Demonstration of leptospiral antigens on tissues using monoclonal antibodies and avidin-biotin peroxidase staining.

Glycolipoprotein (GLP) cytotoxin was extracted from Leptospira interrogans serovar canicola. The silver staining profile of GLP subjected to SDS-PAGE under denaturing conditions showed a number of bands in the mol. weight range of 14-66 kDa. Mouse Monoclonal Antibodies (MAbs) IgG3 recognizing a band near to 24 kDa of leptospiral GLP were produced (clone number MGLP-01). The agglutinating property of MAbs was established by microscopic agglutination test (MAT) using 25 different serovars as antigens. Only the homologous serovar was agglutinated by MAbs suggesting that the recognized epitope is a specific surface-exposed antigen. The MAbs were applied to demonstration of leptospiral antigens in tissue damage by avidin-biotin immunoperoxidase staining. Golden hamsters were experimentally infected with a virulent strain of L. interrogans serovar canicola. Histologically kidneys stained by routine hematoxylin and eosin showed changes characterized by injury of tubular epithelial cells leading to acute tubular necrosis (ATN). Typical, well-defined morphologic leptospires or finely granular deposits were found by immunoperoxidase staining near to blood vessels, within inflammatory infiltrates and intraluminal in proximal and distal parts of the nephron. Binding of leptospiral antigens to capillary endothelial cells, tubular epithelial cells and macrophages were also demonstrated. This entails a basis for further studies either in research or in diagnostic histopathology.

Human immune response to epitopes on the meningococcal outer membrane class 5 protein following natural infection.

Two monoclonal antibodies (mAbs) were produced against a serogroup B Neisseria meningitidis strain. These mAbs recognized two epitopes in the class 5 outer membrane proteins (OMP), designated P5.7 and P5.Bm, and were able to kill the homologous strain through complement activation. Both epitopes were surface exposed and 68% of group B meningococcal clinical isolates had one or both epitopes present in their class 5 OMP. Antibodies to one or both epitopes were demonstrated in 17 patients with meningococcal meningitis using an ELISA inhibition assay. Of the 17 paired sera, 41% and 29% of the acute-phase sera had antibodies to the P5.7 and P5.Bm epitopes, respectively. Immunoglobulin G to P5.Bm were found in all 17 convalescent-phase sera while specific antibodies against P5.7 were only found in 6 of these sera. These results demonstrate the potential importance of the P5.Bm and P5.7 epitopes on the class 5 OMP as candidates for vaccine composition.

Surface antigen analysis of group B Neisseria meningitis outer membrane by monoclonal antibodies: identification of bactericidal antibodies to class 5 protein.

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.