Serological studies on chloridazon-degrading bacteria.

Agglutination tests and immunofluorescence tests with antisera against four strains of chloridazon-degrading bacteria revealed the serological uniformity of a group of 22 chloridazon-degrading bacterial strains. No serological relationship could be found between chloridazon-degrading bacteria and representatives of other Gram-negative bacteria. This was demonstrated by agglutination tests, including testing of the antiserum against Acinetobacter calcoaceticus, and by immunofluorescence tests, including testing of the sera against Pseudomonas and Acinetobacter strains. The tests were performed with 31 representatives of different Gram-negative bacteria, and with 22 strains of chloridazon-degrading bacteria as antigens. Differences in the extent of agglutination reactions and antibody titres among chloridazon-degrading bacterial strains together with cross-adsorption xperiments, suggest a rough classification of chloridazon-degrading bacteria into two subgroups. On the basis of immunofluorescence data, a linkage-map was worked out to represent serological relationships in the group of chloridazon-degrading strains.

Surface immunolabeling of Neisseria gonorrhoeae employing monoclonal antibodies to proteins IA and IB.

The technique of immunomarking intact bacteria with monoclonal antibodies (MAb) and gold-labeled anti-IgG provides a rapid and reliable method for localization of surface-exposed antigens. Cell suspensions of Neisseria gonorrhoeae were incubated with monoclonal antibodies directed against different epitopes of protein I as shown by ELISA inhibition test (unpublished data). The bound IgG molecules were detected with gold labeled anti-mouse IgG or protein A. MAbs against various epitopes of protein I showed differences in their reaction pattern with intact bacteria. We compared the results achieved by the gold immunomarking technique with coagglutination (CoA) and immunofluorescence (IF) tests. The results of the three methods correlated for some antibodies and were ambiguous for other MAbs. The advantage of the immunogold method over immunofluorescence and coagglutination is that distribution and localization of antigen on the surface of gonococci can be exactly determined using electron microscopy.

Surface-exposed antigenic cleavage fragments of Neisseria gonorrhoeae proteins 1A and IB.

Whole bacteria, isolated outer membranes, and purified protein I (PI) from one transparent (O-) and two different opaque (O+) phenotype gonococcal strains (serogroups I, II, and III; PI serotypes 1, 5, and 9b) were each treated with tolylsulfonyl phenylalanyl chloromethyl ketone-trypsin, alpha-chymotrypsin, and proteinase K. Protein IA (PIA) of strain 7122 (O-, serotype 1, serogroup I) was resistant to proteolysis by tolysulfonyl phenylalanyl chloromethyl ketone-trypsin and alpha-chymotrypsin and only slightly affected by proteinase K, as long as it was associated with intact bacteria or isolated outer membranes. Purified PIA however was cleaved by these enzymes, resulting in two to five fragments. In contrast, all preparations of strains 5766 opaque phenotype (O+, serotype 7, serogroup II) and 1955 (O+, serotype 9b, serogroup III) were accessible to proteolysis, resulting in cleavage fragments of PIB compatible to those described previously by O. Barrera and J. Swanson (Infect. Immun. 44:565-568, 1984), M. S. Blake et al. (Infect. Immun. 33:212-222, 1981), and Blake (in G. K. Schoolnik, ed., The Pathogenic Neisseriae, 1985). Our data indicated that the purified PIB fraction was more accessible to proteases than the PIBs of whole bacteria or outer membranes. The fragmentation pattern of PIA cleavage products were quite different from PIB fragments, consistent with the different structure of these two groups of PI molecules. Time-dependent cleavage experiments with proteases, i.e., alpha-chymotrypsin, indicated that PIA was subsequently cleaved into smaller fragments. Highly reactive monoclonal antibodies, each specific for a surface-exposed epitope of PIA of strain 7122 or PIB of strains 5766 and 1955, as assessed by coagglutination, Western blot, and immunofluorescence, were reacted with PIA and PIB cleavage fragments in Western blot experiments. All cleavage fragments of the purified PIA and PIB preparations with molecular weights of greater than or equal to 14,200 showed immune reaction in Western blotting, whereas whole cell and outer membrane PIB fragments were less reactive with the specific monoclonal antibodies.