Purification and characterization of a wall protein antigen from Clostridium botulinum type A

Abstract

A wall surface protein, designated antigen S, was extracted from Clostridium botulinum type A strain 190L with 0.1% Brij 58-2 M LiCl and purified sequentially by acetone pecipitation, ion-exchange chromatography, hydroxyapatite chromatography, chromatofocusing, and gel filtration. Crossed immunoelectrophoresis of the purified antigen S preparation against homologous multispecific antiserum to whole cells revealed only a single precipitin line. Antigen S had an apparent molecular weight of about 195,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was composed predominantly of acidic amino acid residues, and the isoelectric point was estimated to be at pH 4.75. Tryptic digestion of antigen S destroyed antigenic activity and produced one major polypeptide fragment with a molecular weight of about 44,000. Indirect immunoferritin labeling showed that antigen S was located on the outer layer of the cell wall.

Purification and Immunochemical Properties of a Wall Protein Antigen from Clostridium difficile ATCC 11011

“…Bacteria were grown anaerobically in GYPT medium at 37 C as described previously (10,11). For the extraction of antigen, cells grown to the early station- cm) of Polybuffer exchanger PBE 94 (Pharmacia Fine Chemicals, Uppsala, Sweden) as described previously (31). The column was eluted with Polybuffer 74 (Pharmacia Fine Chemicals)-hydrochloride buffer (pH 4.0) according to the supplier's instructions.…”

“…Preparation of cell walls. Cell walls were prepared from log growth phase cells according to the method described previously (31). The finally washed wall pellet was used as the cell wall fraction.…”

“…Antisera. Rabbit polyspecific antiserum against whole cells of strains ATCC 11011 and rabbit monospecific antiserum against the purified 32K antigen were prepared by the same procedure as described previously (31), except that Freund's incomplete adjuvant was used on the second injection.…”

“…Immunological tests. IEP and crossed IEP were carried out with 1% agarose gels in barbital buffer (pH 8.6) as described previously (31). Immunoblot analyses.…”

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

“…difficile wall proteins were lower than those of the other Clostridium species [14,15]. Nakamura et al [16] reported that the toxigenic and nontoxigenic strains of C. difficile were classified for serovars by the agglutination test and a close relationship between toxigenicity and the serovars was found.…”