Epitope analysis of PPARgamma monoclonal antibody Pgamma48.34A and its application for screening PPARgamma ligands.
Peroxisome proliferator-activated receptors (PPARs) are transcription factors that directly modulate gene expression by binding to specific ligands. It has been established that PPARgamma ligands play an essential role in obesity, diabetes, and inflammation. Recently, a great deal of research has focused on the screening of PPARgamma ligands. In this study, both a human peroxisome proliferator-activated receptors gamma2 (PPARgamma2) recombinant protein and a specific monoclonal antibody against PPARgamma2 were produced in order to screen PPARgamma ligands. Analysis of deletion mutants revealed that monoclonal anti-PPARgamma antibody Pgamma48.34A possesses an antigenic determinant in the N-terminal region (31-84 a.a) of human PPARgamma2. The results of Western blot testing revealed that Pgamma48.34A recognized both glutathione S-transferase (GST)- and his-tagged human and mouse PPARgamma recombinant proteins and also identified PPARgamma 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 PPARgamma2 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 PPARgamma in a complex consisting of PPARgamma and SRC-1. Using Pgamma48.34A antibody, an enzyme-linked immunosorbent assay (ELISA) system based on the binding between fPPARgamma2 and SRC-1 has been optimized to screen new PPARgamma ligands. This new antibody, Pgamma48.34A, exhibits higher degrees of both specificity and sensitivity against PPARgamma than do other commercial anti-PPARgamma antibody, and may constitute a profound contribution to the screening of PPARgamma ligands as well as the functional study of PPARgamma.
Lee
Hae-Sook
HS
Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong P.O. Box 115, Daejeon 305-600, South Korea.
Cho
Min-Chul
MC
Baek
Tae-Woong
TW
Choe
Yong-Kyung
YK
Kim
Jong-Wan
JW
Hong
Jin-Tae
JT
Myung
Pyung-Keun
PK
Paik
Sang-Gi
SG
Yoon
Do-Young
DY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D000941:Antigens / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D051548:Histone Acetyltransferases; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D056920:Nuclear Receptor Coactivator 1; D047495:PPAR gamma / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D008024:Ligands; D047495:PPAR gamma; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1; D007213:Indomethacin