IDENTIFICATION AND CHARACTERIZATION OF OUTER-MEMBRANE PROTEINS OF PASTEURELLA-MULTOCIDA SEROTYPE-D BY USING MONOCLONAL-ANTIBODIES

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized wit h outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells . Desirable MAbs were selected by enzyme-linked immunosorbent assay (E LISA) with OMP as the antigen. MAbs MT1 and MT2 identified two differe nt proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with prote olytic enzymes and sodium periodate indicated that the binding sites o f MAbs MT1 and MT2 are of protein and glycoprotein natures, respective ly. The epitopes reactive with MAbs were surface exposed, as visualize d by immunoelectron microscopy. Among field isolates of P. multocida s erotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were d esignated types I and II, In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both t ype I and II isolates. Neither MAb MT1 nor MAb MT2 reacted with either reference strains of capsular serotypes A, B, E, and F or field isola tes of capsular serotype A of P. multocida. This is the first report o f MAbs identifying the serotype D-specific OMP of P. multocida.