Quantitation of Bacillus anthracis by using of soybean agglutinin conjugates.

We examine the possibility of using the soybean agglutinin (SBA) marked by peroxidase (HRP), biotin, FITC, or gold in order to determine the number of Bacillus anthracis cells of vaccine strain STI. It was shown that the technique based on interaction between the lectin and microbial cell walls likely are not inferior in sensitivity to traditional ELISA variants. The sensitivities of methods were 10(4) cells/ml in the case of SBA-biotin, 10(5) cells/ml in the case of SBA-HRP, or 10(6) cells/ml in the cases of SBA-gold and SBA-FITC.

[Study of lectin-binding segments on the surface of Leishmania gymnoductyli reptulii during in vitro differentiation]. / Izuchenie lektinsviazyvaiushchikh uchastkov na poverkhnosti leishmanii gymnoductyli reptulii v techenie differentsirovki in vitro.

Changes in expression of lectin-binding sites, which are complex carbohydrate structures of Leishmania gymnoductyli reptulii, were studied by the classical lectin agglutination test. The results evidence that this test may be used to assess the level of ontogenesis of Leishmania strains, as well as for the detection and isolation of metacyclic (invasion) stage from Leishmania cell culture by lectins.

Comparative studies of the interaction between lectins and Leishmania in agglutination tests and enzyme-linked lectin-biotin assays (ELLBA).

It was demonstrated that soybean agglutinin and peanut agglutinin aggregated all the investigated species of Leishmania, including virulent and avirulent members of L. major in agglutination tests. Concanavalin A and Pisum sativum agglutinin were shown to aggregate L. species ZMA and L. major but showed no effect on L. gymnodactyli and L. mexicana amazonensis which were aggregated by wheat germ agglutinin, an extract from Ulex europaeus and Ricinus communis. There was no correlation between the results of ELLBA studied in agglutination tests. The results indicate that the surfaces of Leishmania strains and species are heterogeneous with respect to lectin binding. There are possibly two subsets of lectin receptors on the surface structures of Leishmania cells.

[Influenza viruses studied by fluorescent immunoassay with temporary resolution]. / Issledovanie virusov grippa metodom fliuorestsentnogo immunoanaliza s vremennym razresheniem.

Monoclonal antibodies, epidemic strains of influenza A and B viruses, conjugates were studied by fluorescence immunoassay with temporary resolution using equipment of different companies. Differences and specificity of monoclonal antibodies to influenza A and B viruses were demonstrated. The highest sensitivity of the method with the test system used was determined, being 20 ng/ml of viral protein. The method was shown to be useful for influenza virus detection both in solutions containing purified virions and in virus-containing allantoic preparations. It was established that virological studies could be carried out using conjugates and knock-down microplates of the national make alongside with foreign ones.

[Immunoglobulin level (G, A and M) in healthy adults]. / Uroven' immunoglobulinov (G, A, i M) u zdorovykh vzroslykh liudei

The simple radial immunodiffusion method was applied to determination of the quantitative content of three principal classes of immunoglobulins (G, A and M) in 987 blood sera of healthy adults, Moscow residents, aged from 19 to 50 years (donors). Monospecific antisera and standard sera were used. Investigations were carried out in men and women of three age groups: 19--29, 30--39 and 40--50 years old. The mean level of immunoglobulins G, A and M was expressed in International Units (IU/ml) and in milligrams per 100 ml of the serum (mg%). The IgG and the IgM concentration proved to be higher in women than in men in all the age groups (the detected differences were statistically significant); as to the average IgA level no statistically significant differences were revealed either by age or by sex. As a result of these studies mean levels of immunoglobulin G, A and M were determined in the blood sera of adult healthy persons, Moscow residents; which could serve as the basis for comparing the immunoglobulin content under normal conditions and in different pathological conditions of the organism.

[Production of rabbit precipitating antisera to subclasses of human IgG]. / Poluchenie krolich'ikh pretsipitiruiushchikh antisyvorotok k subklassam IgG cheloveka.

Precipitating antisera to human subclasses IgG were obtained by immunization of rabbits by whole molecules IgG2, IgG3, IgG4 and gamma 1-chains derived from IgG1H (Pr). Analysis of the antisera obtained demonstrated that rabbits produced specific antibodies to the antigenic subclass determinants IgG3 well, to IgG2, IgG4--much worse, and failed to produce specific antibodies to subclass IgG1 (in immunization with whole molecules of this protein). Antisera contained antibodies to the antigenic determinants common of IgG, and antibodies to light chains which were removed by immunosorption, for which purpose a sorbent on the basis of BrCN sepharose conjugated with IgG of the three other subclasses and Fab-fragment was used.

[Venous immunomorphology in acute experimental thrombosis and human recurrent migrating thrombophlebitis]. / Immunomorfologiia ven pri éksperimental'nom ostrom tromboze i pri retsidiviruiushchem migriruiushcem tromboflebite cheloveka.

The direct immunofluorescence procedure was used to demonstrate immune complexes (fixed immunoglobulins) in the internal and external sheath of veins of a dog with experimental acute thrombosis caused by partial ligation of the ileofemoral vein in the presence of immunization of the animal with extracts of auto- and homologous vein tissue. The results suggest the participation of auto-immune processes in the pathogenesis of acute thrombosis of the main veins.

[Immunoenzyme analysis for determining class G and M antibodies to HBsAg]. / Immunofermentnyi analiz dlia opredeleniia antitel klassov G i M k HBsAg.

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.

[The use of an immunoenzyme method for the rapid diagnosis of legionellosis]. / Primenenie immunofermentnogo metoda dlia ékspress-diagnostiki legionelleza.

An enzyme immunoassay (EIA) system for the detection of L. pneumophila antigen in clinical material (sputum, urine, bronchial washings) has been developed. The use of EIA permits the detection of L. pneumophila antigen in the urine of 75-80% of patients during the first week of the disease. The specificity and sensitivity of EIA makes it possible to recommend this method for the rapid diagnosis of L. pneumophila infection.

[Lipopolysaccharide identification in aqueous extracts of Pseudomonas aeruginosa by an immunoenzyme method]. / Identifikatsiia lipopolisakharida v vodnykh ékstraktakh Pseudomonas aeruginosa immunofermentnym metodom.

To determine the content of lipopolysaccharide (LPS) in P. aeruginosa aqueous extracts the ELISA was used. Membrane filtration and ultracentrifugation followed by precipitation with ammonium sulfate at 80% saturation and gel filtration on Sephadex G-100 have proved to be the most effective methods for purification of the aqueous extract from LPS.

[Use of an immunoenzyme method for detecting specific antibodies to Pseudomonas aeruginosa in patients with acute and chronic lung diseases]. / Ispol'zovanie immunofermentnogo metoda dlia vyiavleniia spetsificheskikh antitel k Pseudomonas aeruginosa u bol'nykh s ostrymi i khronicheskimi zabolevaniiami legkikh.

ELISA in which P. aeruginosa slime preparations are used as antigens permits the detection of antibodies to this microorganism in the blood sera of patients with acute and chronic pulmonary diseases induced by P. aeruginosa. This assay makes it possible to find out the etiology of infection even before the results of bacteriological investigation are obtained.

[Use of the ELISA immunoenzyme method for the detection of the causative agent of tularemia]. / Ispol'zovanie immunofermentnogo metoda ELISA dlia vyiavleniia vozbuditelia tuliaremii.

The conditions of the enzyme-linked immunosorbent assay (ELISA) for the detection of Francisella tularensis were worked out. In the study of 27 strains differing in their biological characteristics, the sensitivity of the assay was determined, varying within the range of 1 X 10(4)--5 X 10(4) million cells/ml and exceeding the sensitivity of the currently used methods for the immunodiagnosis of tularemia by 1-2 orders. ELISA also proved to be a highly effective technique for the detection of the specific antigen in the organs of infected animals. The antigen was regularly detected in the organs of white mice, beginning from day 3 after their infection with the minimal doses of F. tularensis. The method may be recommended both for the identification of isolated cultures and for the early diagnosis of tularemia infection.

[Use of an immunoenzyme method on a solid-phase carrier for determining the tularemia antigen]. / Primenenie immunofermentnogo metoda na tverdofaznom nositele dlia opredeleniia tuliaremiinogo antigena.

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.

[Determination of tularemia antibodies by an immunoenzyme method on a solid-phase carrier (ELISA)]. / Opredelenie tuliaremiinykh antitel immunofermentnym metodom na tverdofaznom nositele (ELISA).

ELISA "sandwich" techniques have been developed and the optimum assay conditions for detecting specific antibodies in human serum samples have been determined. The possibility of using these techniques for the determination of the level of antibodies to tularemia antigens in the sera of persons immunized with live tularemia vaccine has been shown. Statistically significant differences in the level of antibodies to tularemia antigen in the sera of immunized and nonimmunized persons have been established. The comparative study of five serological methods - ELISA, the agglutination test, the passive hemagglutination test, the immunofluorescence test and the defined antigen substrate sera ( DASS ) techniques - has revealed the advantage of ELISA, whose sensitivity has proved to be considerably higher than that of all other methods used in our work.

[Differential affinity of pathogenic species of microorganisms for a set of lectins detectable by the sandwich method using fluorescein isothiocyanate (FITC)]. / Differentsirovannoe srodstvo patogennykh vidov mikroorganizmov k naboru lektinov, proiavliaemykh "séndvich"-metodom s uchastiem fliuorestseinizotiotsionata (FITTs).

The qualitative differences in the affinity of concanavalin A (Con A), wheat-germ agglutinin (WGA) and Phaseolus vulgaris lectin to the surface of 10 microbial strains inducing various diseases in humans and agricultural animals have been demonstrated by means of the indirect immunofluorescence tests. Enterobacteria, Coxiella burnetii and Bacillus anthracis have been found to possess pronounced affinity to Con A and WGA, while Rickettsia prowazekii, Francisella tularensis and Brucella abortus, as well as Treponema pallidum, have proved to be resistant to lectins. WGA has been found to bind specifically to Brucella abortus and Treponema pallidum. Con A and WGA are seemingly suitable for use in the preliminary test for the total capacity of lectin receptors to come in contact with biological macromolecules.

[Utilization of luminescent monospecific sera against human immunoglobulins G, A and M for diagnostic purposes]. / Ispol'zovanie liuminestsiruiushchikh monospetsificheskikh syvorotok protiv immunoglobulinov G, A, M cheloveka dlia diagnosticheskikh tselei

A method for obtaining luminescent sera, consisting in isolation of pure antigens G, A, M from human gamma-globulin and blood serum from patients with myeloma disease is suggested. A native antiserum was obtained by immunization of rabbits with water-insoluble polycondensate of antigens "sewn" with glutaric aldehyde. Adsorption of antisera as well as specific antigens was carried out with antigen- and antibodyimmunosorbent, the latter being obtained with the help of both glutaric aldehyde and sefarose 4B treated with cyanogen bromide. The sera had a specific titre in the precipitation reaction against their own antigens 1:32 and were highly specific. A globulin fraction was obtained by sedimentation with polyethylene-glycol. Marking of the specific protein with fluoresceine isothiocyanate was carried out using the dialysis method with subsequent purification on sefadex and DEAE-cellulose. The application of the abovementioned sera made it possible to ascertain the character of distribution of deposits of immunoglobulins in glomeruli in systemic lupus erythematodes, glomerulonephritis and in the cells of the synovial fluid sediment in rhematoid arthritis.

[Indirect hemagglutination inhibition reactions as a method of titrating immune serums. V. Application of the reaction to the quantitative determination of immunoglobulins A, M and G in human blood sera]. / Reaktsiia tormozheniia nepriamoi gemaggliutinatsii kak metod titrovaniia immunnykh syvorotok. Soobshchenie V. Primenenie reaktsii dlia kolichestvennogo opredeleniia immunoglobulinov A, M i G v syvorotkakh krovi liudei

The authors present the results of using the indirect hemagglutination inhibition test (IHIT) for quantitative determination of A, M and G immunoglobulins in the blood sera of humans in comparison with the method of radial immunodiffusion in agar (RID) after Mancini. The results of IHIT were no less precise and reproducible than those of RID. The significance of the correlation coefficient of grades after Spirman constituted greater than 99.9% for both tests. On this basis a conclusion was made that, having a number of advantages over RID, IHIT could be recommended for quantitative titration of immunoglobulins of various classes.

[Isotachophoretic isolation of IgD and IgE and production of monospecific antisera against them]. / Vydelenie metodom izotakhoforeza IgD i IgE i poluchenie k nim monospetsificheskikh antisyvorotok.

The method of preparative isotachophoresis in acrylamide gel ensuring a high yield of IgD and IgE with insignificant admixtures of IgG, etc. was used for the isolation of IgD and IgE from the blood sera of myeloma patients. As a result of immunization with these antigens, monospecific IgD and IgE antisera were obtained. These antisera, alongside with specific antibodies, contained antibodies to admixtures; the latter were eliminated by the method of immune absorbtion carried out with the use of a sorbent based on Sepharose activated with bromo-cyanogen and conjugated with normal human blood serum. Ig D antisera were also shown to contain antibodies to idiotypical IgD determinants located in the Fab fragment of this immunoglobulin.

[Comparative study of modifications in the immunoperoxidase and immunofluorescent methods of detecting human tissue immunoglobulins]. / Sravnitel'noe izuchenie modifikatsii immunoperoksidaznogo i immunofliuorestsentnogo metodov vyiavleniia tkanevykh immunoglobulinov cheloveka.

The comparison of the data obtained by using different modifications of the immunoperoxidase and immunofluorescent methods for detecting immunoglobulins in intestinal tissues was made. The direct immunoperoxidase method was shown to allow the quantitative study of immunoglobulin-containing cells. The direct immunoperidase method was found to be technically simpler, but the indirect and complex peroxidase-antiperoxidase methods proved to be more sensitive.

[The diagnosis of antileptospiral antibodies in human subjects by solid-phase immunoenzyme analysis]. / Diagnostika protivoleptospiroznykh antitel u liudei metodom tverdofaznogo immunofermentnogo analiza.

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.