改良MTT法测定血清TNF活性

Improved MTT Colorimetric Assay for Serum TNF Activity

为寻求一种简便可靠的测定血清肿瘤坏死因子（ＴＮＦ）活性的方法，作者对经典的四甲基偶氮唑蓝（ＭＴＴ）比色法进行了改良，采用改良的ＭＴＴ法测定ＴＮＦ活性，并与经典的ＭＴＴ法比较。结果显示：经改良后的ＭＴＴ法较经典的ＭＴＴ比色法稳定，在甲瓒溶解过程中无蛋白凝固沉淀形成，溶解后在３～１２小时内不褪色；在靶细胞活性≥９５％，细胞数＞１０２／孔时，细胞数与细胞还原ＭＴＴ产生的甲瓒溶解后的ＯＤ值呈线性相关（ｒ＝０．８７，Ｐ＜０．０１），标准品ＴＮＦ每孔含量与ＯＤ值呈负相关（ｒ＝－０．７９，Ｐ＜０．０１）。用此法检测２０份正常人血清，其ＴＮＦ活性均值为２０．９５±３．２ＩＵ／ｍｌ。本法简便易行，稳定可靠。

The activity of serum tumor necrosis factor(TNF) is known to be related with the mechanism and prognosis of many diseases.The aim of this experiment was to look for an economic and reliable method for assaying the activity of serum TNF.We used the neutrophil solventor 20%SDS50%DMF instead of the acidity solventor 0.04 mol/l acidified isopropy alcohol and established an improved 3(4,5dimethyl thiazoly)2,5diphenyltetrazolium bromide(MTT) colorimetric assay for measuring the amount and function of living cell.We used this improved MTT colorimetric assay for measuring TNF activity of peripheral blood serum of healthy persons.It avoided the deposition of the protein in serum and medium and showed more repeatability,as compared with the conventional MTT colorimetric assay.The results showed:when the activity of target cell(TC)L929 is ≥95%,the density of TC is ≥1×102/well and the number of living cells in each well is positively correlated with the OD volume (OD570nmOD630nmof purple formazan metabolite of MTT solution(r=0.87,P<0.01);when the density of TC is 5×104/well,the level standard of TNF in each well is negatively correlated with the OD volume (OD570nmOD630nm)r=0.79, P<001).Using this method,we measured the TNF activity of 20 healthy persons peripheral blood serum.The [AKx-〗±s of TNF activity is 20.95±3.2 IU/ml.This method is dependable,easytodo and economic;it has good repeatability within 312 hours.