Fractionation of antigen for ELISA of bovine fascioliasis

, Rhee, Jae Ku; , Baek, Byeong Kirl; , Lee, John Hwa

doi:1986.24.2.171

Korean J Parasito > Volume 24(2):1986 > Article

Original Article


Korean J Parasitol. 1986 Dec;24(2):171-176. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1986.24.2.171
Copyright © 1986 by The Korean Society for Parasitology


Fractionation of antigen for ELISA of bovine fascioliasis
Jae Ku Rhee,Byeong Kirl Baek and John Hwa Lee
Department of Veterinary Parasitology, Jeonbug National University, Jeonju, 520, Korea.



Abstract
In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay(ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using Sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: The specificity (95 per cent confidence interval in negative sera of bovine fascioliasis ; Mean+2 × SD of absorbance ) of the first (MW>150,000) and the second antigens (MW 120,000) were 93.7 per cent, but those of others including crude antigen showed 100 per cen.t. The sensitivity (positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6 per cent, 87.5 per cent and 0 per cent, respectively, but those of others showed all 100 per cent. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8.43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the forth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. The wheal size for the bovine infected with F. hepatica was 2.46+-0.15 cm in the intradermal test antigen (saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that of intradermal test antigen, whereas those of the forth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.

Figures


	Fig. 1 Fractionation of Fasciola hepatica extract into 7 fractions by sephadex G-100 gel chromatography. Arrows indicate elution points of blue dextran (BD), hemoglobin (H), cytochrome C (C) and tryptophan (T). Vo: void volume, Vt: total bed volume


	Fig. 2 Precipitation test of crude antigen of Fasciola hepatica against bovine positive sera naturally infected with F. hepatica (1,2), bovine negative sera (3,4), rabbit positive serum with Clonorchis sinensis (5), rabbit negative serum (6) and sera of immunized rabbits by crude antigen of F. hepatica (7,8).


	Fig. 3 Precipitation test of serum from immunized rabbit by crude antigen of F. hepatica against crude antigen (C) and fractionated antigens (1 to 7).

Tables


	Table 1 Molecular weights and protein concentrations of Fasciola hepatica antigens fractionated by Sephadex G 100 gel chromatography


	Table 2 ELISA results by fractionated antigens against positive and negative sera of bovine fascioliasis


	Table 3 Comparison of wheal sizes by the antigen of VRI^* with fractionated antigens form bovine fascioliasis

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