[Immunochromatographic and latex-agglutination systems for diphtheria toxin detection].

The use of two monoclonal antibody types specific to different epitopes of diphtheria toxin systems have been developed to reveal diphtheria corynebacteria toxigenicity rapidly based on immunochromatographic and latex-agglutination detection of the diphtheria toxin. The methods have been tested on a sample of 36 clinical isolates. The possibility of significant detection of the toxigenic properties of the Corynebacterium strain, grown for 1 day, has been demonstrated. The developed methods allow for the detection of diphtheria toxin in concentrations of 3-4 ng/ml. The developed test systems are a perspective tool for diphtheria diagnostics because of significant time shortening as compared to traditional microbiological methods.

[Identification of heterologous antitoxin in sera of patients with diphtheria].

Using immobilized diphtheria toxin and peroxidase conjugate of monoclonal antibodies to light chains of equine immunoglobulin a method of quantification of equine antibodies against diphtheria in sera of patients after serotherapy was developed. The sensitivity of indirect enzyme-linked immunosorbent assay was 0.0005 IU/ml, and coefficient of variation did not exceed 10%. It was shown that in patients with toxic diphtheria heterologous antitoxin is eliminated within 4-6 weeks. Level of anti-diphtheria immunoglobulin under the similar severity of disease and dosage of antitoxin can vary in wide ranges and depends from individual's characteristics.

[The humoral immunity against diphtheria].

Results of the conducted study showed that naturally acquired antibacterial and postvaccinal antitoxic antibodies against diphtheria were found in human blood sera. Challenge of ADT-M toxoid to adults resulted in production of antitoxic as well as antibacterial antibodies in high concentrations. In response to challenge of ADT-M toxoid simultaneously with bacterial vaccine against diphtheria Codivac both antibacterial and antitoxic antibodies were synthesized in blood on optimal physiologic levels. This study revealed dynamics of some specific characteristics of humoral immune response after challenge of two different vaccines against diphtheria--ADT-M toxoid and Codivac vaccine.

[Study of diphtheria anatoxins in immunochemical and tissue culture assays].

Equine diphtheria antitoxins from different manufacturers were studied. Their immunochemical interaction with diphtheria toxin, toxoid, and antigens of Corynebacterium diphtheriae in ELISA and immunoblotting assays as well as biological activity in CHO cell assay were compared. The discovered differences between antitoxin samples with stated equal activity in IU/ml point to heterogeneity of antigen composition in preparations used for immunization. Mentioned methods allow to standardize antitoxins basing on their biological activity and immunochemical characteristics.

[Development of monoclonal latex diagnostic kit for diphtheria toxin and toxoid detection].

Latex diagnostic kit with high specificity and sensitivity of diphtheria toxin and toxoid detection has been developed on the basis of protective monoclonal antibodies to diphtheria toxin and polyacrolein microspheres. Diagnostic kit was stable during 1 year of shelf-life (time of the study).

[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).

[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.

[Determination of tetanus toxin and toxoid by ELISA using monoclonal antibodies]. / Immunofermentnoe opredelenie stolbniachnogo toksina i anatoksina s ispol'zovaniem monoklonal'nykh antitel.

The procedure of obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that both commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can be used an immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed detecting as much as 0.01 EC/ml toxoid and 50 LD50/ml toxin.

[Peroxidase refolding in the presence of specific antibodies]. / Izuchenie refoldinga peroksidazy v prisutstvii spetsificheskikh antitel.

A panel of eight monoclonal antibodies raised against horseradish root peroxidase has been assembled and characterized. Affinity constants were determined for all antibodies, and their specificity for various structural forms of the enzyme (native peroxidase, apoperoxidase, and denatured peroxidase) were assessed by competitive enzyme immunoassay. The effects of the antibodies on the process of refolding of peroxidase after its denaturing with 6.5 M guanidine hydrochloride were studied spectrophotometrically, by the restoration of the enzymatic activity in the reaction of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate). The yield of the active enzyme in the course of the refolding was increased 1.5 to 1.7 times in the presence of antibody H1. Effects of the antibodies constituting the panel on the activity of native peroxidase and the stability of its dilute solutions were analyzed.

[Genetic divergence of Far Eastern dace species belonging to the genus Tribolodon (Pisces, Cyprinidae) and closely related taxa]. / Geneticheskaia divergentsiia vidov dal'nevostochnykh krasnoperok roda Tribolodon (Pisces, Cyprinidae) i blizkikh taksonov.

Based on a biochemical-genetic approach, heterozygosity and divergence of structural genes of 30 enzyme loci were analyzed in six dace species. In addition, intra- and interspecific divergence of gene expression was analyzed based on a sample of 12 to 15 loci. Mean heterozygosities per individual varied as follows: Tribolodon species, Hobs = 0.007 +/- 0.007 and Hexp = 0.007 +/- 0.007; T. ezoe, Hobs = 0.045 +/- 0.016 and Hexp = 0.067 +/- 0.029. Several variants of genetic distances were estimated. Standard Nei's distances (DN) varied from 0.145 to 0.284 in four dace species studied. As related to Tribolodon dace species, the following genetic distances were obtained for two members of other genera: Pseudaspius leptocephalus, DN = 0.269; Leuciscus waleckii, DN = 0.769. Based on the distance matrices, different clustering algorithms were realized. The main feature shared by different dendrograms was a separate position of the cluster joining Far-Eastern dace species, to which P. leptocephalus and L. waleckii are successively added. Among the species studied, the proportion of loci similar by expression (E) varied from 87 to 100%. The greatest difference was found between anadromous and nonanadromous ecotypes of T. hakonensis, E = 67%. The following conclusions can be made: (1) Four studied species of the genus Tribolodon are rather well genetically differentiated. Diagnostic loci are available. (2) A nominal dace species, T. species, should be considered the fourth isolated species of this genus, which is confirmed by its recent zoological acceptance of this species. (3) The origin and divergence of dace species belonging to the genus Tribolodon are relatively late (1 to 3 Myr ago) historical events. (4) Taxonomically, the genus Tribolodon belong to the tribe Pseudaspinini together with P. leptocephalus, which is confirmed by genetic data. (5) Data on heterozygosity and the divergence of structural and regulatory elements of genome, along with the proposed scheme of speciation types, suggest the following speciation modes for the species studied: for four species, adaptive divergence and for two species, genetic transformation.

[Comparative immunochemical characteristics of polyclonal and monoclonal antibodies to aflatoxin B1]. / Sravnitel'naia immunokhimicheskaia kharakteristika poliklonal'nykh i monoklonal'nykh antitel k aflatoksinu B1.

Four hybrid clones (MM-(AB1)-1, MM-(AB1)-2, MM-(AB1)-3, and MM-(AB1)-4) were obtained by hybridoma technology with immunization of BALB/c mice with a BSA conjugate of aflatoxin B1 carboxymethyloxime derivative. Antibodies produced by these clones varied in the ability to recognize the aflatoxin B1 analogues. The sensitivity of enzyme immunoassay based on all monoclonal antibodies was higher compared to analysis based on polyclonal rabbit antibodies (0.1 and 0.4 ng/ml, respectively).

[The estimation of the amount of diphtheria toxin and its activity by various tests in the dynamic cultivation of Corynebacterium diphtheriae PW8]. / Otsenka kolichestva difteriinogo toksina i ego aktivnosti raznymi testami v dinamike kul'tivirovaniia 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.

[An acellular pertussis vaccine based on the natural complex of antigens isolated from the supernatant of a synthetic culture medium]. / Beskletochnaia kokliushnaia vaktsina na osnove prirodnogo kompleksa antigenov, vydelennykh iz supernatanta sinteticheskoi sredy kul'tivirovaniia.

A highly effective technology for obtaining a protective antigenic complex, including the main Bordetella pertussis protective antigens (pertussis toxin, filamentous hemagglutinin, agglutinogens, pertactin and adenylate cyclase) and isolated from the supernatant of B. pertussis cultivation medium, has been developed. A new method for the detoxication of antigenic complex which more available to preserve the protective property was suggested. Experimental batches of monovaccine and adsorbed DPT vaccine with the acellular pertussis component, possessing high protective activity and low histamine-sensitizing properties, have been obtained. The stability of protective properties both in liquid and lyophilized acellular pertussis preparations has been noted.

[Preparation and characteristics of antibodies to a sulfur-containing hapten, mimicking the transition state in lactamization reactions]. / Poluchenie i kharakteristika antitel k serosoderzhashchemu gaptenu, imitiruiushchemu perekhodnoe sostoianie v reaktsiiakh laktamizatsii.

A sulfur-containing hapten was proposed to be an analogue of the tetrahedral transition state of lactamization reactions. The dynamics of the immune response to this hapten upon using different immunization schemes was studied. Sixty-one clones of mouse anti-hapten antibodies were obtained. Three clones efficiently bound not only the transition state analogue (the hapten) but also potential substrates of lactamization reactions. Antibody-induced changes in the optical absorption spectra of substrate solutions were studied for a substrate with stabilized active conformation (2-aminomethylbenzoate) and an unmodified substrate (gamma-aminobutyric acid), which demonstrated that the catalytic antibodies obtained significantly accelerated the lactam ring formation.

[The immune response to Pseudomonas pseudomallei in vitro]. / Immunnyi otvet k Pseudomonas pseudomallei in vitro.

The results of studies made with the aim of selecting conditions for inducing immune response to P.pseudomallei are presented. The study revealed that the dose of the antigen, the amount of macrophages and the development of the process in stages are the decisive factors for inducing immune response. The proliferation of macrophages till their predominance in the culture was found to depend on the dose of the antigen. Some mitogens increased the antigen-induced proliferation of macrophages. The intensive in vitro synthesis of specific antibodies was observed at minimal doses of the antigen and the moderate content of macrophages. Under the action of mitogens and large doses of the antigen which induced the proliferation macrophages antibody synthesis was absent.