Journal of Proteomics ( IF 2.8 ) Pub Date : 2020-06-10 , DOI: 10.1016/j.jprot.2020.103876 Cornelia A Deeg 1 , Roxane L Degroote 1 , Isabella M Giese 1 , Sieglinde Hirmer 1 , Barbara Amann 1 , Maria Weigand 1 , Carmen Wiedemann 1 , Stefanie M Hauck 2
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In life sciences, antibodies are among the most commonly used tools for identifying, tracking, quantifying and isolating molecules, mainly proteins. However, it has recently become clear that antibodies often fall short with respect to specificity and selectivity and in many cases target proteins are not even known. When commercial availability of antibodies is scarce, e.g. for targeting proteins from farm animals, researchers face additional challenges: they often have to rely on cross-reactive antibodies, which are poorly characterized for their exact target, their actual cross-reactivity and the desired application.
In this study, we aimed at identifying the true target of mouse monoclonal antibody 8F2, which was generated against chicken PBMC and used for decades in research, while it's actual target molecule remained unknown. We used 8F2 antibody for immunoprecipitation in chicken PBMC and subsequently identified its true target as CD11d, which was never described in chicken lymphocytes before, by quantitative LC-MSMS. The most abundant interactor of CD11d was identified as integrin beta 2.
The existence of this alpha integrin was therefore clearly proven on protein level and provides a first basis to further assess the role of CD11d in chickens in future studies.
Data are available via ProteomeXchange with identifier PXD017248.
Significance
Our studies determined CD11d as the true target of a previously uncharacterized mouse monoclonal antibody 8F2, generated against chicken peripheral blood derived mononuclear cells (PBMC). This is therefore now first member of alpha integrins in chickens, that existence was now clearly identified on protein level. The additional identification of CD11d interactors provides information on integrin-dependent regulation of signaling networks, allowing further functional studies.
中文翻译：
CD11d是鸡白细胞上的一种新型抗原。
在生命科学中，抗体是最常用于识别，跟踪，定量和分离分子（主要是蛋白质）的工具。然而，最近变得清楚的是，抗体通常在特异性和选择性方面不足，并且在许多情况下甚至不知道靶蛋白。当缺乏用于抗体的商业用途时，例如用于靶向来自家畜的蛋白质，研究人员面临额外的挑战：他们通常不得不依赖交叉反应抗体，而这些抗体的确切靶标，实际交叉反应性和所需的应用却缺乏特征。
在这项研究中，我们旨在鉴定小鼠单克隆抗体8F2的真正靶标，该抗体是针对鸡PBMC产生的，并在研究中使用了数十年，而其真正的靶分子仍然未知。我们使用8F2抗体在鸡PBMC中进行免疫沉淀，随后通过定量LC-MSMS将其真正的靶标定为CD11d，这在鸡淋巴细胞中从未描述过。CD11d最丰富的相互作用因子被鉴定为整合素beta 2。
因此，这种α整联蛋白的存在已在蛋白质水平上得到了明确证明，并为进一步评估CD11d在鸡中的作用提供了基础。
数据可通过ProteomeXchange获得，其标识符为PXD017248。
意义
我们的研究确定CD11d为先前未表征的小鼠单克隆抗体8F2的真正靶标，该抗体针对鸡外周血衍生的单核细胞（PBMC）。因此，这是鸡中α整联蛋白的第一个成员，现在已经在蛋白质水平上清楚地确定了其存在。CD11d相互作用物的额外鉴定提供了有关整合素依赖的信号网络调节的信息，从而可以进行进一步的功能研究。
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