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Korean J Parasitol > 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

References
1. Ahn YK, et al. New Med J 1975;18(9):1195–1201.
2. al-Yaman FM, Knobloch J. Isolation and partial characterization of species-specific and cross-reactive antigens of Echinococcus granulosus cyst fluid. Mol Biochem Parasitol 1989;37(1):101–107.
  
3. Cabrera Z, Parkhouse RM, Forsyth K, Gomez Priego A, Pabon R, Yarzabal L. Specific detection of human antibodies to Onchocerca volvulus. Trop Med Parasitol 1989;40(4):454–459.
 
4. Cho KM, et al. Yonsei Rep Trop Med 1974;5(1):45–56.
5. Cho KM, et al. Yonsei Rep Trop Med 1976;7(1):26–39.
6. Cho SY, Kang SY, Kim SI. Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunoelectrotransfer blot technique. Korean J Parasitol 1987;25(2):159–167.
 
7. Cho SY, Kim SI, Kang SY, Choi DY, Suk JS, Choi KS, Ha YS, Chung CS, Myung HJ. Evaluation of enzyme-linked immunosorbent assay in serological diagnosis of human neurocysticercosis using paired samples of serum and cerebrospinal fluid. Korean J Parasitol 1986;24(1):25–41.
 
8. Choi WY. Korean Central J Med 1975;28(5):533–537.
9. Hahm JH, et al. Korea Univ Med J 1981;21(1):177–184.
10. Jh Han,et al. Korea Univ Med J 1986;23(1):13–25.
11. Hur NJ, et al. Korea Univ Med J 1988;25(1):375–388.
12. Im KI. Yonsei J Med Sci 1974;7(1):194–205.
13. Inatomi S, et al. Jap J Parasit 1968;17(5):395–401.
14. Jeong KH, Rim HJ, Kim CW. [A Study On The Fine Structure Of Clonorchis Sinensis, A Liver Fluke: 1. The Body Wall And The Nervous System]. Korean J Parasitol 1978;16(2):156–164.
 
15. Jeong KH, Rim HJ, Kim WK, Kim CW, Yang HY. [A Study On The Fine Structure Of Clonorchis Sinensis, A Liver Fluke: II. The Alimentary Tract And The Excretory System]. Korean J Parasitol 1980;18(1):81–91.
 
16. Jeong KH, Rim HJ, Kim CW. [A Study On The Fine Structure Of Clonorchis Sinensis, A Liver Fluke: III.The Prostate Gland]. Korean J Parasitol 1980;18(1):93–97.
 
17. Jin SW, et al. Korea Univ Med J 1983;20(1):191–199.
18. Joo-KH JJ, Ahn H, Chung MS, Rim HJ. Demonstration of species-specific and cross reactive components of Paragonimus westermani crude worm antigen by EITB. Korean J Parasitol 1989;27(1):9–14.
 
19. Cho SY, Kang SY, Kong Y. Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody. Korean J Parasitol 1990;28(3):135–142.
 
20. Kim SI, Kang SY, Cho SY, Hwang ES, Cha CY. Purification of cystic fluid antigen of Taenia solium metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization. Korean J Parasitol 1986;24(2):145–148.
 
21. Kim TS, et al. Korea Univ Med J 1987;24(1):197–205.
22. Klatser PR, De Wit MY, Kolk AH. An ELISA-inhibition test using monoclonal antibody for the serology of leprosy. Clin Exp Immunol 1985;62(3):468–473.
 
23. Goh YH. Acquired Immunity In Albino Rats To Clonorchis Sinensis. Korean J Parasitol 1969;7(1):32–41.
 
24. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256(5517):495–497.
  
25. Kwon KH, et al. Korea Univ Med J 1984;21(1):91–100.
26. Laemmli UK. Nature 1970;227:681–685.
 
27. Lee JK, et al. Yonsei J Med Sci 1981;14(1):133–147.
28. Lee JS. Korea Univ Med J 1975;12(2):487–493.
29. Lee OR, et al. J Soon-chunhyang Coll 1987;10(2):409–414.
30. Lee OR, Chung PR, Nam HS. [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]. Korean J Parasitol 1988;26(2):73–86.
 
31. Long EG, Lawrence MC, Augustine T. ELISA for Schistosoma mansoni infection: durability of blood spots on filter paper. Trans R Soc Trop Med Hyg 1981;75(5):740–741.
  
32. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.
 
33. Mattes MJ, Steiner LA. Antisera to frog immunoglobulins cross-react with a periodate-sensitive cell surface determinant. Nature 1978;273(5665):761–763.
  
34. Min DY, Soh CT. Evaluation Of Specific Ige Antibody In Clonorchis Sinensis Infection. Korean J Parasitol 1983;21(1):27–31.
 
35. Min DY, et al. Yonsei J Med Sci 1980;13(2):94–106.
36. Mott KE. Acta Tropica 1987;44(12) Suppl:13–20.
37. Sadun EH, Walton BC, Buck AA, Lee BK. The use of purified antigens in the diagnosis of clonorchiasis sinensis by means of intradermal and complement fixation tests. J Parasitol 1959;45(2):129–134.
  
38. Sawada T, et al. Jap J Exp Med 1964;34(6):315–322.
39. Schantz PM, Shanks D, Wilson M. Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am J Trop Med Hyg 1980;29(4):609–612.
 
40. Seo BS, Lee SH, Cho SY, Chai JY, Hong ST, Han IS, Sohn JS, Cho BH, Ahn SR, Lee SK, Chung SC, Kang KS, Shim HS, Hwang IS. An Epidemiologic Study On Clonorchiasis And Metagonimiasis In Riverside Areas In Korea. Korean J Parasitol 1981;19(2):137–150.
 
41. Sirisinha S, Sahassananda D, Bunnag D, Rim HJ. Immunological analysis of Opisthorchis and Clonorchis antigens. J Helminthol 1990;64(2):133–138.
  
42. Soh CT, et al. Yonsei Rep Trop Med 1976;7(1):3–16.
43. Sho CT, et al. Yonsei Rep Trop Med 1985;16(1):1–10.
44. Sun T. The non-antigenicity of intact ova of Clonorchis sinensis. J Med Microbiol 1969;2(3):358–360.
  
45. Sun T, Gibson JB. Antigens of Clonorchis sinensis in experimental and human infections. Am J Trop Med Hyg 1969;18(2):241–252.
 
46. Sung DR, et al. Korea Univ Med J 1988;25(3):813–828.
47. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76(9):4350–4354.
  
48. Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.
  
49. Weir DM, et al. Blackwell Scientific Publications 1986;4:113.
50. Whang KK. Archives Thesis Med Sci Yonsei Univ 1990;1:365–381.
51. Yang JS, et al. Korea Univ Med J 1983;20(1):201–209.
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