Development Of Sandwich ELISA For The Detection Of Prion Protein In Cattle

PrPsc (Scrapie Prion Protein) is the main causative agent of Bovine Spongiform Encephalopathy (BSE). Prion Protein has two conformation forms,a normal form- PrPc (Cellular Prion Protein), and the other pathogenic form - PrPsc. As the common view, PrPSc is thought to be converted from PrPc, which can trigger prions disease. The main different performance between PrPc and PrPsc is the resistance to the proteinase K digestion. Both of them can response to the antibody, but after proteinase K digestion, normal PrPc will be fully digested, and the abnormal PrPsconly partially digested, still retained section of 27000-30000 fragments which could be identified by antibody. It provides a theoretical basis for the establishment of the normal PrPc detection method.At present,more effective detection method is based on the response between antigen and immune antibody,capillary electrophoresis methods and some method of the enzyme tinked assay,fluorescence scanning methods, in which enzyme-linked immunosorbent assay (ELISA) possess high sensitivity, specificity, reproducibility,and no radioactive contamination. It has the advantage of quantitative measurement and automation, and is fit for high-flux samples screening of BSE. In this experiment, bovine prion protein expressed in Escherichia coli was purified for the establishment of double-antibody sandwich ELISA method to detecte PrPc, which made a foundation on the development of PrPscdetection Kits for BSE.The 660bp fragment of the PRNP coded PrPc was amplified by PCR method which the template of total DNA extracted from cattle livers,the purified target gene frament was cloned into the vector termed pMD18-T Simple, After sequencing the positive clons, the PRNP gene digested by BamH I and Xho I was subcloned into the expression vector pGEX-6p-1 digested by the same two enzymes. After the identification, the positive recombinant plasmid pGEX-6p-1-PRNP was picked up.The recombinant plasmid pGEX-6p-1-PRNP was transformed to the host E.coli termed Rosetta-gami(DE3),the protein was analysed by SDS-PAGE and the specific expression band was detected by Western Blotting assay with the special monoclonal antibodies SAF16.Rabbits were immuned using the prp protein purified by the method of affinity chromatography. After the fifth immune, the titler of the multiclone antibody is 1:6400 and the optimal reaction conditions of the double antibody sandwich ELISA method was established: dilution of MAb (1:8000), monoclonal antibody for optimal concentration of 1.25μg/mL,the coating of MAb 4℃for 12h,37℃SAF16 monoclonal antibody for 1h incubation,the working condition of HRP-coating antigen IgG (1:5000), 1% BSA +1% gelatin as the confining liquid for twice, overnight at 4℃and the OPD was chosen to be enzyme reaction substrate at 37℃for 30 min. The standard curve was obtained and regression equation y =0.0004x+0.4469 was got by a regression analysis. The results indicated that 32.0 ng/mL of antigen was the lowest detectable limit of ELISA.