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

Original Article
Korean J Parasitol. 1986 Dec;24(2):177-186. English.
Published online Mar 20, 1994.  http://dx.doi.org/10.3347/kjp.1986.24.2.177
Copyright © 1986 by The Korean Society for Parasitology
Purification of antigenic proteins of Paragonimus westermani and their applicability to experimental cat paragonimiasis
Won Young Choi,Jae Eul Yoo,Ho Woo Nam and Hyung Rak Choi
Catholic Institute of Parasitic Disease, Catholic Medical College, Korea.
Abstract

This study was designed to evaluate the partially purified antigens which were fractionated from crude extract of Paragonimus westermani and to monitor the enzyme-linked immunosorbent assay (ELISA) in experimental cat paragonimiasis during the course of infection as well as before and after chemotherapy. Crude extract of 6-month-old adult P. westermani was fractionated to 5 antigens by successive applications of ammonium sulfate precipitation, ion exchange chromatography and gel filtration. And the cats, 10 in each group, were infected with 60, 30, 15, and 5 metacercariae, then the half of each group was treated with praziquantel 2 times in one day of 100 mg per kilogram of weight on 150 days after the infection. Sera were collected every 10 days. ELISA was performed with the concentration of 2 µg/ml antigen, 100 times diluted sera and 1,000 times diluted alkaline phosphatase conjugated anti-cat IgG. The results were as follows: Absorbance by ELISA with proteins precipitated by differential concentration of ammonium sulfate was the highest at 51-65 per cent precipitate (PA2), followed by 0-50 per cent precipitate (PA1), 66-80 per cent precipitate (PA3), and 81-90 precipitate (PA4). Unprecipitated protein over 90 per cent ammonium sulfate (PA5) showed the lowest antigenicity. Fractionation of PA1, PA2, and PA3 through the DEAE-cellulose column did not differentiate the antigenic proteins. By passing through the Sephadex G-200 column, PAl and PA2 were fractionated to high molecular weight proteins and those of low molecular weight which showed high absorbance by ELISA (PA1-I, II and PA2-I, II). But PA3 was shown to have a fraction of high molecular weight proteins (PA3-I) which showed high antigenicity. SDS-polyacrylamide gel electrophoresis of PA1-I, PA1-II, PA2-I, PA2-II, PA3-I, and crude extract was performed. Fraction PA1-I was composed of proteins which had the molecular weight of 270 kilodaltons (KD) to 196 KD; of them 220 KD protein was major band. Fraction PA2-I was composed of 255-225 KD, and PA3-I, 255-240 KD, respectively. Fraction PA1-II and fraction PA2-II consisted of 30 KD proteins. Absorbance by ELISA began to increase within 10-20 days after the infection and reached the highest on 140-180 days, then made plateau thereafter. Absorbance by ELISA decreased after praziquantel treatment. In 60 metacercariae infection group, the absorbance had been decreasing, but remained within the positive range during observation period, while those of 30, 15, and 5 metacercariae infection groups turned to negative range. Fraction PA1-II showed the highest antigenicity in ELISA, then fraction PA2-I, fraction PA1-I , fraction PA2-II, fraction PA3-I and crude extract followed. In early phase of infection, the absorbance of fraction PA1-II showed more rapid increase than those of the other fractions and it came to positive range at 20-30 days after infection.

Figures


Fig. 1
Elution profile of PA1 (precipitated by 0~50% ammonium sulfate from crude antigen of P. Westermani) through DEAE-cellulose column and absorbance of ELISA for its fractions against a reference positive serum.


Fig. 2
Elution profile of PA2 (precipitated by 51~65% ammonium sulfate from crude antigen of P. Westermani) through DEAE-cellulose column and absorbance of ELISA for its fractions against a reference positive serum.


Fig. 3
Elution profile of PA3 (precipitated by 66~800% ammonium sulfate from crude antigen of P. Westermani) through DEAE-cellulose column and absorbance of ELISA for its fractions against a reference positive serum.


Fig. 4
Elution profile of PA1' through Sephadex G-200 gel and absorbance of ELISA from its fractions against a reference positive serum. PA1' is the fraction which is filtered PA1 through DEAE-cellulose column.


Fig. 5
Elution profile of PA2' through Sephadex G-200 gel and absorbance of ELISA from its fractions against a reference positive serum. PA2' is the fraction which is filtered PA2 through DEAE-cellulose column.


Fig. 6
Elution profile of PA3' through Sephadex G-200 gel and absorbance of ELISA from its fractions against a reference positive serum. PA3' is the fraction which is filtered PA3 through DEAE-cellulose column.


Fig. 7
SDS-PAGE patterns of crude and fractionated antigens.

C: crude antigen

D: fraction which is fractionated by DEAE-cellulose chromatography

I : fraction of high molecular weight proteins

II: fraction of low molecular weight proteins



Fig. 8
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 60 metacercariae.


Fig. 9
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 30 metacercariae.


Fig. 10
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 15 metacercariae.


Fig. 11
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 5 metacercariae.


Fig. 12
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 60 metacercariae and treated with praziquantel.


Fig. 13
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 30 metacercariae and treated with praziquantel.


Fig. 14
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 15 metacercariae and treated with praziquantel.


Fig. 15
Mean absorbance curves of ELISA against crude and fractionated antigens in the group of cats given 5 metacercariae and treated with praziquantel.

Tables


Table 1
Absorbance values of ELISA for fractionated antigens by ammonium sulfate differential precipitation

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