Chen Xiang, Zhou Qiyang, Zhang Ting, Wang ChunXin, Yu Zheng, Ahamada Hadji, Bai Zhonghu, Huang Xuan
School of Biotechnology, Jiangnan University, Wuxi, China.
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, China.
J Anal Methods Chem. 2019 May 21;2019:5402903. doi: 10.1155/2019/5402903. eCollection 2019.
Folic acid (FA) is an important vitamin for human growth, especially for pregnant women. FA deficiency is associated with megaloblastic anemia, neural tube defects, cardiovascular diseases, irritability, diarrhea, and psychiatric disorders. Normally, FA molecules bind to folate-binding protein (FBP) in the serum as complex. Before quantify the FA concentration, a releasing procedure should be conducted. Alkaline condition and tris(2-carboxyethyl)phosphine (TCEP) are used to release binding FA to freeing state. In this work, a chemiluminescence immunoassay (CLIA) for human serum FA was established by competition model. Streptavidin (SA) was labeled to magnetic beads by an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDAC/NHS) method. Activated biotin molecules were labeled to FBP molecules purified from milk. FA was labeled to horseradish peroxidase (HRP) by EDAC to activate the FA molecules. The pretreated samples or standards were added into the reaction tube with biotin-FBP and FA-horseradish peroxidase (HRP), FA in the sample compete with FA-HRP for binding to biotin-FBP, the signal is inversely proportional to the FA concentration. The method established shows good thermostability and performance. The limitation of detection (LOD) is 0.44 ng/mL. The intra-assay coefficient of variation (CV) is 3.6%-7.1%, the interassay CV is 4.2%-7.5%, and the recovery rate is 92.1%-103.5%. Cross reactivity (CR) was remarkably low with aminopterin, folinic acid, and methotrexate. The method shows good correlation with the FA CLIA product from Beckman Coulter; the equation is = 0.9618-0.1434 while the value is 0.9224. The established method is sensitive, rapid, and accurate which can fully satisfy for the clinical requirement.
叶酸（FA）是人体生长尤其是孕妇所必需的一种重要维生素。叶酸缺乏与巨幼细胞贫血、神经管缺陷、心血管疾病、易怒、腹泻及精神障碍有关。正常情况下，FA分子以复合物形式与血清中的叶酸结合蛋白（FBP）结合。在定量FA浓度之前，应进行释放程序。碱性条件和三（2-羧乙基）膦（TCEP）用于将结合型FA释放为游离状态。在本研究中，通过竞争模型建立了一种用于检测人血清中FA的化学发光免疫分析法（CLIA）。采用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺/ N-羟基琥珀酰亚胺（EDAC / NHS）法将链霉亲和素（SA）标记到磁珠上。将活化的生物素分子标记到从牛奶中纯化得到的FBP分子上。通过EDAC将FA标记到辣根过氧化物酶（HRP）上以活化FA分子。将预处理后的样品或标准品加入含有生物素-FBP和FA-辣根过氧化物酶（HRP）的反应管中，样品中的FA与FA-HRP竞争结合生物素-FBP，信号与FA浓度成反比。所建立的方法具有良好的热稳定性和性能。检测限（LOD）为0.44 ng/mL。批内变异系数（CV）为3.6%-7.1%，批间CV为4.2%-7.5%，回收率为92.1%-103.5%。与氨蝶呤、亚叶酸和甲氨蝶呤的交叉反应率（CR）极低。该方法与贝克曼库尔特公司的FA CLIA产品具有良好的相关性；方程为 = 0.9618 - 0.1434， 值为0.9224。所建立的方法灵敏、快速且准确，能够充分满足临床需求。
