ESTIMATION OF ANTIGENIC TETANUS TOXOID EXTRACTED FROM BIODEGRADABLE MICROSPHERES

Microspheres made from poly (lactic/glycolic acid) polymers have been considered as a new delivery system for single-dose tetanus toroid (TT ) vaccines. One of the most critical properties of the proposed vaccin es is the loading and distribution of PT as this will have a profound effect on immunogenicity. As the concentration of PT in microspheres i s very low sensitive assay methods are required. An assay incorporatin g monoclonal antibody (MAb) recognizing a neutralizing epitope and cro ss-reacting with TT was developed (MAb capture ELISA) which provided a sensitivity of 0.001 Lf/ml. An extraction procedure was devised which did not destroy the antigenicity and gave a recovery of 90.6 +/- 3.39 % when applied to different preparations. The extracted TT was then qu antified by MAb capture ELISA which was estimated to be 250-fold more sensitive than single-site ELISA for toroid. The loading of 20 microsp heres preparations (12 filled and 8 placebo) was determined by both pr otein micro-BCA assay and the developed assay for TT. The TT content o btained for the 12 filled microspheres preparations from different sou rces varied up to 400-fold (range 0.01-4.0 Lf/mg microspheres). The ut ility of the MAb capture ELISA for detection of total antigenic conten t in microspheres was confirmed by the observation that the determined TT loading correlated with the theoretical loading predicted from the protein content for the best preparations. Preparations with high loa ding gave the greatest peak response. There was no relationship betwee n dose and the in vivo immunogenic response, suggesting that encapsula ted vaccines with differential loading, release properties and presenc e of excipients will have different response curves in vivo. Hence, th e present assay, when combined with informatation on toroid release ra te and presence and effect of excipients may be of value in predicting in vivo response. (C) 1996 The International Association of Biologica l Standardization