Development of antitoxin with each of two complementary fragments of Clostridium botulinum type B derivative toxin

Abstract

Two fragments with molecular weights of 111,000 (fragment I) and 59,000 (fragment II) were separated from each other by gel filtration of dithiothreitol and urea-treated, trypsinized derivative toxin (molecular weight, 170,000) of the proteolytic Okra strain of Clostridium botulinum type B on a column of Sephadex G-200 (superfine) with a buffer containing dithiothreitol and urea. Upon removal of dithiothreitol and urea by dialysis, the two fragments reassembled to reconstruct the derivative toxin molecule. Both fragments were immunogenic, and both anti-fragments neutralized type B toxin. The neutralizing activities of both anti-fragment I and anti-fragment II were, however, lower than that of the anti-derivative toxin, suggesting that the molecular integrity of derivative toxin is essential for sufficient production of the neutralizing antibody. The immunological difference found between type B toxin from a proteolytic strain and that from a nonproteolytic strain was ascribed to the antigenic difference of fragment I.