Preparation purification and characterization of neisseria meningitidis group a capsular polysaccharide tetanus toxoid conjugates

ABSTRACT

Conjugation of Neisseria meningitidis A Capsular polysaccharide [CPS] to tetanus toxiod [TT] was aimed at in this work, with the intention of developing a new vaccine which may be more protective, specially to the age group below 18 months. The CPS was prepared and applied to sepharose CL-48 column. Fractions were collected and monitored for their carbohydrate content. They were divided according to their Kd into two pools [low and high molecular weight pools]. Tetanus toxoid was applied to sephacryl S-300 column and fractions were pooled into three pools. The second pool, having a molecular weight of 150 KD, as determined by SDS-PAG was used as the protein carrier. Conjugation was carried out, using cyanogen bromide activated, adipic acid dihydrazide derivative of the CPS. Binding to TT was achieved by a carbodiimide-mediated reaction. The obtained high and low molecular weight conjugates were subjected to gel filtration on sepherose CL-4B column, and the carbohydrate and protein content in the fractions were monitored. In the low molecular weight conjugates, there was a shift in the elution pattern the left. This indicates an increase in the molecular size. Fractions were pooled and the pools were tested for their antigenicity, against a standard horse anticapsular polysaccharide antibodies. The ratio of carbohydrate to protein in the conjugate was also determined