Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

, Cho, S Y; , Kang, S Y; , Kong, Y

doi:1990.28.3.135

Korean J Parasito > Volume 28(3):1990 > Article

Original Article


Korean J Parasitol. 1990 Sep;28(3):135-142. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1990.28.3.135
Copyright © 1990 by The Korean Society for Parasitology


Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody
S Y Cho,S Y Kang and Y Kong
Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.


Abstract
The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.

Figures


	Fig. 1 Fractionation of crude saline extract of sparganum through Sephacryl S-300 Super-fine. 1.6(ϕ)×40cm sized column was used. Eluent: 0.15 M PBS containing 0.01% merthiolate. Flow rate: 30 ml·cm^-2·hour. Collection volume: 35 drops/tube.


	Fig. 2 Disc-PAGE findings of the crude and fractions at 10% gel concentration. C: Crude extract, 1: Fraction 1, 2: Fraction 2, 3: Fraction 3, 4: Fraction 4, 5: Fraction 5.


	Fig. 3 SDS-PAGE findings of the crude and fractions. Ten-15% linear gradient gel was used for protein separation. M_r: Molecular weight in kDa, C, 1-5: Same as described in Fig. 2.


	Fig. 4 SDS-PAGE/immunoblot finding of SP-53-2 monoclonal antibody to sparganum antigen.


	Fig. 5 Elution profile of immunoaffinity chromatography. UB: Unbound protein, B: Bound protein.


	Fig. 6 Findings of SDS-PAGE of the crude and purified proteins. M_r: Molecular weight in kDa, C: Crude extract, 1: UB of crude extract, 2: Fraction 3, 3: UB of crude Fraction 3, 4: B of crude extract, 5: B of Fraction 3.

Tables


	Table 1 Mean absorbance for sparganum-specific IgG antibody in human sparganosis and other conditions against the crude and processed antigens of sparganum


	Table 2 Comparison of sensitivity and specificity in 28 sparganosis and 99 other conditions in different antigens

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