Studies on the immunodiagnosis of rabbit clonorchiasis II. Immunoaffinity purification of whole worm antigen and characterization of egg, metacercaria and adult antigens of Clonorchis sinensis

Abstract

The sensitivity and specificity of crude and affinity-purified antigens of Clonorchis sinensis obtained from the infected rabbits were studied. Stage-specific antigenic proteins from the eggs, metacercariae and adult worms were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) The results were as follows: The antibody-binding antigen (ABA) purified from whole worm crude antigen (WWA) by CNBr-activated Sepharose 4B affinity chromatography made 4 specific bands against rabbit anti-sera on Ouchterlony gel diffusion plate, while WWA made 7 bands. Major WWA protein bands by SDS-PAGE were found at 16,300-18,500 and 28,000-29,000 daltons, while major ABA protein bands were at 18,000-21,000 and 29,000-31,000 daltons. The reactivity of ABA with rabbit anti-sera in ELISA was remarkably less sensitive than that of WWA. Molecular weights of egg antigen (EGA), metacercarial antigen (MEA) and adult worm antigen (WWA) of C. sinensis ranged from 15,000-200,000 daltons, 15,000-100,000 daltons and 11,000-80,000 daltons, respectively. Major WWA proteins consisted mainly of polypeptide bands of low molecular weight, less than 31,000 daltons, while those of EGA and MEA consisted of higher molecular weights than 30,000 daltons. The ELISA reactivities of WWA to rabbit anti-sera were remarkably greater than those of MEA. EGA showed negative reaction throughout the experiments. WWA showed higher optical density (O.D.) than 1.0, when reacted with rabbit anti-sera obtained at 4-6 weeks after the infection. In the rabbit anti-sera later than 12 weeks after the infection, the O.D. reacting with WWA showed a plateau without variation. MEA showed relatively low O.D. values (<0.6), when reacted with anti-sera from lightly infected groups throughout the experiments, although there were some weak positive cases (O.D.>0.6) in heavily infected groups. MEA reacted with rabbit anti-sera showed negative results on Ouchterlony gel diffusion plates. Summarizing the above results, it is suggested that the whole worm antigen prepared from the adult worms of C. sinensis is most highly antigenic. However, this antigen might reveal cross reactions with other trematodes such as Paragonimus westermani, therefore, purification of antigenic proteins from the crude antigen is essential to increase the sensitivity and specificity for the immunodiagnosis of clonorchiasis.