Analysis of <i>Clonorchis sinensis</i> antigens and diagnosis of clonorchiasis using monoclonal antibodies

, Yong, T S; , Im, K; , Chung, P R

doi:1991.29.3.293

Korean J Parasito > Volume 29(3):1991 > Article

Original Article


Korean J Parasitol. 1991 Sep;29(3):293-310. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1991.29.3.293
Copyright © 1991 by The Korean Society for Parasitology


Analysis of Clonorchis sinensis antigens and diagnosis of clonorchiasis using monoclonal antibodies
T S Yong,K Im and P R Chung
Department of Parasitology & Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul 120-752, Korea.


Abstract
Clonorchis sinensis is a common parasite of man in Korea. Researches on the specific antigens of C. sinensis would be valuable not only because those elucidate the molecular characteristics of this fluke but also because it is applicable to immunodiagnosis. Although many monoclonal antibodies have been used in the field of parasite immunology, few articles on monoclonal antibodies against C. sinensis have been published so far. The aim of this study was to analyze C. sinensis antigens recognized by monoclonal antibodies, and to set up ELISA-inhibition test using C. sinensis specific monoclonal antibodies for improved specificity of immunodiagnostic tests. By fusion between spleen cells of the mice immunized with C. sinensis water-soluble crude adult worm antigens and plasmacytoma cells of mouse origin, 29 hybridoma clones secreting anti-C. sinensis monoclonal antibodies were made, and 8 clones among those were found specific. After cell cloning, isotypes of 6 selected specific monoclonal antibodies were determined to be IgG1, IgG2b and IgA. Four exposed antigenic determinants of natural infection were recognized by different specific monoclonal antibodies. By enzyme-immunoelectrotransfer blot, 10 KD, 34 KD antigenic determinants were found to be reacted with CsHyb 0714-20, CsHyb 0605-10 monoclonal antibodies, respectively. The antigenic determinant recognized by CsHyb 0714-20 monoclonal antibody was revealed to be located at the surface and parenchyme of a parasite by indirect immunofluorescent antibody technique, and those reacted with CsHyb 0605-10, CsHyb 0714-25 monoclonal antibodies were found at the parenchyme and intestine. The antigenic determinant reacted with CsHyb 0605-23 monoclonal antibody was found mainly around the uterine eggs. Four antigenic determinants recognized by specific monoclonal antibodies were all found to be present in the early eluted fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration. By conventional ELISA, 75% of clonorchiasis cases were found positive, but 7.1% of normal controls and 37.5% of paragonimiasis cases showed false positives. However, by ELISA-inhibition test using C. sinensis specific monoclonal antibody (CsHyb 0605-23), 77.1% of clonorchiasis cases were found positive, and there were no false positives in normal controls or paragonimiasis cases, indicating 100% specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Figures


	Fig. 1 Immunization schedule for BALB/c mice with C. sinensis water-soluble crude adult worm antigens.


	Fig. 2 Test form exposed antigenic determinants of natural infection by ELISA-inhibition test: (a) exposed antigenic determinant of natural infection, (b) non-exposed antigenic determinant of natural infection.


	Fig. 3 ELISA-inhibition test suing specific monoclonal antibody to detect anti-C. sinensis antibodies in the human sera.


	Fig. 4 SDS-PAGE finding of C. sinensis watersoluble crude adult worm antigens.


	Fig. 5 Enzyme-immunoelectrotransfer blot pattern of C. sinensis antigen fractions against monoclonal antibodies: (1) CsHyb 0714-20, (2) CsHyb 0605-10, (3) CsHyb 0605-23, (4) CsHyb 0714-25, (5) immune mouse serum.

Fig. 6
Distribution of C. sinensis antigens reacted with monoclonal antibodies by indirect fluorescent antibody technique. Frozen sections of C. sinensis adult worm was reacted with:

1) immune mouse serum as a positive control,

2) normal mouse serum as a negative control,

3) CsHyb 0605-23 monoclonal antibody showing positive reactions strongly around the uterine eggs, intestinal epithelium(IE) reacting rather weakly. But the worm surface showed no fluorescence at all,

4) CsHyb 0714-20 monoclonal antibody showing strong positive reactions on the surface, ventral sucker(VS) and parenchyme.


	Fig. 7 Elution profile of C. sinensis water-soluble crude adult worm antigens separated by Sephadex G-200 gel filtration.


	Fig. 8 Distribution of absorbance values of the sera in clonorchiasis and paragonimiasis cases against C. sinensis antigens by conventional ELISA.


	Fig. 9 Distribution of absorbance values of the sera in clonorchiasis and paragonimiasis cases against C. sinensis antigens by ELISA-inhibition test using specific monoclonal antibody ("CsHyb 0605-23").

Tables


	Table 1 Efficiency of making hybridomas secreting anti-C. sinensis antibodies


	Table 2 Specificity of anti-C. sinensis antibodies secreted by 29 hybridoma clones by ELISA


	Table 3 Isotypes of anti-C. sinensis monoclonal antibodies assayed by ELISA^*


	Table 4 Evaluation of antigenic determinants recognized by monoclonal antibodies for exposed antigens of natural infection


	Table 5 Distribution of antigens reacted with monoclonal antibodies


	Table 6 The fractions of C. sinensis antigens separated by Sephadex G-200 gel filtration reacted with monoclonal antibodies by ELISA

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