微孔板酶底物检测尿酸酶活性方法的建立及验证  

Establishment and validation of an enzyme-substrate assay using microplate for detecting uricase activity

作　　者：张慧颖 江筠 张林焱 刘敏 秦荣浦 田石华 林惠娟 何成 ZHANG Hui-ying;JIANG Yun;ZHANG Lin-yan;LIU Min;QIN Rong-pu;TIAN Shi-hua;LIN Hui-juan;HE Cheng(DepartmentⅡof Research,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]上海生物制品研究所有限责任公司研发部第二研究室,上海200051

出　　处：《微生物学免疫学进展》2022年第1期30-34,共5页Progress In Microbiology and Immunology

基　　金：上海市科委基金(15431906200)。

摘　　要：目的建立石英微孔板法酶底物检测尿酸酶活性的方法,并对方法进行验证。方法摸索尿酸浓度与吸光度之间的线性关系,找到合适的范围,并验证方法的专属性、线性、重复性、中间精密度、准确性和耐用性。尿酸酶加入固定浓度尿酸溶液,酶促反应5 min后,通过酶标仪检测A_(292 nm)值变化计算尿酸的含量,根据单位时间内尿酸减少量计算出尿酸酶的活性。结果专属性验证结果显示,空白对照尿酸浓度未检出,25μg/mL尿酸样品的回收率为102.0%,50μg/mL尿酸用等体积的硼酸缓冲液稀释后的样品回收率为103.2%,表明专属性较好。尿酸质量浓度在0~50μg/mL范围内线性关系良好,R^(2)>0.99。该方法重复性验证的RSD为0.6%;中间精密度验证的RSD为1.4%;准确性验证的3个样品回收率分别为101.3%、101.3%、101%,RSD分别为1.1%、1.2%、0.7%;不同温度条件下,耐用性验证的RSD为4.8%。结论本实验建立了微孔板酶底物检测尿酸酶活性的方法,该方法的专属性、线性、重复性、中间精密度、准确性和耐用性良好,可用于尿酸酶活性的定量检测。Objective To establish and verify an enzyme substrate method for detecting uricase activity in a quartz microplate.Methods To explore the linear relationship between uric acid concentration and absorbance at 292 nm,find out the appropriate linear range.The established method was verified for specificity,linearity,repeatability,intermediate precision,accuracy and durability.Uricase was added into fixed-concentration uric acid solution,and the enzymatic reaction was lasted for precise five minutes.The reduced content of uric acid was calculated by the changing absorbance detected at 292 nm and the uricase activity was calculated based on the reduction of uric acid per unit time.Results Specificity verification was qualified that since uric acid was not detected in the blank control group.The recovery rate of 25μg/mL uric acid samples was 102.0%,and the recovery rate of 50μg/mL uric acid sample which diluted by one-fold dilution of boric acid buffer was 103.2%.Uric acid concentration had a good linear relationship in the range of 0~50μg/mL,R^(2)>0.99.The RSD of repeatability verification of this method was 0.6%,and the RSD of intermediate precision verification was 1.4%.The recovery rates of the three samples verified for accuracy were 101.3%,101.3%and 101%,with the RSD of 1.1%,1.2%and 0.7%,respectively.Under different temperature conditions,the RSD for durability verification was 4.8%.Conclution In this study,a method for detecting uricase activity by quartz microplate has been established.The method was perfectly qualified for specificity,linearity,repeatability,intermediate precision,accuracy,durability and could be used for the quantitative detection of uricase activity.

关 键 词：尿酸酶 尿酸 酶活性检测 石英微孔板 酶标仪 

分 类 号：R392[医药卫生—免疫学]