title: The generation of monoclonal antibodies against human peroxisome proliferator-activated receptors (PPARs).
abstract: Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins. In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent. In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens. The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs). Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form. This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step.
authors.affiliation: Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Tanaka
authors.forename: Tetsu
authors.initials: T
authors.lastname: Takeno
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Uchiyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murakami
authors.forename: Hisahiko
authors.initials: H
authors.lastname: Yamashita
authors.forename: Akifumi
authors.initials: A
authors.lastname: Suzuki
authors.forename: Rie
authors.initials: R
authors.lastname: Aoi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Iwanari
authors.forename: Shu-Ying
authors.initials: SY
authors.lastname: Jiang
authors.forename: Makoto
authors.initials: M
authors.lastname: Naito
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tachibana
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Shulman
authors.forename: David J
authors.initials: DJ
authors.lastname: Mangelsdorf
authors.forename: Raphael
authors.initials: R
authors.lastname: Reiter
authors.forename: Johan
authors.initials: J
authors.lastname: Auwerx
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D017924:Nucleopolyhedroviruses / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D018411:Spodoptera; D014157:Transcription Factors / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors