Detection, quantification, and glycotyping of prion protein in specifically activated enzyme-linked immunosorbent assay plates.
The conversion of a normal glycoprotein, prion protein (PrP(C)), to its abnormal protease-resistant isoform (PrP(Sc)) seems to be one of the main factors underlying the pathogenesis of spongiform encephalopathies. There are many studies indicating that PrP interacts with glycosaminoglycans, and we exploited this interaction to develop a sensitive solid phase assay for detection of both PrP forms. Glycosaminoglycans, such as chondroitin sulfate and heparin, were immobilized by their negative charge to enzyme-linked immunosorbent assay (ELISA) plate wells activated by glutaraldehyde and spermine. PrP in the samples examined (recombinant PrP or tissue homogenate) was allowed to interact with glycans. The interaction of recombinant PrP was more efficient against immobilized chondroitin sulfate of type A, and a linear correlation with concentration was demonstrated. From this curve, the concentration of each one of the PrP isoforms in biological samples can be determined. In addition, and taking into account that glycosylation of prion protein is species specific, we used similarly activated ELISA plate wells to determine different PrP glycoforms. A monoclonal antibody against PrP was immobilized, and PrP present in the samples (brain homogenates) was bound and visualized by various lectins. The most interesting outcome of the study is the differential binding of ricinus communis agglutinin I to the normal and scrapie brain homogenates. Dattura stramonium lectin and wheat germ agglutinin seem to bind almost equally to both samples, and all three have an increased sensitivity to PrP(Sc) after proteinase K digestion.
Triantaphyllidou
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IE
Department of Chemistry, University of Patras, 26500 Patras, Greece.
Sklaviadis
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Vynios
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DH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001681:Biological Assay; D001921:Brain / Q000473:pathology; D001923:Brain Chemistry; D002417:Cattle; D016643:Encephalopathy, Bovine Spongiform / Q000175:diagnosis* / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006031:Glycosylation; D018621:PrPC Proteins / Q000032:analysis / Q000737:chemistry; D018620:PrPSc Proteins / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D014020:Tissue Extracts / Q000737:chemistry
D000911:Antibodies, Monoclonal; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011994:Recombinant Proteins; D014020:Tissue Extracts; D019286:Endopeptidase K