Production and Characterization of l\/lonoclonal Antibodies Specific for Prion Protein
Masanori Morita^'^, Akimasa Ohmizu^'^, Hideki Maeno^'^, Takato Ma- tsuo^'"*, Yoichi Ogata^'^, Mitsuru Naiki^'^, Shouji Suzuki^'^ and Isao Na- kata''^
^Committee on Creutzfeldt-Jacob disease, Japanese Ketsueki-Seizai (Blood Products) Association ^Benesis Corp., 2-1 Seiryo, Aoba-ku, Sen- dai 980-8575 Japan ^Japanese Red Cross Plasma Fractionation Center
"^Nihon Pharmaceutical Co. Ltd. ^The Chemo-Sero-Therapeutic Research Institute Nippon Zoki Pharmaceutical Co. Ltd.
<e-mail> [email protected]
Abstract
Creutzfeldt-Jacob disease (CJD) is a neurodegenerative disorder caused by piron, which mainly consists of abnormal prion protein (PrP^^). As PrP^^
was detected recently in two patients who received transfusion from pre- clinical variant CJD (vCJD), concerns about the theoretical risk of trans- mission of vCJD through blood products increased. In order to contribute the blood products-safety against prion, we aimed at obtaining monoclonal antibodies (mAbs) eligible for screening procedures.
By immunizing prion protein (PrP) gene ablated (Pmp-/-) or normal (Pmp+/+) mice with insoluble form of recombinant human PrP, expressed in E. coli, we obtained a panel of hybridoma. Using purified mAbs we studied their immunoreactivity against PrP^^ and recombinant PrP by Western blotting or by immunohistochemistry. Epitope mapping was performed using forty 15mer-peptides representing human PrP sequence, and using fusion proteins between glutathione-S-transferase and PrP-deletion mutants.
All mAbs recognized both the recombinant PrP and the proteinase K- resistant PrP (PrP"^^^). mAb 5A1, which bound oligopeptides correspond- ing amino acid residues 138-157, recognized two fragmented PrP'^^^ bands around 16.5 kDa. mAb 12A1, which bound oligopeptides of 158-172 residues, recognized one fragmented PrP^^^ band around 12 kDa, but poorly bound to di-glycoform of PrP^^^ mAb 4A1, which bound deleted PrP fu-
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sion proteins but not any of oligopeptides, recognized three fragmented PrP'"' bands around 12, and 16.5 kDa.
These mAb will help to establish the screening tests for plasma or blood products as well as to investigate PrP^^. We thank Prof T. Kitamoto, as- sistant Prof. T. Muramoto (Tohoku Univ.), and Prof S. Mohri (Kyushu Univ.) for cooperation and advices.
