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Hybridomas producing monoclonal antibodies (McAb) to group A streptococcal polysaccharide (A-PS) were obtained. Of these, 3 clones were selected: 2 clones producing IgG3, precipitating McAb and 1 clone producing IgM nonprecipitating McAb. The results of the competitive inhibition in the enzyme immunoassay suggested that precipitating and nonprecipitating McAb reacted with nonidentical epitopes of A-PS, though determinants, specifically reacting with the given McAb, had a common site which included N-acetyl-D-glucosamine. On the surface of bacteria, in addition to protein M, the presence of the given determinants of A-PS was established in the direct immunofluorescence test. The newly developed method of direct immunofluorescence with the use of specially selected precipitating McAb was the basis for the development of rapid diagnosticum, permitting the identification of group A streptococci.

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A panel of monoclonal antibodies (McAb) to different determinants of group A Streptococcus polysaccharide (A-PS) has been studied. As revealed in this study, A-PS contains at least 4 determinants, common with different epidermal antigens. McAb, cross-reacting (CR) with different mammalian tissue antigens, have not been found to be group-specific. Experiments on the inhibition of the immunoenzyme reaction of McAb with A-PS indicate that CR determinants include rhamnose and beta-N-acetyl-D-glucosamine of rhamnose alone. In addition, we have confirmed our earlier suggestion that group-specific high-affinity precipitating antibodies acted on different sites of A-PS. Contrary to earlier opinions, antibodies to group-specific determinants of A-PS have been found not to react with epidermal antigens.

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The protein-cellulose complex prepared by covalent immobilization of single protein molecules on insoluble cellulose particles was used for priming C57BL/6 and BALB/c mice. The serum antibody content and the number of spleen AFC were assayed after animals' boosting with the soluble protein. Such a complex was shown to have marked advantages over the same protein injected both in complete Freund's adjuvant and in a soluble form, in particular. The immune response of BALB/c mice was more than 10-fold higher than that of C57BL/6 animals.

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The role of surface antigens in the density-dependent inhibition of primary immune response in mouse spleen cell cultures was investigated. For this purpose Fab-fragments of rabbit IgG obtained after immunization with mouse splenocytes were used. Such Fab-fragments alone had no effect on immune response in both optimal and dense cultures. However, successive treatment of cells with Fab-fragments and with ass antibodies against rabbit IgG dramatically augmented the density-dependent inhibition of antibody formation.

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Incorporation of uridine into the cells was studied under conditions of rapid increase in density of the cell suspension either freshly-prepared from spleen of mice strain C57BI/6 or subjected to precultivation. Absorption of uridine by the cells after an increase in their density was rapidly decreased within several minutes. The phenomenon was not due to presence of dead cells in the culture. An inhibitor of protein synthesis puromycin partially removed the suppression of uridine absorption in dense cultures.

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The authors previously reported local intercellular interactions suppressing the increase of the antibody-forming cell (AFC) count in the suspension of the spleen cells of nonimmunized mice and sharply elevating with increase in density of the cultivated suspensions. This work showed that preliminary immunization of mice with an antigen eliminated or sharply reduced the accretion inhibition of the AFC cells against the given antigen, but failed to influence the accretion inhibition of cells producing antibodies against another antigen, and the proliferation inhibition of the dividing bulk cells in the culture.

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There was a sharp decrease in the number of antibody producing cells formed in induction of primary immune reaction in vitro with increase of surface density (number of cells per 1 cm2 of the bottom of the incubation vessel); the cause of this phenomenon was studied. It was shown that depression of antibody formation in dense cultures was associated not with disturbance of the induction of the clone of antibody-forming cells, but with the inhibition of the subsequent proliferation of the cells of this clone. Disturbance of proliferation was reversible: a reduction of the density of the cultures 1 or 3 days after the beginning of the incubation led to the restoration of the growth of the number of antibody-producing cells. The depressive effect could not be transferred by humoral way from the cultures with a high density to the cultures with a low density; it could not be eliminated by daily replacement of the incubation medium in dense cultures for a fresh nutrient medium.

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A relationship was revealed between the number of cells in the suspension of the splenic tissue taken for induction of primary immunization of the reaction outside the organism and the number of the plaque-forming cells (PFC) formed by the end of incubation. It was shown that with increase of the cell density in the culture in the bottom of the incubation flask there was a 10-100 fold decrease of the PFC response; as to the cell viability--it was not affected or decreased but slightly. This effect was observed in using sheep red blood cells and water-soluble antigen extracted from them as an antigen. The effect was independent of shortage of the antigen or of the nutrient substances; it was accompanied by a general reduction of the 3H-thymidine incorporation into cultured cells.

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