Immunoreactivity of a new generation of horse F(ab′) 2 preparations against European viper venoms and the tetanus toxin

Abstract

The immunoreactivity of the current and the new more purified, pasteurized preparations of horse F(ab′) 2 against the tetanus toxin and Vipera aspis venom was investigated with a biosensor based on technology using the optical phenomenon of surface plasmon resonance. Immunoreactivity data were compared with seroneutralization titres to investigate immunoreactivity-immunoprotection efficacy relationships. The association-dissociation rate and affinity constants of the current and the new tetanus toxin-specific F(ab′) 2 preparations were similar, at about 10 4 M −1 sec −1, 10 −4 sec −1 and 10 8 M −1, respectively. Similar values were found using a solid immunoradiometric assay. To assess the immunoreactivity of V. aspis venom-specific horse F(ab′) 2, the mol. wt and percentage of the antigenic fractions of V. aspis venom were determined. Western blotting of electrophoresis gels showed four antigenic fractions of V. aspis venom (mol. wts 17,500, 28,500, 32,000 and 60,000), which represented 6, 3.4, 17.7 and 5% of total venom, respectively. Association and dissociation rate constants were in the same range as those of the tetanus toxin-F(ab′) 2 interactions for each of the four antigenic fractions. Seroneutralization of both tetanus toxin and V. aspis by the corresponding specific F(ab′) 2 showed that the ld 50 mg −1 protein was 1.76-fold and 1.51-fold higher with the new than with the current preparations, respectively. These improvements in efficacy were in close agreement with the higher immunoreactive fraction ratios, which were 2-fold and 1.8-fold higher with the new preparations. These results demonstrate that the removal of non-IgG T immunoglobulins and the pasteurization treatment have no overall influence on F(ab′) 2 affinity but improve the specific activity of these new antitoxin horse F(ab′) 2.