Physicochemical and immunological characterization of meningococcal group A polysaccharide-tetanus toxoid conjugates prepared by two methods

Abstract

Two methods have been applied for the covalent binding of high molecular weight Neisseria meningitidis group A polysaccharide to tetanus toxoid. The first method used cyanogen bromide as the coupling reagent and the second used both glycine and 6-amino-n-hexanoic acid as spacers and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide as the coupling reagent. Each conjugation was performed twice. The conjugates were analysed by biochemical, physicochemical and immunochemical techniques. The immunogenic activities of the conjugates were studied in mice. The IgG responses to both components of the conjugates were measured by the enzyme-linked immunosorbent assay (ELISA). After the first dose differences in immunogenic activity of the tetanus toxoid component of the conjugates were seen, whereas after the second dose differences in activity of the polysaccharide component were observed. The second dose of all conjugates produced clear booster effects in the responses to both components. AIPO 4 potentiated both the primary antibody responses and did not influence the booster effects. These data indicate that both components of the conjugates behaved as thymic-dependent immunogens. The second method of preparation resulted in conjugates with higher immunogenic activities. Significant differences in activity, however, were seen between conjugates prepared by the same method. These data indicate that the reproducibility of the method of preparation needs further consideration.