SEQUENTIAL AFFINITY CHROMATOGRAPHY FOR PURIFICATION OF ANTIGENS EXTRACTED FROM ONCHOCERCA-VOLVULUS ADULT WORMS

  • 1 January 1978
    • journal article
    • research article
    • Vol. 29  (1) , 39-48
Abstract
Adult O. volvulus worms were extracted sequentially with buffers of various ionic strengths. The extract was incubated with purified human onchocerciasis immunoglobulin[Ig]G covalently coupled to Sepharose. Antigens were 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. Double immunodiffusion and double crossed immunoelectrophoresis showed at least 3 antigens. 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 3 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 identical with those isolated with human onchocerciasis IgG. The isolated antigens and their corresponding artificial antibodies generated in rabbits were successfully applied in the enzyme linked immunosorbent assay. Host material and onchocerciasis IgG remaining after the purification procedure interfered in the assay system.