Spleen cells from mice infected with the rough strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of strain B115 and whole B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth strain 99 and the smooth strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane. Immunogold labelling with the R-LPS specific mAb was observed at the outer membrane, outside the cells and on R-LPS vesicles. These results indicate that O-chain expressed in the rough strain B115 is immunogenic in mice, not exposed at the cell surface but present in the cytoplasm and most probably at the cytoplasmic membrane.


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