The generation of monoclonal antibodies against human peroxisome proliferator-activated receptors (PPARs).
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.
Tanaka
Toshiya
T
Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The Tokyo University, Tokyo, Japan.
Takeno
Tetsu
T
Watanabe
Yuichiro
Y
Uchiyama
Yasutoshi
Y
Murakami
Takeshi
T
Yamashita
Hisahiko
H
Suzuki
Akifumi
A
Aoi
Rie
R
Iwanari
Hiroko
H
Jiang
Shu-Ying
SY
Naito
Makoto
M
Tachibana
Keisuke
K
Doi
Takefumi
T
Shulman
Andrew I
AI
Mangelsdorf
David J
DJ
Reiter
Raphael
R
Auwerx
Johan
J
Hamakubo
Takao
T
Kodama
Tatsuhiko
T
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*
D000911:Antibodies, Monoclonal; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors