In this study, a chemiluminescence immunoassay (CLIA) for human serum 25-hydroxyvitamin D (25(OH)D) was established by a competition model. In serum, more than 99% of total circulating 25(OH)D binds to protein and less than 1% of 25(OH)D is in free form (Jassil et al., 2017). Before measuring concentration of 25(OH)D in serum, a releasing procedure should be conducted. A new reagent is used to release binding 25(OH)D to free form. Streptavidin (SA) was labeled to magnetic beads by a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) method. Biotinylated VD was used as a competitor of 25(OH)D in samples. Anti-VD antibody (aby) was labeled to horseradish peroxidase (HRP) by EDC to react with 25(OH)D and biotinylated-VD molecules. The pretreated samples or standards were added into the reaction tube with biotin-VD and anti-VD aby-HRP, free 25(OH)D in the sample competes with biotinylated VD for binding to anti-VD aby-HRP, an SA-labeled magnetic particle is added to isolate the signal-generating complex, and the signal is inversely proportional to the 25(OH)D concentration in the sample. The method established shows good thermostability and performance. The limitation of detection (LoD) is 1.43 ng/mL. The intra-assay coefficient of variation (CV) is 3.66%–6.56%, the interassay CV is 4.19%–7.01%, and the recovery rate is 93.22%–107.99%. Cross-reactivity (CR) was remarkably low with vitamin D2, vitamin D3, 1, 25-dihydroxyvitamin D3, and 1, 25-dihydroxyvitamin D2. At the same time, the cross-reaction values with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 97% and 100%, respectively. The developed method shows good correlation with the total VD product from Roche and DiaSorin. 1096 clinical patient samples were measured with developed reagent kit in this study. 7 types of disease were involved, and the concentration of 25(OH)D is less than 30 ng/mL in 94.98% of patients.

中文翻译：

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化学发光免疫分析法在人血清中定量25-羟基维生素D的开发。

在这项研究中，通过竞争模型建立了人血清25-羟基维生素D（25（OH）D）的化学发光免疫分析法（CLIA）。在血清中，总循环中超过99％的25（OH）D与蛋白质结合，而少于1％的25（OH）D以游离形式结合（Jassil et al。，2017）。在测量血清中25（OH）D的浓度之前，应进行释放程序。一种新的试剂用于释放结合的25（OH）D为游离形式。链霉亲和素（SA）通过1-乙基-3-（3-二甲基氨基丙基）碳二亚胺/ N-羟基琥珀酰亚胺（EDC / NHS）方法标记在磁珠上。生物素化的VD用作样品中25（OH）D的竞争剂。抗VD抗体（aby）通过EDC标记为辣根过氧化物酶（HRP），与25（OH）D和生物素化VD分子反应。将预处理的样品或标准品与生物素-VD和抗VD aby-HRP加入反应管中，样品中的游离25（OH）D与生物素化的VD竞争结合SA标记的抗VD aby-HRP添加磁性颗粒以分离产生信号的复合物，并且该信号与样品中的25（OH）D浓度成反比。建立的方法显示出良好的热稳定性和性能。检测限（LoD）为1.43 ng / mL。批内变异系数（CV）为3.66％–6.56％，批间变异系数为4.19％–7.01％，回收率为93.22％–107.99％。与维生素D2，维生素D3、1、25-二羟基维生素D3和1、25-二羟基维生素D2的交叉反应（CR）非常低。同时，与25-羟基维生素D2和25-羟基维生素D3的交叉反应值为97％和100％，分别。所开发的方法与Roche和DiaSorin的总VD产品显示出良好的相关性。在这项研究中，使用开发的试剂盒测量了1096个临床患者样品。涉及7种疾病，在94.98％的患者中25（OH）D的浓度低于30 ng / mL。