf/ml. The possibility of detecting toxin in Corynebacterium diphtheriae strains is shown.     

A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins

The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.     

Detection of Legionella pneumophila antibodies in the blood serum of pneumonia patients by an immunoenzyme method

The conditions of making the enzyme immunoassay (EIA) for the detection of antibodies to L. pneumophila have been optimized. The use of L. pneumophila purified serotypic antigen at a concentration of 0.25 micrograms/ml for the sensitization of polystyrene plates has been shown to increase the sensitivity and specificity of the assay. 220 patients with severe pneumonia have been examined. As revealed in this investigation, antibodies to L. pneumophila can be detected in 12.2% of cases. A high degree of correlation (94.4%) between the results of EIA and the indirect immunofluorescence test has been shown.     

Specific antibodies and their role in the formation of antidiphtheria immunity

In human sera, studied with the use of the enzyme immunoassay, antidiphtheria postvaccinal antitoxic IgG and naturally acquired antibacterial IgG, IgM and IgA were detected. In the blood of children and adults aged up to 50 years antitoxic IgG were present at normal and high concentrations. In 50% of children antibacterial IgA were absent, while specific antibacterial IgM could be found at high concentrations. Changes in the content of antibacterial antibodies of different classes in sera were observed with age. More than 90% of adults had antibacterial IgA and IgG at normal and hig concentrations, while the level of IgM decreased. Under the influence of ecological, social, anthropogenic and other environmental factors the optimum levels of specific antibodies were replaced by anomalous ones, which led to an increased number of persons susceptible to diphtheria infection and in the intensity of the circulation of the infective agent. The deficiency of antidiphtheria antibacterial antibodies in the blood determined the necessity of correcting immunity by means of not only toxoid, but also bacterial antigens.     

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and anatoxins

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).     

The choice of a method of isolating human IgG F(ab)2 fragments for determining antibodies to staphylococcal protein A]

The comparative study of two methods of the proteolysis of IgG with the aim of obtaining F(Ab)2-fragments from the blood sera of patients and healthy donors has been made. Different methods for the isolation of F (ab)2-fragments, such as gel filtration on Sephadex G-200, affinity chromatography on protein A-BrCN-sepharose, reprecipitation with zinc sulfate, have been analyzed. For further work the method of the peptic decomposition of whole serum with subsequent salting out with ammonium sulfate and gel filtration on Sephadex G-200 has been chosen.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.     

The estimation of the amount of diphtheria toxin and its activity by various tests in the dynamic cultivation of Corynebacterium diphtheriae PW8]

Direct correlation between the results of tests for the biological activity of diphtheria toxin, carried out in vivo guinea pigs and in vitro in the microcytotoxicity test in CHO cell culture, has been established, which makes it possible to use the latter as one of the methods for the rapid, reproducible and economic evaluation of diphtheria toxin. The qualitative and quantitative evaluation of diphtheria toxin in the enzyme immunoassay with the use of monoclonal antibodies and in the microcytotoxicity test demonstrates that these two tests, when used for controlling cultivation processes, have essential advantages over the flocculation test as regards their specificity and information content.     

Detection of toxin and evaluation of the degree of toxin-formation of Corynebacterium diphtheriae using immunoenzyme analysis

Toxin production and the intensity of toxin formation in 265 C. diphtheriae strains circulating in different areas of the USSR have been studied by the method of the enzyme-linked immunosorbent assay (ELISA). This study has been carried out with the use of the assay system consisting of monoclonal antibodies to the COOH-area of the B-fragment of the toxin molecule adsorbed onto the surface of polystyrene plates, affinity-purified polyclonal antidiphtheria antibodies labeled with horse-radish peroxidase and substrate indicator mixture (5-aminosalicylic acid and hydrogen peroxide). Some specific features of using ELISA for the detection of C. diphtheriae toxin directly in liquid culture medium are presented. High sensitivity, specificity and good reproducibility of this method permitting the detection of C. diphtheriae toxin and the determination of the intensity of toxin formation in the C. diphtheriae strains under study are shown. The method may be recommended for practical use at health institutions.     

Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection

Sensitive and specific electrochemiluminescence (ECL) assays were used to detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in undiluted human serum, undiluted human urine, assay buffer, and selected food matrices (whole milk, apple juice, ground beef, pastry, and raw eggs). These novel assays used paramagnetic bead-based electrochemiluminescent technology in which biotinylated serotype-specific antibodies were bound to streptavidin-coated paramagnetic beads. The beads acted as the solid support and captured analyte from solution. Electrochemiluminescent detection relied on the use of ruthenium chelate-labeled anti-serotype antibodies and analysis with a BioVeris M-Series M1R analyzer. The sensitivities of the assays in clinically relevant matrices were 50 pg/ml for serotypes A and E, 100 pg/ml for serotype B, and 400 pg/ml for serotype F. The detection limits in selected food matrices ranged from 50 pg/ml for serotype A to 50 to 100 pg/ml for serotypes B, E, and F. The antibodies used for capture and detection exhibited no cross-reactivity when tested with the other serotypes. When purified native toxin was compared with toxins complexed to neurotoxin-associated proteins, no significant differences in assay response were noted for serotypes A, B, and F. Interestingly, the native form of serotype E exhibited reduced signal and limit of detection compared with the complexed form of the protein. We suspect that this difference may be due to trypsin activation of this particular serotype. The assays described in this article demonstrate limits of detection similar in range to the gold standard mouse bioassay, but with greatly reduced time to data. These rapid sensitive assays may have potential use in clinical settings, research studies, and screening of food products for botulinum toxins.     

Specificity of antibodies to diphtheria toxin subunits in children with various forms of diphtheria infections

In order to study the specificity of serum antibodies to separate subunits of diphtheria toxin, SDS-electrophoresis of diphtheria toxin preliminary disintegrated on the subunits via trypsin treatment was performed, followed by immunoblotting assay. 86 blood serum samples of children with diphtheria carriers of toxigenic and non-toxigenic strains of Corynebacterium diphtheriae as well as children with other infectious diseases similar to diphtheria in their clinical manifestation, and healthy ones immunized with DTP-vaccine were tested. A special computer program was written and applied for results processing and assumption. The data obtained showed that there were particular differences in frequency of predominating the antibodies to one or another subunit of diphtheria toxin among various groups of the children. We consider that the different specificity of antibodies of sick children and children-carriers is capable to predetermine the different course of infectious process.     

Effectiveness of methods of isolating specific class-G rabbit antibodies for the immunoenzyme determination of human placental alkaline phosphatase

Four different chromatographic methods of IgG isolation from rabbit antisera to placental alkaline phosphatase (HPAP) have been compared. The antibodies were obtained by ion-exchange chromatography and affinity chromatography on protein-A-sepharose, on the sepharose with immobilized antigen. IgG samples were characterized by the content of specific antibodies to HPAP and checked in enzyme immunoassay (EIA). IgG purified on immobilized antigen were found to be the optimal both from the point of view of the specific antibodies content and EIA sensitivity, but satisfactory results could be also obtained with ion-exchange and protein-A-chromatography purified IgG. The last two isolation methods are simpler and provide 3-10 ng/ml sensitivity of HPAP detection, which is lower, as compared with the test employing affinity antibodies (1 ng/ml), but allows the detection of HPAP in serum samples.     

Fibrinogen-fibrin degradation products: hemagglutination inhibition immunoassay in plasma.

Immunoassay for fibrinogen and/or fibrin degradation products (FDP) is generally in the clot and hence assay of serum may not reveal the true concentration of FDP in blood. We have developed a hemagglutination inhibition immunoassay for FDP in human plasma. D fragment appears to possess an antigenic determinant, called D-neoantigen, not found in native fibrinogen. Rabbit antiserum produced against D fragment was absorbed with immunosorbent columns coupled with fibrinogen and normal human serum, respectively, so that it contained only those antibodies directed against the neoantigenic determinant of D fragment. In this immunoassay, sheep erythrocytes (SRBC) were stabilized with glutaraldehyde and subsequently sensitized with D fragment by means of tannic acid. Hemagglutination of absorbed anti-D-neoantigen serum against SRBC sensitized with D fragment was titered to be 1:256. The hemagglutination was inhibited by D fragment but not by fibrinogen; the sensitivity of detecting D fragment was 8 mug/ml. Human plasma from normal subjects did not inhibit. This appears to be the first report of a hemagglutination inhibition immunoassay for FDP in plasma.     

Detection of the antigens of the causative agent of brucellosis by an immunoenzyme method

The conditions of the enzyme immunoassay for the detection of Brucella antigens have been selected, making it possible to detect these antigens both in solutions and in biological material within 3-4 hours. In guinea pigs infected with B. abortus 99 in a dose of 1,000 microbial cells, brucellar antigen has been detectable in the organs and blood serum of the animals as early as 24 hours after infection. This assay, if carried out under the optimal conditions, detects soluble brucellar polysaccharide antigen at a concentration of 1 ng/ml and Brucellae at a concentration of 5 X 10(4) microbial cells/ml in the presence of 200-fold surplus of other bacterial cells.     

A monoclonal antibody-based enzyme immunoassay for the detection of T-2 toxin at picogram levels

Thirteen monoclonal antibodies reactive with HT-2 were prepared by using a HT-2 hemisuccinate coupled to human serum albumin as antigen for the immunization of BALB/c mice. In a competitive enzyme immunoassay on a double antibody solid phase using HT-2 hemisuccinate coupled to horseradish peroxidase as enzyme linked toxin all antibodies reacted much better with T-2 toxin and acetyl T-2 than with HT-2. Eleven antibodies showed almost the same sensitivity and specificity, and one of these, designated 3E2, is extensively described. Its cross-reactivities with HT-2, T-2 toxin, acetyl T-2, iso T-2, T-2 tetraol tetraacetate and T-2 triol were 1·0, 140·2, 161·2, 0·32, 0·14 and 0·016, respectively. Two other antibodies, designated 2A4 and 2A5, behaved quite differently. The cross-reactivities of antibody 2A4 with these toxins were: 1·0, 113·9, 374·4, 1·35, 0·34 and 0·023, respectively; for antibody 2A5 they were 1·0, 46·1, 155·4, 8·31, 0·9 and 0·08, respectively. All antibodies proved to be IgGl. By using the antibody 3E2 a highly sensitive and very specific enzymc immunoassay for the detection of T-2 toxin was developed. The detection limit for T-2 toxin was 5 pg/ml (0·25 pg/assay).     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 235–242.Original Russian Text Copyright © 2005 by Titova, Sviridov.     

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.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.