Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis.
We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.
Nagano
Isao
I
Department of Parasitology, Gifu University School of Medicine, Gifu, 500-8705 Japan. isao@cc.gifu-u.ac.jp
Pei
Fuquan
F
Wu
Zhiliang
Z
Wu
Jun
J
Cui
Huier
H
Boonmars
Thidarut
T
Takahashi
Yuzo
Y
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D015153:Blotting, Western; D003003:Clonorchiasis / Q000175:diagnosis* / Q000276:immunology*; D003004:Clonorchis sinensis / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005399:Fishes; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
D000909:Antibodies, Helminth; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases