Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies.
Clostridium difficile toxins A and B were shown to share immunochemical and structural features, including shared sequential epitopes. Nineteen hybridomas generated after immunization of mice with a mixture of toxoids produced monoclonal antibodies, all IgM(x), which bound to toxin A and toxin B in a solid-phase radioimmunoassay (RIA). None of the antibodies neutralized the cytotoxicity of either toxin, alone or in pairs, nor did they neutralize mouse lethality. The antibodies did not inhibit hemagglutination by toxin A, and none of those tested neutralized the toxin's enterotoxic activity. Studies of binding of antibodies to native toxins in the RIA showed that the antibodies differed in their recognition of the toxins. Many of the antibodies bound with higher avidity to toxin A than to toxin B. In Western blots, all the antibodies recognized both toxins in the native state; in addition, some antibodies recognized the minor cytotoxic species of toxin B. When the toxins were denatured and reduced, five antibodies bound to both toxins, five to A only, and nine to neither, demonstrating that the antibodies had different epitope specificities. Further structural comparisons were made by investigation of mol. wts, subunit structures and amino acid compositions. The native mol. wts of toxin A and toxin B, as determined by electrophoresis to equilibrium in 4-30% polyacrylamide gel electrophoresis (PAGE), were 430,000 and 368,000, respectively. Denatured and reduced toxins each had a single subunit of 315,000. Both toxins had about 50% hydrophobic amino acids.
Rothman
S W
SW
Department of Biological Chemistry, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
Gentry
M K
MK
Brown
J E
JE
Foret
D A
DA
Stone
M J
MJ
Strickler
M P
MP
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000032:analysis / Q000276:immunology*; D004768:Enterotoxins; D000939:Epitopes / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011489:Protein Denaturation; D011863:Radioimmunoassay
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile