A luminescent oxygen channeling immunoassay for the determination of insulin in human plasma
A luminescent oxygen channeling immunoassay for the determination of insulin in human plasma
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DOI：
10.1177/1087057106297566
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发表时间：
2007-03-01
影响因子：
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通讯作者：
Jensen, Kirsten Borres
中科院分区：
文献类型：
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作者：
Poulsen, Fritz;Jensen, Kirsten Borres
作者描述了一种均匀、灵敏和快速的基于微珠的夹心免疫分析方法，具有广泛的分析范围，可以定量测定人血浆中的胰岛素。用生物素标记的抗胰岛素抗体与共价包被抗胰岛素抗体的小球孵育1h，然后与共价包被链霉亲和素的小球孵育30min，进行两步反应。在孵化步骤之后，对珠子内的化学发光反应产生的光进行定量。色谱柱为384孔板，进样量为5 mU L，分析范围为1~10000 pM。不同浓度胰岛素的批内精密度(%变异系数)在1.9%至3.8%之间。批间精密度为4.6%~7.3%。检测下限为0.3 pM。中度溶血(血红蛋白高达375毫克/分升)、胆红素(高至至少50毫克/分升)、甘油三酯(高至至少1000毫克/分升)、生物素(高达7.7毫克/毫升)或抗坏血酸(高达100毫克/分升)不会产生干扰。然而，严重的溶血确实影响了化验。血浆(乙二胺四乙酸、柠檬酸盐和肝素处理)和血清也得到了类似的结果。与酶联免疫吸附试验(EL ISA)相关性良好(y=1.25x+1.19，R2=0.98)。该方法简便，是一种替代ELISA法的方法。
The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for I h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 mu L. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dQ, biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R-2 = 0.98). This method is convenient and represents an alternative to ELISA.