Two outer-membrane proteins (OMPs) of serotype D, designated H and W, possess potentially important serotype D-specific antigens. Antigenicity as well as toxigenicity of 55 strains of representing various serotypes, geographic origins and host species were studied by SDS-PAGE, enzyme-linked immunosorbent assay (ELISA), immunoblot and polymerase chain reaction (PCR) assays. Based on the electrophoretic mobility of protein H, different OMP patterns were observed within different capsular serotypes. Three monoclonal antibodies (MAbs) designated MT1, MT2 and MT3 were produced against H and W proteins of in BALB/c mice. MAbs MT2 and MT3 reacted with two distinct epitopes on W protein of serotype D in competitive ELISA. MAb MT1 reacted with all serotype D-I strains but not with D-II strains, whereas MAb MT2 reacted with both serotype D-I and D-II strains in dot-ELISA and immunoblot assay. MAb MT3 reacted with all strains belonging to different capsular serotypes in dot-ELISA. None of the MAbs reacted with other gram-negative bacteria tested, indicating that protein H has a serotype D-I specific epitope and protein W has both serotype and species-specific epitopes. PCR assay was used to identify toxigenic strains of ; 92% of strains possess both gene and MAb MT2 reacting epitope, suggesting a strong association between MAb MT2 reacting epitopes and gene. Rapid dot-ELISA with MAb was found to be specific, sensitive and easy to perform and thus suitable for routine serotyping of serotype D strains which might be potentially pathogenic.


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