葡萄糖磷酸异构酶酶显色定量测定方法的建立及性能评估

Establishment and performance evaluation of enzymic colorimetric Quantitative Method for the Determination of glucose phosphate isomerase

目的建立葡萄糖磷酸异构酶(GPI)的酶显色定量测定方法。方法 D-果糖-6-磷酸二钠在GPI作用下生成葡萄糖-6-磷酸,葡萄糖-6-磷酸与烟酰胺腺嘌呤二核苷酸在葡萄糖-6-磷酸脱氢酶(G6PDH)作用下生成还原型烟酰胺腺嘌呤二核苷酸(NADH),NADH与水溶性四氮唑盐(WST-3)反应,反应产物在550nm处有最大吸光度值,吸光度值与GPI水平呈正比。利用此原理对39例类风湿关节炎患者、52例其他自身免疫性疾病和32例健康对照者血清中GPI进行检测。同时评价该方法的线性、精密度、准确度、稳定性,并与GPI酶联免疫吸附试验(ELISA)试剂盒进行一致性分析。结果该方法线性良好(r2=0.992 3)、精密度批内和批间变异系数均小于10%、准确度t检验显示各水平测试结果偏倚均可接受。试剂稳定性大于12个月,测试结果相对偏差在±20%以内均满足试验要求,且与GPI ELISA试剂盒相关性良好,测定结果偏差无统计学意义(P>0.05)。结论 GPI的酶显色定量测定方法的各项指标均符合临床要求,可用于临床GPI水平检测。

Objective To establish a quantitative detection method for enzymic colorimetric of glucose phosphate isomerase(GPI).Methods D-fructose-6-disodium phosphate(F6P)produces glucose-6-phosphate(G6P)under the action of GPI.Glucose-6-phosphate and nicotinamide adenine dinucleotide(NAD)react with water-soluble tetrazolium salt(WST-3)to form reduced nicotinamide adenine dinucleotide(NADH)under the action of glucose-6-phosphate dehydrogenase(G6PDH).The maximum absorbance of the reaction product was at550nm,and the absorbance value was proportional to the level of GPI.Using this principle,serum GPI was detected in39patients with rheumatoid arthritis,52patients with other autoimmune diseases and32healthy controls.At the same time,the linearity,precision,accuracy and stability of the method were evaluated,and the consistency with the GPI ELISA kit was analyzed.Results The linearity of this method was well(r2=0.9923),the coefficient of variation(CV)values of intra and inter assay were all less than10%and accuracy t test showed that the bias of each concentration was acceptable(P>0.05).The period of validity was more than12months,the relative deviation of the test results was within±20%which met the experimental requirements.This reagent was well correlated with GPI ELISA kit and the deviation had no statistical significance(P>0.05).Conclusion This method was valuable for the clinical application and could be used for detection of the level of GPI in the clinics.