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ISOLATION AND PURIFICATION OF Clostridium perfringens ENTEROTOXIN BY AFFINITY CHROMATOGRAPHY
Author(s) -
BARNHART H. M.,
BULLERMAN L. B.,
BALL ELLEN M.,
WAGNER FRED W.
Publication year - 1976
Publication title -
journal of food science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.772
H-Index - 150
eISSN - 1750-3841
pISSN - 0022-1147
DOI - 10.1111/j.1365-2621.1976.tb00749_41_4.x
Subject(s) - enterotoxin , clostridium perfringens , toxin , chromatography , antibody , affinity chromatography , chemistry , elution , protein a , antigen , microbiology and biotechnology , biochemistry , biology , bacteria , escherichia coli , immunology , enzyme , genetics , gene
Sporulating cells of certain strains of Clostridium perfringens produce an intracellular enterotoxin believed to be the causative agent of C. perfringens food poisoning. Rabbit antibody preparations to both vegetative and sporulating cell extracts were used to purify toxin by differential immuno‐affinity chromatography. Solid phase immune adsorbents were prepared by attaching antibody via α‐amino groups to succinylaminoethyl Sepharose‐4B to give two resins: one binding anti‐vegetative immunoglobulin G (IgG), (V‐resin), the other, antisporulating IgG, (S‐resin). Sporulating cell extract was passed through resin with bound IgG to vegetative cell extract. A large protein fraction representing antigens common to both C. perfringens forms was retained, but toxin was not measurably absorbed to the resin as determined by erythemal activity in rabbits. The toxic fraction was then passed through resin with bound IgG to sporulating cell extract. The majority of the protein did not adhere to this resin; however, that which did, showed erythemal activity in rabbits. Disc‐gel electrophoresis of the protein fraction eluted from resin with bound IgG to sporulating cell extract demonstrated the presence of five components. One elicited erythemal activity in rabbits. The maximum capacity of the V‐resin column was 102 μ g of bound protein/ml of resin, and of the S‐resin column, 32 μg of bound protein/ml of resin. A 150‐fold purification was achieved by the procedure. Resins could be used repeatedly.