Development and Characterization of three anti-PrPSc IgM Monoclonal Antibodies

• Main Authors: Ying-Yu Lin, 林盈妤
• Other Authors: Yi-Ming A. Chen
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/rfn5c6

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 100 === Prion disease is a neurodegenerative disease characterized by spongiform change in the brain of cattle and caused by aggregation of misfolded wild-type cellular prion protein (PrPc), i.e., PrPSc. The emergence of variant Creutzfeld-Jakob Disease (vCJD) and the compelling evidence linking it to bovine spongiform encephalopathy (BSE) have raised concern about a possible epidemic in human due to the consumption of PrPSc-contaminated foods. Since there is an urgent need to develop diagnostic reagents that can specifically recognize PrPSc, the specific aim of this study was to generate and characterize monoclonal antibodies against PrPSc. In this study, we used a recombinant mutant mouse β PrP generated from E. coli as antigen to immunize BALB/c mice. This mouse β PrP contained 4 amino acid mutations which removed its disulfide bond and resulted in the formation of β oligomers. Splenocytes harvested from mice immunized with mutant mouse β PrP for 5 times were used to fuse with NS1 myeloma cells. Enzyme immunoassay was used to screen hybridomas secreting monoclonal antibodies (mAbs) against β PrP. After several rounds of limited dilution and screening, 3 mAbs were obtained and their isotypes were IgM. Their reactive epitopes were mapped using both peptide scan and phage display methods and the results showed that these 3 mAbs against different epitopes located at the N-terminal, α helix 1 andαhelix 2. Furthermore, two of three mAbs can identify PrPSc from normal mouse brain homogenates without pre-treatment with protease K using both dot blot and Western blot assays. In summary, we found that β oligomers can induce IgM Ab production preferentially. Besides, these mAbs can be used to develop more specific assay for prion disease.