Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology
Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology
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DOI：
10.1371/journal.pone.0192615
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发表时间：
2018-02-09
期刊：
影响因子：
3.7
通讯作者：
Haberland, Annekathrin
中科院分区：
文献类型：
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作者：
Wenzel, Katrin;Schulze-Rothe, Sarah;Haberland, Annekathrin
用于检测 β1-肾上腺素受体自身抗体 (β1-AAB) 的细胞分析是有效的，但难以处理，应被易于应用的常规实验室方法所取代。目前正在努力开发基于固相的检测方法，例如 ELISA，以利用表位部分捕获自身抗体。然而，与动物来源的自身抗体相比，这些基于固相的测定在用于人类患者材料时通常不可靠。因此，我们测试了基于免疫原肽的 ELISA 来检测 β1-AAB，并将市售的针对人 β1-肾上腺素受体第二胞外环 (ADRB1-AB) 的山羊抗体与从患者材料中富集的自身抗体进行了比较。在基于细胞的测定中测试了这些自身抗体的功能以进行比较，并使用 2D 凝胶电泳研究了它们的结构外观。 ELISA 显示，当掺入人对照血清中时，ADRB1-AB 的检测限约为 1.5 nmol 抗体/L，而当掺入物种相同（山羊）基质材料时，检测限仅为约 25 nmol/L。当应用于人类来源的样本时，ELISA 未能识别特定的 β1-AAB。低浓度的 β1-AAB，以及从 2D 凝胶外观可见的患者来源样品的结构不一致，可能导致基于肽的 ELISA 技术检测人类 β1-AAB 失败。
Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.