Development of an enzyme-linked immunosorbent assay for the quantification of O-Phosphoethanolamine in human plasma
Development of an enzyme-linked immunosorbent assay for the quantification of O-Phosphoethanolamine in human plasma
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开发用于定量人血浆中 O-磷酸乙醇胺的酶联免疫吸附测定法
DOI：
10.1016/j.ab.2022.114952
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发表时间：
2022
影响因子：
2.9
通讯作者：
Chisato Shimanoe
中科院分区：
文献类型：
--
作者：
Tetsuya Saita;Hiroto Kataoka;Rintaro Sogawa;Tadashi Hayama;Ryoko Tomita;Akira Monji;Yoshito Mizoguchi;Chisato Shimanoe
O-磷酸乙醇胺（PEA）是一种内源性物质，作为抑郁症的生物标志物引起了人们的兴趣，因此需要开发一种专门测量PEA的简单分析方法。本研究以间马来酰亚胺苯甲酰-N-羟基磺基琥珀酰亚胺酯（MBS）为标记物，将巯基琥珀酰牛血清白蛋白（BSA）与PEA偶联，免疫小鼠，获得抗PEA抗体，建立一种简便、特异的PEA酶联免疫吸附试验（ELISA）。在该测定中，待定量的PEA通过苯甲酰氯化学修饰，使其与PEA-MBS-HRP缀合物竞争结合有限量的抗PEA抗体，所述抗PEA抗体用于包被微量滴定板的威尔斯孔。该ELISA的检测线性范围为0.11-27 μM，定量限为0.144 μM。抗PEA抗体对苯甲酰PEA显示出高亲和力。未发现与苯甲酰基2-氨基乙醇、O-磷酸-L-酪氨酸或苯甲酰基鞘氨醇-1-磷酸的可检测交叉反应性。通过该ELISA测量的血浆PEA水平的值与通过HPLC测量的值相当，并且在通过两种方法测定的值之间观察到强相关性。开发的ELISA应该提供一个有价值的新工具，PEA在人血浆中的定量。
O-Phosphoethanolamine (PEA) is an endogenous substance that is attracting interest as a biomarker for depression, and thus there is a need to develop a simple analytical method that specifically measures PEA. Therefore, this study aimed to develop a simple and specific enzyme-linked immunosorbent assay (ELISA) for PEA.Anti-PEA antibody was obtained by immunizing mice with an antigen conjugated with mercaptosuccinyl bovine serum albumin usingm-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (MBS). In this assay, the PEA to be quantified is chemically modified by benzoyl chloride that is allowed to compete with a PEA-MBS-HRP conjugate for binding to a limited amount of an anti-PEA antibody, which was used to coat the wells of a microtiter plate. This ELISA shows a linear range of detection of 0.11–27 μM, and a limit of quantification of 0.144 μM. The anti-PEA antibody showed high affinity for benzoyl PEA. No detectable cross-reactivity was found with benzoyl 2-aminoethanol,O-phospho-l-tyrosine or benzoyl sphingosine-1-phosphate. The values of plasma PEA levels measured by this ELISA were comparable to those measured by HPLC, and a strong correlation was observed between the values determined by the two methods. The developed ELISA should provide a valuable new tool for the quantification of PEA in human plasma.