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酶联免疫吸附测定(ELISA)中用于检测 G 蛋白偶联受体自身抗体的无抗原对照孔-正确数据评估的必要条件。

Antigen-free control wells in an ELISA set-up for the determination of autoantibodies against G protein-coupled receptors-a requisite for correct data evaluation.

机构信息

Berlin Cures GmbH, Robert-Rössle-Str. 10, 13125, Berlin, Germany.

出版信息

Anal Bioanal Chem. 2018 Aug;410(21):5101-5105. doi: 10.1007/s00216-018-1172-x. Epub 2018 Jun 11.

Abstract

First functional acting autoantibodies against G protein-coupled receptors such as the beta2-adrenoceptor in e.g. asthmatic patients have already been discovered in the early 1980s of the last century using assays that show their functional activity. Today, almost 40 years later, the measurement of such autoantibodies is still a challenge. Bioassays able to show the functional activity of such autoantibodies against G protein-coupled receptors are still the ne plus ultra for their detection and also classification when additionally exploiting specific receptor blockers for the neutralisation of the effect. Bioassays based on living cells make specific demands on the laboratories and are, therefore not suitable for every routine laboratory. Routine diagnostics, therefore, ideally requires different assays based on e.g. solid-phase technology, such as enzyme-linked immunosorbent assay (ELISA) technology. Here, endeavours are going on, using either the exact epitopes of such autoantibodies, if known, for trapping the autoantibodies, or the complete receptor in biological or artificial membranes that are immobilised onto a plastic carrier (ELISA principle). Here, we question and discuss the outcome of such tests, especially, if no controls such as the non-coated plastic carrier or the corresponding receptor-free membrane coat is offered as control in parallel, in light of the manifold experiences already collected with even non-agonistic acting autoantibodies by Güven et al. (J Immunol Methods 403:26-36, 2014).

摘要

早在上个世纪 80 年代初,人们就已经使用能够显示其功能活性的检测方法,发现了例如哮喘患者中β2-肾上腺素能受体等 G 蛋白偶联受体的具有功能活性的自身抗体。时至今日,近 40 年后,此类自身抗体的测量仍然是一个挑战。能够显示此类自身抗体针对 G 蛋白偶联受体的功能活性的生物测定法,仍然是检测此类自身抗体及其分类的终极方法,当同时利用特定的受体阻滞剂来中和其作用时更是如此。基于活细胞的生物测定法对实验室有特殊要求,因此不适合每个常规实验室。因此,常规诊断理想情况下需要基于固相技术的不同检测方法,例如酶联免疫吸附测定(ELISA)技术。目前,正在使用此类自身抗体的精确表位(如果已知的话)来捕获自身抗体,或者使用固定在塑料载体上的完整受体(ELISA 原理),进行此类努力。在这里,我们根据 Güven 等人已经积累的多种经验(J Immunol Methods 403:26-36, 2014),对这些测试的结果,特别是在没有提供非涂覆塑料载体或相应的无受体膜涂层作为对照的情况下,提出质疑并进行讨论。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97ea/6061717/e2496553afea/216_2018_1172_Fig1_HTML.jpg

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