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Tissue origin of soluble component proteins in saline extract of adult Paragonimus westermani
Y Kong,C Y Park,**S Y Kang and S Y Cho
Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
***Junior medical student of Chung-Ang University at the time of the study
Abstract
Tissue origin of individual component proteins in crude extract of adult Paragonimus westermani was investigated. Major soluble component proteins were separated by disc-PAGE in 8% slab gel. By predefined Rf values, strips of gel containing each band protein was cut out. Each band protein was eluted by electrophoresis. Monospecific antibodies were prepared by immunizing rabbits with each band protein. When peroxidase-antiperoxidase (PAP) staining was done, antiserum to Band 1 reacted to content of eggs both in the worm and in the infected lung tissue. Antiserum to Band 2 reacted to parenchymal tissue of the worm. Antiserum to Band 4 showed the positive reaction at intestinal content while that to Band 5 reacted to the intestinal epithelial border. Antiserum to combined proteins of Bands 6/7 and that to Band 8 reacted to parenchymal tissue of the worm respectively. From the results, the origin of individual proteins in crude extract of adult P. westermani could be differentiated.
Figures
Fig. 1 Purity of the band proteins of adult P. westermani after the disc-PAGE and electrophoretic elution. The proteins were visualized in 8% disc-PAGE. Coomassie stained. Numericals stand for an each band protein.
Mr : Molecular mass in kDa, C: Crude extract.
Fig. 2 SDS-PAGE fingings of the band protein. A gradient separationg gel of 10~15% sas used. Markings and numericals are the same as described in Fig. 1.
Figs. 3-8 Figs. 3 & 4. Negative control I & II (×100). The worm sections were treated with PBS (I) and rabbit serum collected before immunization (II) as primary antibodies. No structure showed the positive reactions.
Fig. 5. Positive control (×100). Antiserum against the crude extract was reacted at intestinal epithelial border, intestinal content, eggs and parenchymal tissue. Cvary and vitellarian follicles showed equivocal reaction. Tegument, testes, basal layer of intestine and suckers were not stained positively with PAP.
Fig. 6. The rabbit antiserum to Band 1 reacted to the cells within egg shells in uterus (×100).
Fig. 7. Another PAP staining of antiserum to Band 1 (×100). The eggs which were scattered in cat lung tissue showed the stronger positive reaction.
Fig. 8. When the antiserum to Band 2 was reacted, positive reaction was observed at the parenchymal tissue of the worm (×100).
Figs. 9-12 Fig. 9. Intestinal content was stained positively with antiserum to Band 4(×100).
Fig. 10. Antiserum to Band 5 reacted to the intestinal epithelial border. Basal part of epithelium was not stained with chromogen (×100).
Fig. 11. Rabbit antiserum to Bands 6/7 showed the diffuse positive reaction to parenchymal tissue of the worm (×200).
Fig. 12. Parenchymal tissur of the worm was stained with antiserum to Band 8 (×100).
Tables
Table 1 Recovery of each Band protein when 45 mg of protein in the crude extract of adult P. westermani was purified by disc-PAGE and electrophoretic elution
Table 2 Specific IgG antibody levels (abs. by ELISA) in differently diluted rabbit antisera against respective antigens
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