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Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1
Histochemistry and Cell Biology ( IF 2.1 ) Pub Date : 2021-08-27 , DOI: 10.1007/s00418-021-02025-5
Leyre Echeazarra 1, 2 , Gontzal García Del Caño 3, 4 , Sergio Barrondo 4, 5, 6 , Imanol González-Burguera 3, 4 , Miquel Saumell-Esnaola 4, 5 , Xabier Aretxabala 3 , Maider López de Jesús 4, 5 , Leire Borrega-Román 4, 5 , Susana Mato 7, 8, 9, 10 , Catherine Ledent 11 , Carlos Matute 7, 8, 9 , María Aranzazu Goicolea 12 , Joan Sallés 4, 5, 6
Affiliation  

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.



中文翻译:

针对大麻素受体 1 的氨基和羧基末端结构域的抗体的基于适合目的的测试和验证

特异性和选择性抗 CB 1抗体是揭示 CB 1受体在生理和病理条件下介导的复杂生物学过程的最强大的研究工具之一。然而,抗体的低性能仍然是不同实验室结果不一致的主要原因。使用多种技术,包括一些最普遍接受的抗体特异性测试技术,我们确定了针对 CB 1受体不同区域的五种商业抗体中的三种作为特定最终用途的最佳选择。具体来说,一种针对 CB 1胞外氨基尾长片段的抗体受体(但不是一种针对末端氨基末端的短序列)在应用于活细胞时检测到强烈的表面染色,而针对 CB 1受体末端羧基末端的相同片段的两种不同抗体(但没有一种针对上游肽)在所有平台上都表现出可接受的性能,尽管它们在免疫组织化学分析中表现不同,具体取决于所使用的组织固定程序,并且在蛋白质印迹分析中表现出不同的特异性,这使得它们中的每一个都特别适合其中一种技术。我们的结果提供了一个框架来解释在有关 CB 1的当前知识的背景下使用不同的抗 CB 1抗体得出的过去和未来结果分子水平上的受体,并强调需要对特定目的进行充分验证,不仅在抗体投放市场之前,而且在决定停止使用它们之前。

更新日期:2021-08-29
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