Tetrodotoxin (TTX) is a haptenic, highly toxic neurotoxin with no specific antidote available yet. Anti-TTX vaccine is being studied for antitoxin development. The effectiveness of the carrier protein in eliciting TTX-specific antibody response was comparatively studied. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH), Limulus polyphemus hemocyanin (LPH), tetanus toxoid (TT), diphtheria toxoid (DT), and bovine serum albumin (BSA) chemically to form artificial antigens TTH-TTX, LPH-TTX, TT-TTX, DT-TTX, and BSA-TTX, respectively, with which BALB/c mice were immunized, and the antibody response and antitoxic efficacy were detected. The serum anti-TTX antibody response and antitoxic efficacy varied markedly with adopted carrier protein. TTH-TTX elicited the best and BSA-TTX the worst TTX-specific antibody response. The proportion of the immunized mice surviving a 3x lethal dose (LD) dose of TTX challenge was 92%, 75%, 42%, 8%, and 0% for TTH-, TT-, LPH-, DT-, and BSA-TTX conjugates, respectively. The rank order of total efficacy of carrier protein for both anti-TTX antibody response and antitoxic effect was TTH > TT > LPH > DT > BSA. As a result of formaldehyde treatment in coupling of TTX carriers, the relative immunogenicity of TTX vs carrier, that is, the ratio of TTX- to carrier-specific antibody response, evidently varied with respective carrier adopted, in a rank order of TT > BSA > TTH > DT > LPH. The results suggest that the carrier protein used in haptenic TTX vaccine is greatly important in eliciting potent anti-TTX antibody, and both TTH and TT are the preferred carriers for development of excellent experimental TTX vaccine.
