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 used in an immunoaffinity procedure to purify a serotype 1-specific polysaccharide epitope from Pasteurella haemolytica. The P haemolytica serotype 1-specific antibody was precipitated from peritoneal ascitic fluid, dialyzed, and covalently attached to cyanogen bromide-activated Sepharose 4B beads. Retention of purified antibody activity and coupling 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 agglutination and was titrated for the lowest effective concentration. Immunobead activity was observed microscopically by immobilization of encapsulated P haemolytica serotype 1 and its reversible dissociation after elution with 0.4M potassium thiocyanate. Specificity of immobilization was visualized, using P haemolytica serotypes 2 and 5, which were not bound, and by blocking serotype-1 binding with homologous capsular material. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-specific polysaccharide epitope, the product was dialyzed and analyzed, using chemical and immunologic methods. The immunoaffinity product contained no detectable protein and greater than half the original hexosamine content. Using defined monoclonal antibodies in ELISA, titration of the original capsular material and the immunoaffinity product revealed specific retention of lipopolysaccharide, a 10- to 30-kd polysaccharide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epitope sharing among polysaccharide antigens of P haemolytica serotype 1.
Austin
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FW
Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803.
Corstvet
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RE
D000371:Agglutination; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000302:isolation & purification*; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D016978:Mannheimia haemolytica / Q000276:immunology*; D051379:Mice; D011135:Polysaccharides, Bacterial / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D011135:Polysaccharides, Bacterial