Conformation-dependent high-affinity monoclonal antibodies to prion proteins.
Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP(Sc), an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP(Sc) commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP(Sc) from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K(D) values for mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.
Foodborne Contaminants Research Unit, U.S. Department of Agriculture Agricultural Research Service, Albany, CA 94710, USA.
Larry H
LH
Stanker
Ana V
AV
Serban
Elisa
E
Cleveland
Robert
R
Hnasko
Azucena
A
Lemus
Jiri
J
Safar
Stephen J
SJ
DeArmond
Stanley B
SB
Prusiner
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001923:Brain Chemistry / Q000276:immunology; D002417:Cattle; D045744:Cell Line, Tumor; D006224:Cricetinae; D003670:Deer; D005260:Female; D006801:Humans; D008647:Mesocricetus; D051379:Mice; D016513:Mice, SCID; D010446:Peptide Fragments / Q000008:administration & dosage / Q000276:immunology* / Q000378:metabolism; D000072002:Prion Proteins; D011328:Prions / Q000008:administration & dosage / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012756:Sheep
D000911:Antibodies, Monoclonal; C512656:PRNP protein, human; D010446:Peptide Fragments; D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse; D011994:Recombinant Proteins; C507417:prion protein (90-231)