生物学杂志

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25-羟基维生素D化学发光检测试剂盒的研发

  

  1. 1. 江南大学 粮食发酵工艺与技术国家工程实验室, 无锡 214122; 2. 江南大学 工业生物技术教育部重点实验室, 无锡 214122; 3. 江南大学 糖化学与生物技术教育部重点实验室,无锡 214122
  • 出版日期:2017-10-18 发布日期:2017-10-18
  • 通讯作者: 杨艳坤,副教授,硕士生导师,主要从事微生物学、基因工程、分子遗传、蛋白质的分离纯化及特性研究等相关领域科研工作,E-mail:yangyankun@jiangnan.edu.cn
  • 作者简介:宋翠翠,硕士研究生,主要从事体外诊断试剂研发工作,E-mail: 906861617@qq.com
  • 基金资助:
    江苏省政策引导类计划(产学研合作)——前瞻性联合研究项目(BY2015019-17)

The development of chemiluminescent immunoassay detection kit for 25-hydroxy vitamin D

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology; 2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology; 3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2017-10-18 Published:2017-10-18

摘要: 研制了用于定量检测人血清中25-羟基维生素D(25-OHD)水平的化学发光法检测试剂盒,并对试剂盒各项性能指标进行验证。试剂盒建立的方法为:将25-OHD单克隆抗体包被于微孔板中,将血清样品与释放剂同时加入微孔板中进行孵育,使样品的25-OHD充分释放出来并与包被板上的25-OHD抗体相结合。再加入酶结合物(生物素化25-OHD和链霉亲和素化过氧化物酶(SA-HRP)的混合物),使之与25-OHD抗体相结合,形成竞争免疫复合物,随后洗涤除去未结合的物质,加入化学发光底物,使用化学发光免疫分析仪读取相对发光强度(RLU)。制备的25-OHD检测试剂盒最低检出限为4.9 ng/mL,准确度相对偏差在±10%以内,批内变异系数小于10%,批间变异系数小于15%,2℃~8℃存放有效期为1年。对200份临床血清样本进行检测,与参比试剂盒检测结果的相关系数R=0.9717。试剂盒各项性能指标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

Abstract: In this paper, a chemiluminescent immunoassay (CLIA) kit for quantitative assay of 25-hydroxyvitamin D(25-OHD)in serum was developed and evaluated. The kit is established as follows: first, 25-hydroxyvitamin D monoclonal antibodies was coated with the 96 well microplate, then the serum sample and the release reagent were added into the microplate. After incubation, the 25-OHD of the sample was fully released and combined with the 25-OHD antibody coated on the microplate. HRP conjugate (mixture of biotinylated 25-OHD and streptavidin-labeled HRP ) compete with the 25-OHD released from sample to antibodies. After washing and adding the CLLIA substrate, the relative luminous intensity was read with chemiluminescent immunoassay analyzer. The minimum detection limit of the kit is 4.9 ng.mL-1, with the bias of less than 10%. The intra-assay and inter-assay coefficients of variation were less than 10%, and 15%, respectively. Its preserving duration could be as long as 12 months at 2-8℃. Two hundred clinical serum specimens were tested with this kit and a foreign made serum ferritin kit, the correlation coefficient was 0.9717. 25-hydroxy vitamin D (CLIA) quantification detection kit was successfully prepared, and all the technical parameters reached the industrial standards. The developed kit is sensitive, stable, fast and simple.

Key words: 25-hydroxyvitamin D, chemiluminescent immunoassay, kit