No abstract

0.1 ml aliquots of serial dilutions of sera in physiologic saline, starting at 1: 10, were mixed with 0.1 ml of 1 x 108/ml suspensions of organisms previously washed 3X in physiologic saline; the mixtures were agitated 30 min on a Boerner type rotating machine at approximately 120 rotations per min at room temperature and agglutination read at 100 X magnification. Known negative and positive serum controls were always run concurrently. 482OO37-9727/78/ l583-O482$O 1 .OO/O

Acclimatized rabbits were passively immunized with graded volumes of antisera against endotoxins from either smooth wild type Gram-negative bacteria possessing intact O-antigenic side chains, or rough mutants deficient in these O-antigens. The recipients were challenged with test doses of endotoxins that evoked fever within the sensitive dose-response range. Pyrogenic tolerance conferred with antisera to smooth endotoxins was consistently directed toward the O-specific antigen; minimal protection extended to endotoxins from heterologous Gram-negative bacterial species, or from homologous O-deficient rough mutant strains. In contrast, antisera to O-deficient rough mutant strains transferred significantly more effective protection against heterologous endotoxins. The level of such heterologous protection, however, was markedly less than the O-specific protection conveyed by the smooth anti-endotoxin sera. The protective factors in antiserum to smooth endotoxin were separable by either pyrogen-free Sephadex G-200 columns or ammonium sulfate precipitation into 2-mercaptoethanol (2-ME) resistant and sensitive O-antibody-containing fractions. In the latter, 2-ME virtually abolished O-agglutinating, but not O-combining or protective activities. Both O-combining and protective activities could be reduced by absorption of antiserum with spleen cells but not with erythrocytes. It is concluded that: 1) the humoral components of endotoxin tolerance are antibodies to both O-specific and common core antigens. Such antibodies are both IgM and IgG, protect by primary (binding) rather than secondary (agglutinating) activity, and are in part cytophilic; 2) the relative importance of O-specific and common core antibodies is dependent upon both the immunizing and test endotoxin preparations. Antisera to smooth endotoxins afford the highest levels of passive tolerance, but such protection is restricted primarily to the O-specific endotoxin. Antisera to rough O-deficient endotoxins provide less tolerance but afford the better approach to broad passive endotoxin protection; 3) the findings support the hypothesis that common core antigens are partially masked by the O-specific antigens in the endotoxin molecule.