ANAL METHODS FOR THE DETERMINATION OF TRANSMISSIBLE SPON

这项工作包括测定乙酰胆碱酯酶和酪氨酸羟化酶的活性，以及相关方法的开发，在项目编号Z01 BR05008-02 LMD的合作支持下，“分化神经细胞培养中传染性海绵状脑病（TSE）病原体传染性的潜在测定”。乙酰胆碱酯酶测定采用G. Ellman （Biochem）的方法。《药学》，7,88-95,1961)。以乙酰硫代胆碱为底物，酶产物硫代胆碱与二硫代双硝基苯甲酸酯反应后，在412nm处紫外吸附量增加，从而测定酶的活性。采用高效液相色谱法测定酶产物DOPA（二羟基苯丙氨酸）测定酪氨酸羟化酶活性。1998年和1999年，在T. Nagatsu （J. Chrom 163, 247-252, 1979）和D. Goldstein （J. Chrom 275, 174-177, 1983）的研究基础上发展了一种方法。在此过程中，在酪氨酸底物和6-甲基四氢蝶啶辅助因子的饱和浓度下进行孵育；DOPA产物经酸性氧化铝萃取后，采用反相高效液相色谱-安培电化学检测法测定。在2000年，基于d.h ooper （J. Chrom B, 694, 317-324, 1987）的研究对该方法进行了改进，其中通过添加甘油来降低空白值和添加二氢蝶啶还原酶和NADPH来再生四氢蝶啶辅助因子来提高信噪比。总蛋白的测定，为了使酶活性正常化到细胞培养的浓度，正在通过微BCA（双膦酸）程序进行。

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.