AH6 Western Blot Western Blot RIPA 90V 160V 100V 1.5 3%BSA 15 AH Prionics -Check WESTERN 100% 99.4% 100% 100% Western Blot

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1 WESTERN BLOT * AH6 Western Blot Western Blot RIPA 90V 160V 100V 1.5 3%BSA 15 AH Prionics -Check WESTERN 100% 99.4% 100%100% Western Blot (Transmissible Spongiform Encephalopathy, TSE) ScrapieBovine Spongiform EncephalopathyBSE Chronic Wasting Disease,CWDFeline spongiform Encephalopathy, FSE [2] Cellular Prion Protein PrP C PrP [456] [3] [789] PrP [10] PrP SC PrP C α- β- [1211] PrP C PrP SC [10], [12] (TSE) TSE OIE A FAO WTO Western Blot OIE *No zhaodm@ cau.edu.cn

2 Western Blot Western WTO Western Blot Marker 14,400-97,400biolab CDP StarTM Boehringer Maninheim -20 Bio-Rad Nonidet P-40 N-N- Fluka Tris Promega SDS Sigma AH6 BE12 TSE Resource Center PVDF German PMSF Triton X-100 Amresco k MerckBSA vdtt RocheX Kodak 1.2 XH-P X- OMAT1000 Kodak Mini Trans-Blot Mini- PROTEAN PowerPac TM HC Bio-Rad 1.3 Prionics -Check WESTERN obex 10% obex Western Blot 0.45~0.70g 50ml µl g 5 12% SDS-PAGE Marker PVDF TBST20mmol/L Tris HCl ph7.5500mmol/l NaCl0.05%Tween TBST CDP-StarTM 5 X Np-40150mM/L Nacl1% Np-4050mM/L Tis-Hclph7.5)RIPA150mM/L Nacl1% Np mM/L Tis5%1% SDSph7.5 2

3 Triton X % Triton X-100, 0.5% SDS150Mm/L Nacl5%, Tris-Hclph SDS-PAGE 90V 90V 160V 90V 200V SDS-PAGE PVDF 30V 100V1 100V V2 30 Marker % TBST5%+3%BSA TBST3%BSA TBST 1% PVDF AH TBST PVDF TBST Prionics -Check WESTERN 161 Prionics -Check WESTERN 10ul 50µg/ml K48 40min 10µl 5mM/ml PMSF SDS-PAGE V 90V 160V 90V 160V 200V 90V 160V V 100V 1 100V 3

4 V 100V 1 100V X 4 5% 3%BSA 3%BSA : Prionics -Check WESTERN Western Blot Prionics -Check WESTERN Prionics -Check WESTERN Western Blot A 159 A Prionics -Check WESTERN 100%100% Western Blot 100%99.4% 4

5 3 3.1 PrP C PrP Sc K [131415] K PrP K PrP C PrP Sc 27-30KD PrP PrP C 3.2 Western Blot 205 PrP C 99.9% PrP C 99.9% Prionics -Check WESTERN OIE [16] 100% 100% OIE OIE Western Blot 800 Western Blot Western Blot Western Blot [1]Prusiner S B. Prion encephalopathies of animals and humans [J]. Dev Biol, 1993, 80, [2]Prusiner S B. Novel proteinaceous infectious particles cause scrapie [J]. Science, 1982, 216: [3]Stahl N, Borchelt D R & Prusiner S B. Differential release of cellular and scrapie prion protein from cellular membranes by phosphatidylinositol-specific phospholipase C [J]. Biochemistry, 1990, 29: [4] [5]...., 2004, 35 (6), [6] Yang J M, Zhao Deming, Liu Hongxiang el at. Comparative analysis of the prion protein open reading frame nucleotide sequences of Keet and Peacock, VIRUS GENES, 30:1,193~196, [7]Brown D R, Besinger A, Herms J W, et al. Microglial expression of the prion protein [J]. Neuroreport, 1998,9:

6 [8]Diomede L, Sozzani S, Luini W, et al. Activation effects of a prion protein fragment [PrP-( )] on human leucocytes [J]. Biochem J, 1996, 320: [9]Brown D R, Schmidt B, Groschup M H, et al. Prion protein expression in muscle cells and toxicity of a prion protein fragment [J]. Eur J Cell Biol, 1998, 75: [10]Prusiner S B. Prions. Proc Natl Acta Sci USA, 1998, 95: [11]Brandner S, Isenmann S, Raeber A. Normal host prion protein necessary for scrapieinduced neurotoxicity [J]. Nature, 1996, 379: [12] Kimberlin R H, Walker C A. Pathogenesis of scrapie (strain 263K) in hamsters infected intracerebrally, intraperitoneally or intraocularly [J].J Comp Pathol, 1986, 67: [13]McKinley M P, Bolton D C, Prusiner S B. A protease-resistant protein is a structural component of the scrapie prion [J]. Cell, 2002, 35 (1): [14]Meyer R K, McKinley M P, Bowman KA, et al. Separation and properties of cellular and scrapie prion proteins[j]. Proc Natl Acad Sci USA 2001, 83: [15]Gabizon R, McKinley M, Prusiner S, Purified prion proteins and scrapie infectivity copartition into liposomes[j]. Proc Natl Acad Sci USA, 1987,84 (12): [16]Moynagh J, Schimmel H. The evaluation of tests for the diagnosis of transmissible spongiform encephalopathy in bovines[r]. Report. European Commission, 1999, July. Development of Western Blot for Detecting BSE and Scrapie Wang Huinuan Zhao Deming Ning Zhangyong Yang Jianmin Wu Changde Hao Junfeng Bai Yu Ma Yun Wang Chuanwu Han Caixia Liu Meili Meng Liping (National Animal TSE Lab, China Agriculture University, Beijing, , China) ABSTRACT: Western Blot for detecting BSE and scrapie was developed by using monoclonal antibody (mab) AH6 and horse anti-mouse IgG antibodies coupled to alkaline phosphates (AP). The various affected factors and conditions of Western Blot were explored, and the optimal reaction conditions of Western Blot were determined. It was shown that when used the homogenization buffer RIPA, the optimal voltage of resolving gel electrophoresis was 90Vand in stacking gel was up to 160V, the optimal voltage and time of blotting was 100V for 1.5h, the optimal PVDF blocking buffer was 3%BSA in TBST, the optimal dilution of monoclonal antibody (mab) AH6 was 1:4000 and incubated for h at 4 C, the optimal dilution of horse anti-mouse IgG antibodies coupled to alkaline phosphates (AP) was 1:1000 and incubated 30 min at room temperature. Following the determination of conditions of Western Blot, Samples were detected and the result of it was compared with that of Prionics -Check WESTERN kit. It show that the sensitivity and the specificity of detection respective are 100% and 99.4%, and those of Prionics -Check WESTERN kit respective are 100% and 100%. There was no significant difference these two detection ways. The development of Western Blot will provide help for the Western Blot kit of us

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