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Partial characterization of monoclonal antibodies which bind to disease-associated prion protein in immunoprecipitaion assay

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Partial characterization of monoclonal antibodies which bind to disease-associated prion protein in immunoprecipitaion assay

Yuko K. Ushiki^'^, Ryo Endo^'^, Yoshihisa Shimizu^, Yoshifumi Iwamaru^, Takuji Yamamoto\ Shunji Hattori^ Shinkichi Irie^ and Takashi Yoko- yama^

Nippi Research Institute of Biomatrix, 1-1-1 Senju Midori-cho, Ada- chi-ku, Tokyo 120-8601 Japan Prion Disease Research Center NIAH

^HITEC Co.,Ltd <e-mail> y-ushiki@nippi-inc.co.jp

Abstract

Conformational conversion of cellular prion protein (PrP^) to scrapie-form prion protein (PrP^^) is thought to be the central event in prion pathogene- sis. Several studies have shown that their distinct conformation should cause different immunogenic properties, however, almost all mAbs gener- ated against PrP are unable to recognize each ones separately. Such char- acters of mAbs make it diffcult to study conformational difference be- tween PrP^ and PrP^^ or mechanism of prion replication. Therefore the purpose of this study is to generate such mAbs that would react with PrP

Sc

but not with PrP^.

PrP knockout mouse was immunized with purified PrP^^ without pro- teinase K (PK) treatment and hybridomas were prepared with common procedure. Supematants from hybridomas were screened with ELISA, which was designed to detect native PrP^^ preferably from scrapie-infected mouse brain homogenate. We obtained 9 mAbs that could react PrP^^

with relatively high affinity. Among these mAbs, we selected 4 mAbs that failed to react with guanidine-treated PrP^^, whose intrinsic conforma- tion should be lost. As a result of immunoprecipitation assay, these mAbs could precipitate PrP^^ from scrapie-infected mouse brain homoge- nate, but they brought no precipitation from normal mouse one. These mAbs were able to precipitate PrP^^ in PK-treated homogenate similarly.

Immunoblot and pepspot analysis of these mAbs showed no signals.

Here, we obtained the new mAbs which distinguish scrapie-infected mouse brain homogenate from normal mouse one. As these mAbs could not recognize linear sequence of PrP, it is possible that they recognize

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conformational epitope. Therefore these mAbs may be valuable tools for

conformational analysis of PrP^ and PrP^^.

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