The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin.
Six monoclonal antibodies against Clostridium botulinum type E derivative toxin were prepared. Three of the five binding to the heavy chain neutralized the derivative toxin; the other one binding to the light chain did not. Immunoblotting analysis with the monoclonal antibodies showed that the fragment obtained by tryptic digestion consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond(s) and that the antigenic determinants common between type E and F derivative toxins were located on both the heavy and light chains. The fragment induced by chymotrypsin treatment, like the tryptic fragment, bound to four monoclonal antibodies. The mild tryptic treatment and reduction resulted in separation of the chymotryptic fragment into two smaller fragments corresponding to the light chain and H-1 fragment. These results indicate that H-1 fragment contains the amino-terminal portion of the heavy chain. The monoclonal antibody neutralizing the toxin and probably recognizing the epitope on the carboxyl-terminal portion (H-2 fragment) of the heavy chain effectively competed for binding of 125I-labeled derivative toxin to synaptosomes. Of the two monoclonal antibodies neutralizing the toxin and recognizing the epitopes on H-1 fragment, one partially inhibited binding, but the other did not. This suggests that the binding of 125I-labeled derivative toxin depends mainly on the carboxyl-terminal region of the heavy chain and that interference with binding is not the only means of toxin neutralization.
S
S
Kozaki
Y
Y
Kamata
T
T
Nagai
J
J
Ogasawara
G
G
Sakaguchi
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000276:immunology; D001905:Botulinum Toxins / Q000276:immunology*; D002918:Chymotrypsin / Q000378:metabolism; D003014:Clostridium botulinum / Q000276:immunology*; D000939:Epitopes; D007163:Immunosorbent Techniques; D051379:Mice; D009500:Neutralization Tests; D013574:Synaptosomes / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D002918:Chymotrypsin; D014357:Trypsin; D001905:Botulinum Toxins