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Component proteins in crude extract of adult Paragonimus westermani purified by immunoaffinity chromatography using monoclonal antibodies
S Y Kang,Y Kong and S Y Cho
Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
Abstract
The nature of 2 component proteins in crude saline extract of adult Paragonimus westermani was investigated. By immunoaffinity chromatography using monoclonal antibodies (MAb) as ligands, the proteins were purified from the crude extract. Band 1 protein in disc-polyacrylamide gel electrophoresis (PAGE) was purified by PFCK-136 MAb. The protein, known to have molecular mass of 440 kDa, was composed of 23, 46 and 92 kDa subunits when observed by reducing SDS-PAGE and SDS-PAGE/immunoblot. This protein was originated from eggs of the worm as revealed by immunohistochemical staining with PFCK-136 Mab. Another affinity purified protein utilizing PFCK-44 MAb was the band 4 protein of 17 kDa in disc-PAGE. This was a monomer protein in reducing SDS-PAGE and SDS-PAGE/immunoblot. The protein was produced at intestinal epithelium of the worm.
Figures
Fig. 1 Findings of SDS-PAGE/immunoblot of MAbs reacted to crude saline extract of adult P. westermani.
A: PFCK-21, B: PFCK-44, C: PFCK-136, D: PFCK-189
Fig. 2 Findings in disc-PAGE of the component proteins of adult P. westermani obtained in affinity chromatography.
C: Crude extract, 1: Unbound protein(UP) from affinity chromatography using PFCK-44, 2: Bound protein(BP) of PFCK-44, 3: UP of PFCK-136, 4: BP of PFCK-136, 5: UP of PFCK-44 and PFCK-136
Fig. 3 SDS-PAGE findings of the component and purified proteins of P. westermani in 10~15% gradient gel.
Mr: Molecular mass in kDa, C: Crude extract, 1: UP of PFCK-44, 2: BP of PFCK-44, 3: UP of PFCK-136, 4: BP of PFCK-136, 5: UP of PFCK-44 and PFCK-136
Figs. 4-9 Immunohistochemical stainings of P. westermani and its surrounding granuloma with MAbs and paragonimiasis patient serum.
Fig. 4. Negative control (×100). Normal human serum was reacted as a primary antibody. No organs were reacted with AEC chromogen.
Fig. 5. Negative control reacted with normal human serum on granuloma wall with eggs (×100).
Fig. 6. A patient serum of paragonimiasis was used as a primary antibody. Strong reactions were shown (see text).
Fig. 7. Section of eggs in granuloma wall reacted with a patient serum (×100).
Fig. 8. Reactivity of PFCK-136 MAb to eggs in granuloma wall (×100).
Fig. 9. Reactivity of PFCK-44 MAb to intestinal wall of the worm (×200).
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