Immunoblot patterns of clonorchiasis

, Hong, S T; , Kho, W G; , Lee, M; , Lee, J S; , Lee, S H

doi:1997.35.2.87

Korean J Parasito > Volume 35(2):1997 > Article

Original Article


Korean J Parasitol. 1997 Jun;35(2):87-93. English. Published online Jun 20, 1997. http://dx.doi.org/10.3347/kjp.1997.35.2.87
Copyright © 1997 by The Korean Society for Parasitology


Immunoblot patterns of clonorchiasis
S T Hong,^*¹W G Kho,²M Lee,¹J S Lee,³ and S H Lee¹
Department of Parasitology and Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul 110-799, Korea.

Received May 29, 1997; Accepted June 05, 1997.
Abstract
Clonorchis sinensis is a liver fluke which is the most prevalent helminth of humans in Korea. The better diagnostic measure of clonorchiasis is required for its nationwide control program. The present study observed antigenic bands of C. sinensis and reacting immunoglobulins in serum of infected residents. Adult C. sinensis were recovered from experimentally infected rabbits and soluble crude extract of the worms was used as the antigen after supplementation of E-64, a cysteine proteinase inhibitor. SDS-PAGE of the crude antigen resolved more than 20 protein bands between 200 and 14 kDa. The sera of infected humans collected at an endemic village showed specific IgG and IgE antibodies but little IgM and IgA antibodies. The protein bands of 94, 80, 72, 68, 52, 47, 43, 37, 34, and 28-25 kDa strongly reacted with serum Ig(GMA) or IgG antibody and 64, 62, 52, 47, 44, 34, 28, and 26 kDa bands reacted with serum IgE antibody. However, the 94, 80, 72, 68, 64, 62, 52, 47, and 40 kDa bands of C. sinensis antigen were found non-specific. The protein bands of 43, 34, and 28-25 kDa of C. sinesis are primary target molecules of further analysis.

Figures

Fig. 1
The influence of endogenous proteinase on electrophoresis patterns of C. sinensis crude antigen. The lysis of protein bands was prevented by supplementation of a cysteine proteinase inhibitor, E-64, before homogenizing the worms and before boiling the samples. The crude antigen was treated with 2 inhibitors in several combinations. About 50 µg of each sample was loaded and separated in a 10%. acrylamide gel using SDS-discontinuous buffer system. Protein bands were stained with Coomassie blue. Lanes 0-3, inhibitors were not supplemented before homogenizing; lane 0, no supplements, this lane was contaminated with molecular weight marker (SM).; lanes 1-3, inhibitors were added only before boiling; lane 4-7, PMSF and E-64 were treated before homogenizing; lanes 5-7, inhibitors were treated also before boiling; lanes 1 & 5, PMSF (1 mM); lanes 2 & 6, E-64 (10 µm); lanes 3 & 7, PMSF and E-64.


	Fig. 2 Absorbance levels of IgG antibody by ELISA to crude antigen of C. sinensis. Egg positive cases (P) and negative cases (N). The positive margin was 0.38.


	Fig. 3 Immunoblot of human Ig(GMA) antibodies to C. sinensis crude antigen in 4 groups. Lanes 1-5, P cases; lanes 6-10, IE cases; lanes 11-13, E case; lanes 14-15, I cases; lanes 16-20, N cases.


	Fig. 4 Immunoblot of human IgG (A), IgG (B), IgG (C), and IgA (D) antibodies to C. sinensis crude antigen in 4 groups. Lanes 1-5, P cases; lanes 6-10, IE cases; lanes 11-13, E cases; lanes 14-15, I cases; lanes 16-20, N cases.

Tables


	Table 1 Prevalence of clonorchiasis in residents of a village, Sangju-gun, Kyongbuk, 1994


	Table 2 Experimental groups of subjected residents by 3 examinations

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