Identification and characterization of outer membrane proteins of Pasteurella multocida serotype D by using monoclonal antibodies

Abstract

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized with outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) with OMP as the antigen. MAbs MT1 and MT2 identified two different proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with proteolytic enzymes and sodium periodate indicated that the binding sites of MAbs MT1 and MT2 are of protein and glycoprotein natures, respectively. The epitopes reactive with MAbs were surface exposed, as visualized by immunoelectron microscopy. Among field isolates of P. multocida serotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were designated types I and II. In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both type 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 isolates of capsular serotype A of P. multocida. This is the first report of MAbs identifying the serotype D-specific OMP of P. multocida.

Detection of Antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen

“…Further studies are needed to determine when antibodies to the P37 antigen appear in rabbit serum after infection and whether they disappear after elimination of infection. Several monoclonal antibodies have been developed for the purpose of classifying different Pasteurella species, serotypes, and toxins (1,19,24,25,29,31,34). Lu et al (24) developed a monoclonal antibody directed against an outer membrane protein of P. multocida estimated to be 37.5 kDa.…”

Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida

“…Production of MAbs. Two MAbs, designated MT1 and MT4.1, both of the IgG2b isotype and directed against a 32-kDa major OMP (OmpH) and a 37-kDa major heat-modifiable OMP (OmpA) of P. multocida, respectively, were produced and characterized as described earlier by Vasfi Marandi and Mittal (41,42). A hybridoma producing IgG1 MAb 3E1 against avian reovirus was always used as an irrelevant control (34).…”

“…The absorbance of the peroxidase reaction product in the ELISA was read with an automated microplate reader (EAR 400 AT; SLA-Lab Instruments). An immunoblot assay was carried out as described by Vasfi Marandi and Mittal (41).…”

“…The negative control consisted of ascitic fluid of MAb 3E1 and bacteria with no added complement. A heat-inactivated rabbit antiserum to rabbit P. multocida serotype D strain P210 was used as a positive control (41).…”

“…Based on the electrophoretic migration of OmpH, different OMP patterns were identified among capsular serotype strains of P. multocida, representing various host species and geographic origins, whereas the electrophoretic mobility of OmpA of P. multocida varied slightly among different strains (40,42). OmpH possessed both specific and cross-reacting epitopes which are abundantly expressed on the bacterial surface, whereas OmpA possessed cross-reacting epitopes which are not exposed on the cell surface, as shown by immunoelectron microscopy (41,42). In the present investigation, the backpack hybridoma tumor system was used to con-tinuously deliver MAbs to the serum and peritoneal cavity.…”