Validation of antibodies for the specific detection of human TRPA1.
Validation of antibodies for the specific detection of human TRPA1.
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验证用于特异性检测人 TRPA1 的抗体。
DOI：
10.1038/s41598-019-55133-7
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发表时间：
2019
影响因子：
4.6
通讯作者：
Virk HS
中科院分区：
文献类型：
--
作者：
Virk HS
瞬时受体电位阳离子通道家族成员锚蛋白1（TRPA 1）是几种疾病的潜在靶标，但由于缺乏严格的抗体验证，在细胞和组织中检测人TRPA 1（hTRPA 1）蛋白是有问题的。我们在TRPA 1阴性细胞系中表达hTRPA 1，通过Western印迹、免疫荧光、免疫细胞化学和流式细胞术评估5种市售抗体。三种最常引用的抗TRPA 1抗体缺乏灵敏度和/或特异性，但两种小鼠单克隆抗TRPA 1抗体在上述测定中特异性地检测hTRPA 1。这使得能够开发流式细胞术测定，其证明TRPA 1在人肺肌成纤维细胞、人气道平滑肌细胞而不是肺肥大细胞中的强表达。最常引用的抗TRPA 1抗体缺乏对hTRPA 1的敏感性和/或特异性。我们已经鉴定了两种特异性检测hTRPA 1的抗TRPA 1抗体。以前发表的关于人类TRPA 1蛋白表达的数据可能需要重新审视。
The transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.