PURIFICATION OF A PASTEURELLA-HAEMOLYTICA SEROTYPE-1-SPECIFIC POLYSACCHARIDE EPITOPE BY USE OF MONOCLONAL-ANTIBODY IMMUNOAFFINITY

A murine IgM monoclonal antibody causing bacterial agglutination was u sed in an immunoaffinity procedure to purify a serotype 1-specific pol ysaccharide epitope from Pasteurella haemolytica. The P haemolytica se rotype 1-specific antibody was precipitated from peritoneal ascitic fl uid, dialyzed, and covalently attached to cyanogen bromide-activated S epharose 4B beads. Retention of purified antibody activity and couplin g efficiency were > 99% when evaluated by ELISA, agglutination testing , and protein determination. Potassium thiocyanate was selected as an eluant on the basis of reversible dissociation of bacterial agglutinat ion and was titrated for the lowest effective concentration. Immunobea d activity was observed microscopically by immobilization of encapsula ted P haemolytica serotype 1 and its reversible dissociation after elu tion with 0.4 M potassium thiocyanate. Specificity of immobilization w as visualized, using P haemolytica serotypes 2 and 5, which were not b ound, and by blocking serotype-1 binding with homologous capsular mate rial. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-spec ific polysaccharide epitope, the product was dialyzed and analyzed, us ing chemical and immunologic methods. The immunoaffinity product conta ined no detectable protein and greater than half the original hexosami ne content. Using defined monoclonal antibodies in ELISA, titration of the original capsular material and the immunoaffinity product reveale d specific retention of lipopolysaccharide, a 10- to 30-kd polysacchar ide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epit ope sharing among polysaccharide antigens of P haemolytica serotype 1.