LAUSR

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.