Epitope analysis of PPARγ monoclonal antibody Pγ48.34A and its application for screening PPARγ ligands

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Epitope analysis of PPARγ monoclonal antibody Pγ48.34A and its application for screening PPARγ ligands
Hae Sook Lee; Min Chul Cho; Tae Woong Baek; Yong Kyung Choe; J W Kim; J T Hong; P K Myung; S G Paik; Do Young Yoon
Bibliographic Citation
Journal of Immunological Methods, vol. 296, no. 1, pp. 125-134
Publication Year
Peroxisome proliferator-activated receptors (PPARs) are transcription factors that directly modulate gene expression by binding to specific ligands. It has been established that PPARγ ligands play an essential role in obesity, diabetes, and inflammation. Recently, a great deal of research has focused on the screening of PPARγ ligands. In this study, both a human peroxisome proliferator-activated receptors γ2 (PPARγ2) recombinant protein and a specific monoclonal antibody against PPARγ2 were produced in order to screen PPARγ ligands. Analysis of deletion mutants revealed that monoclonal anti-PPARγ antibody Pγ48.34A possesses an antigenic determinant in the N-terminal region (31-84 a.a) of human PPARγ2. The results of Western blot testing revealed that Pγ48.34A recognized both glutathione S-transferase (GST)- and his-tagged human and mouse PPARγ recombinant proteins and also identified PPARγ in adipocytes and mouse tissues. Compared to some commercially available antibodies, this antibody does not bind with skimmed milk or BSA and exhibits a higher degree of specificity. An in vitro binding assay revealed that PPARγ2 was bound to steroid receptor coactivator-1 (SRC-1) in a dose-responsive manner in the presence of indomethacin, and Pr48.34A was able to detect PPARγ in a complex consisting of PPARγ and SRC-1. Using Pγ48.34A antibody, an enzyme-linked immunosorbent assay (ELISA) system based on the binding between fPPARγ2 and SRC-1 has been optimized to screen new PPARγ ligands. This new antibody, Pγ48.34A, exhibits higher degrees of both specificity and sensitivity against PPARγ than do other commercial anti-PPARγ antibody, and may constitute a profound contribution to the screening of PPARγ ligands as well as the functional study of PPARγ.
ELISAMonoclonal antibodyPPARγSRC-1
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