Production and characterization of monoclonal antibodies against potentially diagnostic antigens from the liver fluke (Opisthorchis viverrini)

Abstract

Monoclonal antibodies were produced against soluble antigens of the human liver fluke Opisthorchis viverrini. The mice were immunized with total crude somatic extract spotted onto nitrocellulose and solubilized with DMSO to increase the immunogenicity and conserve antigen. Good responses to low-doses were obtained and a number of monoclonals reactive with somatic antigen (s) were selected by ELISA. Five monoclonals immunoprecipitated an antigen of M (,r) 89-90 kd from iodinated somotic extracts and another 6 reacted with the lower MW member of the extremely abundant 16/17 kd doublet of tegumental proteins. Mice immunized with nitrocellulose cut from the 80-95 kd region of an SDS-gel Western blot of total somatic antigen, gave an additional 8 monoclonals. Only one immunoprecipitated the same 89-90 kd protein, however, another 3 reacted against it on Western blot. By indirect immunofluorescence on thin sections of frozen worms, all monoclonals reactive with the 16 kd polypeptide identified the membrane surface of the tegument. The 89-90 kd antigen localized to the various muscles of the worm, most strikingly to the circular and longitudinal layer, but also the oral and ventral sucker, pharynx and a thin layer surrounding caeca. They also identified some small, often paired, monogamously-staining structures widely distributed throughout the body, probably also muscle-associated. This 90 kd somatic antigen is confined exclusively to the somatic extract and appears to be distinct from a specific-species 89 kd exo-antigen identified previously in both somatic extracts and in the metabolic products or excretory-secretory fraction released from living cells. The epitopes recognized on the 16 kd tegumental protein appears to be similar in a number of related fulkes, however, one anti-tegument monoclonal antibody exhibited no crossreaction even with a extract from the every close relative Clonorchis. All monoclonals reactive with the muscle-associated 90 kd somatic antigen show moderate to strong reaction with the homologous Clonorchis antigen. These monoclonals, particularly the specific anti-tegumental antibodies can be used to look for the corresponding antigens or their peptides in various possible autigen sources such as serum, urine, in the hope of developing a more practical and suitable diagnostic test for opisthorchiasis.