ABSTRACT
For the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate the reactive reagent N-ethyl-N'-(dimethylaminopropyl) carbodiimide is used. The application of this reagent results in a number of stable linkages (viz. "peptide" linkages between the polysaccharide and tetanus toxoid, intrachain ester linkages in the polysaccharide component and binding of the N-acylurea derivative of the reagent) and less stable ones (viz. anhydride linkages). As a consequence of the reaction, the reagent is converted to a non-reactive urea derivative. The toxic properties of the reagent and of the converted reagent were studied. These properties do not contraindicate the use of the coupling reagent for the preparation of vaccines for human use. In addition analytical methods have been developed for the quantitative evaluation of the coupling reagent, the reaction products and for the N-acylurea derivative of the reagent and of the residual reactivity of conjugates for primary aminogroups. Although no test was performed for the assay of ester linkages in the polysaccharide component of the conjugate, evidence is presented that such linkages may be present. The results of the test for residual reactivity indicated a spontaneous rearrangement of linkages after the preparation of the conjugate. In addition the influence of the ratio of coupling reagent-to-polysaccharide and tetanus toxoid on antigenic and immunogenic activities of the conjugate was studied. An increase of the ratio resulted in a decrease of the antigenic activity of the polysaccharide component but in an increase of its immunogenic activity as to the induction of IgG antibodies to the polysaccharide. The immunogenic activity of the polysaccharide component correlated rather well with the antigenic activity measured in heterologous enzyme-linked immunosorbent assay using antibodies to both components.
