The "clearance" factor produced in the peritoneal cavity of mice immunized with killed vaccines prepared from Salmonella typhimurium or S. enteritidis was identified as the specific antibodies elicited by the 0 side chain of the cell wall polysaccharides in the organisms used as immunogens. After immunization of mice with vaccines prepared from virulent Salmonella strains, complement-dependent antibacterial antibodies in the serum and "clearance" factors in the peritoneal cavity were found to appear coincidentally, to last for more than one year, and to have the same specificity against the virulent bacterial strains. The relationship between the complement-dependent antibacterial antibodies and "clearance" factor, and the mechanisms of bactericidal action of these antibacterial agents in experimental typhoid were discussed.

In previous papers [3,4], we described a micro-quantitative spot method for in vitro assay of anti-bacterial antibodies to Salmonella which involved growth inhibition in agar-gel by immune sera with the aid of complement. We also reported on the characterization and specificity of the bacterial antibodies assayed by this method. In the present study, we have investigated the spot method for applicability to the assay of anti-bacterial antibodies to other bacterial genera. We also attempted to ascertain whether the spot method was more sensitive than conventional agglutination tests.Immune sera were obtained from DK1 mice [5] which had been intraperitoneally injected with heat killed (100 C, 30 min) or with live organisms. To examine the effect of 2-mercaptoethanol (ME) on the antibody titers, a portion of the sera was incubated with an equal volume of 0.2 M ME at 37 C for 30 min in a water bath just before the tests. The titration of anti-bacterial antibodies (plaque titers) was conducted as described previously [3]. Agglutinin titers in the same sera were determined by conventional methods using formalin-killed bacteria as antigens.The results are shown in Table 1. In the spot method, the immune sera to Shigella, Vibrio, Escherichia coli (except strain 0124: K72), Klebsiella and Proteus vulgaris effectively inhibited the growth of the corresponding bacteria, and were found to make clear plaques in the layer of growing bacteria. The plaque titer of each serum was always higher than the agglutinin titer, indicating that the spot method is perhaps more sensitive than the agglutination test with these particular bacterial strains. When sera were treated with ME, both the plaque-and agglutinin-titers were significantly reduced. On the other hand, in spite of their rather high agglutinin titers, the immune sera to E. coli O124:K72, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans did not produce any plaques with their homologous bacterial strains.Muschel [1] has demonstrated that the resistance of gram negative organisms to the antibody-complement systems is associated with the content of capsular or K antigens. To examine the possibility that the presence of the K antigen on the surface of E. coli 0124:K72 organisms might affect the results in the spot method, immune sera against several E. coli strains possessing K antigens were tested for the plaque titer to the homologous strain. As shown in Table 306