宋翠翠， 李昕， 白仲虎， 杨艳坤^*

（  江南大学粮食发酵工艺与技术国家工程实验室，工业生物技术教育部重点实验室，糖化学与生物技术教育部重点实验室，江苏无锡 214122；  ）

摘要： 目的 研制25-羟基维生素D（化学发光法）定量检测试剂盒，并进行验证。方法 用25-羟基维生素D单克隆抗体包被微孔板，酶结合物（生物素化25-羟基维生素D与链酶亲和素标记辣根过氧化物酶的混合物）与校准品及样本中的25-羟基维生素D竞争性结合到包被抗体上，采用竞争化学发光法建立了25-羟基维生素D定量检测方法，并对试剂盒的各项技术参数进行验证。结果 制备的试剂盒最低检出限为4.90 ng/mL，准确度相对偏差在±10%以内，分析内变异系数为6.5%，批间变异系数为8.9%，2-8℃存放有效期为1年。对200份临床血清样本进行检测，本试剂盒与参比试剂盒的检测结果的相关系数R=0.9717。结论成功制备了25-羟基维生素D（化学发光法）定量检测试剂盒，各项技术参数均符合行业标准要求，具有灵敏，稳定，快速，简便等特点。
关键词： 25-羟基维生素D；化学发光法；试剂盒

Song Cuicui， LI Xin， Bai Zhonghu， Yang Yankun^*

（  National Engineering Laboratory for Cereal Fermentation Technology, the Key Laboratory of Industrial Biotechnology, Ministry of Education, the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122；  ）

Abstract： To develop and evaluate a chemiluminescent immunoassay (CLIA) test kit for quantitative determination of 25-hydroxyvitamin D（25-OHD）in serum. Methods The 96 well microplate was coated with 25-hydroxyvitamin D monoclonal antibodies, HRP conjugate( the mixture of biotinylated 25-OHD and streptavidin-labeled HRP ) compete with the 25-OHD released from sample to bind sites of antibodies. And evaluate the kit. Results The minimum detection limit of the kit was 4.90 ng/mL, the bias was less than 10％, the intra-assay and inter-assay coefficients of variation were 6.5%, and 8.9%. Its preserving duration could be 12 months at 2-8℃. 200 clinical serum specimens were detected with this method and a foreign serum ferritin kit (Roche), the correlation coefficient was 0.9717. Conclusion Successfully prepared 25-hydroxy vitamin D (CLIA) quantification kit .And all the technical parameters reached the industrial standards. The kit was sensitive, stable, fast and simple.
Keywords： 25-hydroxyvitamin D; chemiluminescent immunoassay; kit

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