title: Enhancement of antibody responses to native G protein-coupled receptors using E. coli GroEL as a molecular adjuvant in DNA immunization.
abstract: Antibody-based drug research involves the preparation of polyclonal and monoclonal antibodies, especially those that are reactive with native G protein-coupled receptors (GPCRs) on the cell membrane. Here, we report that DNA immunization of mice with a plasmid that encodes endothelin A receptor (ETAR) fused to Escherichia coli (E. coli) GroEL at its C-terminus (ETAR-GroEL) induced very strong and specific antibody responses to native ETAR. Co-injection of plasmids that expressed ETAR and GroEL (ETAR+GroEL) induced significantly lower antibody responses compared with the ETAR-GroEL plasmid. Monoclonal antibodies that are prepared by using GroEL as a molecular adjuvant could be used in immunoassays, such as flow cytometry, western blotting, and immunoprecipitation, to detect both exogenous and endogenous ETAR. The adjuvant activity of GroEL might involve inflammatory cytokine mediators via Toll-like receptor 4 in addition to the anticipated carrier effect. DNA immunization using GroEL might become a standard method for producing antibodies that are useful for the functional analysis of GPCRs.
authors: Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan. f_akira@rs.noda.tus.ac.jp
authors: Akira
authors: A
authors: Fujimoto
authors: Nanae
authors: N
authors: Kosaka
authors: Hikaru
authors: H
authors: Hasegawa
authors: Hiroshi
authors: H
authors: Suzuki
authors: Sumio
authors: S
authors: Sugano
authors: Joe
authors: J
authors: Chiba
mesh_terms: D000276:Adjuvants, Immunologic / Q000378:metabolism*; D000818:Animals; D000917:Antibody Formation / Q000235:genetics / Q000276:immunology*; D016207:Cytokines / Q000276:immunology; D004247:DNA / Q000235:genetics / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology*; D005260:Female; D057809:HEK293 Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D010957:Plasmids / Q000235:genetics; D044022:Receptor, Endothelin A / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
chemical_list: D000276:Adjuvants, Immunologic; D016207:Cytokines; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D044022:Receptor, Endothelin A; D011993:Recombinant Fusion Proteins; D051197:Toll-Like Receptor 4; D019444:Vaccines, DNA; D004247:DNA