Purification of cystic fluid antigen of <i>Taenia solium</i> metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization

, Kim, Suk Il; , Kang, Shin Yong; , Cho, Seung Yull; , Hwang, Eung Soo; , Cha, Chang Yong

doi:1986.24.2.145

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

Original Article


Korean J Parasitol. 1986 Dec;24(2):145-148. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1986.24.2.145
Copyright © 1986 by The Korean Society for Parasitology


Purification of cystic fluid antigen of Taenia solium metacestodes by affinity chromatography using monoclonal antibody and its antigenic characterization
Suk Il Kim,¹Shin Yong Kang,¹Seung-Yull Cho,¹Eung Soo Hwang,² and Chang Yong Cha²
¹Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 151, Korea.
²Department of Microbiology, College of Medicine, Seoul National University, Seoul 110, Korea.


Abstract
This study was undertaken to purify cystic fluid (CF) antigen of Taenia solium metacestodes by affinity chromatography using specific monoclonal antibody (McAb) and to characterize the antigenicity of the purified antigen. The hybridoma cell lines, prepared by fusion between mouse plasmacytoma and spleen cells from BALB/c mice immunized with CF, secrected antibodies reacting to various helminthic antigens. Majority of cell lines reacted to CF only but some also reacted to parenchymal antigen of T. solium metacestodes, adult T. saginata, sparganum, hydatid cystic fluid, Paragonimus westermani and Clonorchis sinensis, either in combination with CF, other antigens or independently. Cloned cells derived from monoclonal lines also produced antibodies reacting either to CF only or to other helminthes in combination or independently. These results indicated that CF of T. solium metacestodes contained proteins which possessed antigenic determinants not only specific to CF but also cross reactive with the afore-mentioned helminthes. CF of T. solium metacestodes was purified by affinity chromatography using the McAb which reacted to CF and parenchymal antigens. The affinity-purified antigen(A-Ag) and unbound pool(U-Ag) were separated. A-Ag showed 2 protein bands by disc-PAGE whereas CF exhibited 6 bands and U-Ag consisted of all bands CF had. The diagnostic significance of A-Ag was evaluated by ELISA in human neurocysticercosis and other helminthic and neurologic diseases. By A-Ag, the levels of the specific IgG antibody, as shown by absorbance in sera and CSF, were lower than those of CF and U-Ag. Accordingly, the sensitiviy was about 70 % of CF and U-Ag. However, the nonspecific positive reactions to CF and U-Ag, observed in sparganosis, T. saginata infection and paragonimiasis did not occur when A-Ag was used. These results indicated that the affinity-purified A-Ag had the higher specificity but the lower sensitivity as a diagnostic antigen in cysticercosis, probably because it only detected a single or limited numbers of monospecific antibodies among the diverse polyclonal antibodies produced in the patients with neurocysticercosis.

Figures


	Fig. 1 Titration of sera from 10 immunized BAKB/c mice form CF-specific IgG antibody by ELISA.


	Fig. 2 Profile of affinity chromatography of CF of T. solium metacestodes using immunoadsorbent of CFCc-298.18 McAb. Unbound protein(U) was washed away with PBS (first arrow). The first population of bound protein molecules(A) was eluted with 0.1M glycine-HCl buffer (pH 2.5) (2nd arrow), the second population (a) was eluted with same buffer containing 10% dioxane (3rd arrow).


	Fig. 3 Banding patterns of protein in crude and purified antigen by disc-PAGE. 1: CF, cystic fluid of T. solium metacestodes, 2: A-Ag, affinity-purified pool (1st run) from CF, 3: A-Ag, affinity-purified pool (2nd run) from CF, 4: U-Ag, pool of unbound proteins from affinity chromatography of CF.


	Fig. 4 Scattergram of absorbance by ELISA in sera (○) and CSF (△) of confirmed neurocysticercosis patients using crude and purified antigens. CF: Cystic fluid of T. solium metacestodes, A-Ag: Affinity-purified pool of CF (1st rnn), U-Ag: Unbound pool from affinity chromatography.

Tables


	Table 1 Frequency of wells with growing hybird cells in HAT media after cell hybridization and those with secretion antibodies reacting to CF of T. solium metacestodes


	Table 2 Antigen-binding specificity of antibodies in culture supernatant of 71 wells with hybrid cells in HT medium as determined by ELISA using 7 different helminthes antigens


	Table 3 Antigen-binding specificity and subclass of immunoglobulin G of monoclonal antibodies secreted by 83 hybridoma lines


	Table 4 Yields of fractionated antigen by affinity chromatography using CFCc-298.18 McAb


	Table 5 Relative composition of protein bands in disc-PAGE of crude and affinity-purified antigens of CF of T. solium metacestodes as measured by densitometric tracing


	Table 6 Specificity of crude and purified antigens of T. solium metacestodes ad determined by ELISA with sera from other helminthic infections, other neurologic diseases and normal control


	Table 7 Absorbance of sera in cross-reacted patients against 3 antigens as measured by ELISA

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