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

, Lee, Ok Ran; , Chung, Pyung Rim; , Nam, Hae Seon

doi:1988.26.2.73

Korean J Parasito > Volume 26(2):1988 > Article

Original Article


Korean J Parasitol. 1988 Jun;26(2):73-86. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1988.26.2.73
Copyright © 1988 by The Korean Society for Parasitology


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
Ok Ran Lee,Pyung Rim Chung,^* and Hae Seon Nam
Department of Parasitology, Soonchunhyang University College of Medicine, Cheon-An City, Ssangyong-dong, Chungnam 330-090, Korea.
^*Department of Parasitology, College of Medicine, Yonsei University, Seoul 120-749, Korea.


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.

Figures


	Fig. 1 Standard curve for the determination of molecular weight of polypeptide fractions. [1: phosphorylase, 2: bovine serum albumin (BSA), 3: ovalbumin, 4: carbonic anhydrase, 5: soybean trypsin inhibitor, 6: lysozyme]


	Fig. 2 Affinity chromatography of C. sinensis worm homogenate crude antigen (WWA) on CNBr-activated Sepharose 4B. WWA of 70mg protein was loaded onto the column and eluted with 0.04M-PBS buffer(pH 7.2) for unbound Ag., and eluted with 0.1M-glycine/HCI buffer (pH 2.8) in 1 drop/18sec. (4ml/tube).


	Fig. 3 Agar-gel immunodiffusion analysis(Ouchterlony reaction) of C. sinensis antigen. [center well: C. sinensis anti-rabbit serum (S), outer wells: 1. whole worm-saline homogenate crude antigen(WWA), 2. affinity-purified antibody-binding antigen(ABA), 3. metacercaria antigen(MEA), and 4. egg antigen(EGA)]


	Fig. 4 SDS-polyacrylamide gel electrophoretic analysis of C. sinensis antigens. [WWA: whole worm-saline homogenate crude antigen, WNA: whole worm NP-40 extract crude antigen, ABA: affinity-purified antibody-binding antigen, MEA: metacercaria NP-40 extract antigen, EGA: egg NP-40 extract antigen, M: marker protein]


	Fig. 5 Absorbance scan of SDS-PAGE gels(10% acrylamide) loaded with C. sinensis WWA and ABA(stained with silver). [Rm: electrophoretic mobility of fractions (=bands)]


	Fig. 6 Optical density(O.D.) values obtained by C. sinensis crude antigen(WWA) and affinity-purified antigen(ABA), detected by ELISA with rabbit sera, from preinfection (control: Co.) to the 24th week of infection, in different worm burden groups (I-IV).


	Fig. 7 Absorbance scan of SDS-PAGE gels(10% acrylamide) loaded with C. sinensis WWA and WNA(stained with silver).


	Fig. 8 Absorbance scan of SDS-PAGE gels(10% acrylamide) loaded with C. sinensis WWA, MEA and EGA (stained with silver).


	Fig. 9 Optical density (O.D.) values obtained by C. sinensis crude antigen (WWA), metacercaria antigen (MEA) and egg antigen (EGA), detected by ELISA rabbit sera, from preinfection (control: Co) to the 24th week of infection in different worm burden groups (I-IV).


	Fig. 10 Optical density (O.D.) distributions by ELISA of eight paragonimiasis human sera to P. westermani antigen (PWA) and various C. sinensis antigens.

Tables


	Table 1 Numbers of worms recovered in each group of experimental rabbit clonorchiasis


	Table 2 Molecular weight of standard protein and relative mobility (Rf)


	Table 3 Polypeptide band numbers and relative mobility (Rf) of C. sinensis antigens (WWA, ABA, MEA, EGA) on 10% acrylamide gels (SDS-PAGE)

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