Sequential affinity chromatography for the purification of antigens extracted from Onchocerca volvulus adult worms

Adult Onchocerca volvulus worms were extracted sequentially with buffers of various ionic strengths. The extract was incubated with purified human onchocerciasis immunoglobulin-G (IgG) convalently coupled to Sepharose. Antigens were then eluted with 8 M urea and 7.5 M guanidine-HCl. IgG contained in the eluates was removed by incubating the eluates with rabbit anti-human IgG covalently coupled to Sepharose. As demonstrated by double immunodiffusion and "double" crossed immunoelectrophoresis at least three antigens were isolated. Most of the antigens were totally excluded from Sephadex G-200 but entered the included volume of Sepharose 6B. In electrofocusing they focused in acid regions. Antibodies were generated in rabbits against the three antigens isolated together. The antibodies were covalently coupled to Sepharose, which was subsequently used as a new affinity medium for the isolation of antigens. Such isolated antigens appeared to be identical with those with human onchocerciasis IgG. The isolated antigens and their corresponding artificial antibodies generated in rabbits were successfully applied in the enzyme linked immunoadsorbent assay. Host material and onchocerciasis IgG left behind during the purification procedure interfered in the assay system.