Division of Gastroenterology and Nutrition, Children's Hospital Boston, Boston, MA, USA.
J Immunol Methods. 2011 Oct 28;373(1-2):192-9. doi: 10.1016/j.jim.2011.08.018. Epub 2011 Aug 28.
The aim of this study was to develop a standardized enzyme-linked immunosorbent assay (ELISA) for detection of human soluble Fc-epsilon-RI (sFcεRI), a serum isoform of the high affinity IgE receptor. A recombinant version of sFcεRI was produced in baculovirus and used as standard. ELISA plates were coated with anti-mouse IgG followed by incubation with the monoclonal capture antibody CRA1. This FcεRI-alpha-specific antibody binds to the stalk region of the protein and does not inhibit IgE-binding. After incubation with standards or serum samples, plates were incubated with chimeric IgE followed by detection with horseradish peroxidase conjugated anti-human IgE. Enzymatic activity was visualized with (3,3',5,5')-tetramethylbenzidine. Specificity was demonstrated by omission of capture or detection reagents. Units (U) of detection were established and the dynamic range of the assay was defined as 10-640 U/ml for a 1/5 serum dilution. Parameters of linearity (R(2)>0.999), matrix interference test (recovery of 70-110%), intra-assay variability (coefficient of variation (CV) <20%) and inter-assay variability (CV <20%) met acceptance criteria for immunoassay validation. Correlation analysis of serum units of sFcεRI measured with the new ELISA and serum IgE levels confirmed earlier published data describing a weak correlation of the two parameters in patients with elevated serum IgE while no correlation in patients with normal serum IgE or the total patient group was found. In summary, we established and validated a standardized ELISA for the detection of sFcεRI. This novel method now allows for comparative analysis of sFcεRI levels in health and disease.
本研究旨在开发一种用于检测人可溶性 Fc-epsilon-RI（sFcεRI）的标准化酶联免疫吸附测定（ELISA），sFcεRI 是高亲和力 IgE 受体的血清同工型。使用杆状病毒产生了 sFcεRI 的重组版本，并将其用作标准。ELISA 板用抗小鼠 IgG 包被，然后用单克隆捕获抗体 CRA1 孵育。这种 FcεRI-α特异性抗体结合蛋白的茎部区域，并且不抑制 IgE 结合。与标准品或血清样品孵育后，用嵌合 IgE 孵育平板，然后用辣根过氧化物酶偶联的抗人 IgE 检测。用（3,3',5,5'）-四甲基联苯胺可视化酶活性。通过省略捕获或检测试剂来证明特异性。建立了检测单位（U），并且测定的动态范围定义为 1/5 血清稀释度的 10-640 U/ml。线性参数（R(2)>0.999）、基质干扰测试（回收率为 70-110%）、内试验变异性（变异系数（CV）<20%）和间试验变异性（CV<20%）符合免疫测定验证的接受标准。用新 ELISA 测量的血清 sFcεRI 单位与血清 IgE 水平的相关分析证实了先前发表的数据，即在血清 IgE 升高的患者中，这两个参数之间存在弱相关性，而在血清 IgE 正常或总患者组中未发现相关性。总之，我们建立并验证了用于检测 sFcεRI 的标准化 ELISA。这种新方法现在允许在健康和疾病中比较分析 sFcεRI 水平。
