Detection of spongiform encelphalopathy

总结：这项工作涉及乙酰胆碱酯酶和酪氨酸羟化酶活性的测定以及相关方法的开发，以协同支持项目编号Z 01 BR 05008 -02 LMD，“在分化的神经元细胞培养物中传染性海绵状脑病（TSE）病原体感染性的潜在测定”。 乙酰胆碱酯酶测定采用G. Ellman（Biochem.Pharm.7，88-95，1961）。使用乙酰硫代胆碱作为底物，通过由酶产物硫代胆碱与二硫代双硝基苯甲酸酯反应产生的412 nm处的UV吸收的增加来测量酶活性。 酪氨酸羟化酶活性通过HPLC测定酶产物DOPA（二羟基苯丙氨酸）来测量。 在FYs 98和99中，基于T. Nagatsu（J. Chrom 163，247-252，1979）和D. Goldstein（J Chrom 275，174-177，1983）。 在该程序中，在饱和浓度的酪氨酸底物和6-甲基四氢蝶啶辅因子的存在下进行孵育;在用酸性氧化铝本体萃取后，通过反相HPLC结合安培电化学检测来测定DOPA产物。 2000年在D. Hooper（J. Chrom B，694，317-324，1987），其中通过添加甘油以降低空白值和添加二氢蝶啶还原酶和NADPH以再生四氢蝶啶辅因子来改善信噪比水平。 为了使酶活性相对于细胞培养物的浓度标准化，通过微量BCA（二次膦酸）程序进行总蛋白的测定。

Summary: This work involves determinations of acetylcholinesterase and tyrosine hydroxylase activities and related methods development in collaborative support of Project Number Z01 BR05008-02 LMD, "Potential assay of transmissible spongiform encephalopathy (TSE) agent infectivity in differentiated neuronal cell cultures". Acetylcholinesterase determinations are performed by the method of G. Ellman (Biochem. Pharm. 7, 88-95, 1961). Using acetylthiocholine as a substrate, enzyme activity is measured by the increase in UV adsorption at 412nm resulting from the reaction of the enzyme product thiocholine with dithiobisnitrobenzoate. Tyrosine hydroxylase activity is measured by determination of the enzyme product DOPA (dihydroxyphenylalanine) by HPLC. In FYs 98 and 99, a method was developed based on studies by T. Nagatsu (J. Chrom 163, 247-252, 1979) and D. Goldstein (J Chrom 275, 174-177, 1983). In this procedure, incubation was performed in the presence of saturating concentrations of tyrosine substrate and 6-methyltetrahydropteridine cofactor; DOPA product was determined by reverse phase HPLC with amperometric electrochemical detection after bulk extraction with acidic alumina. In FY 00, the method was modified based on the studies of D. Hooper (J. Chrom B, 694, 317-324, 1987) in which signal-to-noise levels were improved with the addition of glycerol to reduce blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. Determination of total protein, for the purpose of normalizing enzyme activities to the concentration of cell culture, is being performed by a micro BCA (bichoninic acid) procedure.