Chen Xiang, Ren Feng, Xu Jingjing, Yu Zheng, Lin Xuemei, Bai Zhonghu, Gong Fang
Clin Lab. 2020 Mar 1;66(3). doi: 10.7754/Clin.Lab.2019.190604.
Vitamin B12 is very important for the human body. Early diagnosis of vitamin B12 deficiency is essential because it is associated with many problems such as fatigue, lethargy, depression, poor memory, breathlessness, headaches, pale skin, mania, psychosis, etc. Most of the vitamin B12 molecules in serum bind to specific proteins as a complex; so, although researchers have developed some sensitive methods to quantify vitamin B12, few can be used for human serum detection of vitamin B12 because of disturbed releasing process. In this study, we are aiming to develop a rapid and accurate chemiluminescence immunoassay (CLIA) for human serum testing.
In this study, we studied and optimized labelling of biotin molecules to vitamin B12 binding protein which had been extracted from hog gastric mucus, labelling of vitamin B12 molecules to horse radish peroxidase (HRP), releasing of vitamin B12 from its bound state in serum, concentrations of reagents, and incubation times.
The developed method shows good performance and thermo-stability. The limit of detection (LOD) is 41 pg/mL, the recovery rate is 90.7% - 107.4%. The intra-assay CV is 4.4% - 8.0% and the inter-assay CV is 4.5% - 12.1%. Cross reactivity (CR) values are 49.4% for hydroxocobalamin and lower than 0.1% for vitamin B1, B2, B3, B9, and C. The method comparison results show that the correlation coefficient (R2) is 0.91, with a slight negative bias of -4.1% [95% limits of agreement (± 1.96 SD); -39.6% to 31.6%].
The developed method shows high sensitivity and specificity and good correlation using a Beckman Coulter instrument while the whole test needs only 40 minutes. It can fully satisfy the very busy clinical labs.
维生素B12对人体非常重要。维生素B12缺乏的早期诊断至关重要，因为它与许多问题相关，如疲劳、嗜睡、抑郁、记忆力减退、呼吸急促、头痛、皮肤苍白、躁狂、精神病等。血清中的大多数维生素B12分子与特定蛋白质结合形成复合物；因此，尽管研究人员已经开发出一些灵敏的方法来定量维生素B12，但由于释放过程受到干扰，很少有方法可用于人血清中维生素B12的检测。在本研究中，我们旨在开发一种用于人血清检测的快速、准确的化学发光免疫分析（CLIA）方法。
在本研究中，我们研究并优化了生物素分子与从猪胃黏液中提取的维生素B12结合蛋白的标记、维生素B12分子与辣根过氧化物酶（HRP）的标记、血清中维生素B12从其结合状态的释放、试剂浓度和孵育时间。
所开发的方法表现出良好的性能和热稳定性。检测限（LOD）为41 pg/mL，回收率为90.7% - 107.4%。批内变异系数（CV）为4.4% - 8.0%，批间变异系数为4.5% - 12.1%。羟钴胺的交叉反应（CR）值为49.4%，维生素B1、B2、B3、B9和C的交叉反应值低于0.1%。方法比较结果显示，相关系数（R2）为0.91，存在-4.1%的轻微负偏差[95%一致性界限（± 1.96 SD）；-39.6%至31.6%]。
所开发的方法使用贝克曼库尔特仪器显示出高灵敏度和特异性以及良好的相关性，而整个检测仅需40分钟。它可以充分满足非常繁忙的临床实验室的需求。
