title: Generation of polyclonal antibodies directed against G protein-coupled receptors using electroporation-aided DNA immunization.
abstract: BACKGROUND
  The generation of antibodies against G protein-coupled receptors (GPCRs) can be technically challenging. A modified DNA immunization protocol was employed in order to generate polyclonal antibodies against two herpes virus-encoded GPCRs, i.e. Epstein-Barr virus (EBV) pBILF1 and rat cytomegalovirus (RCMV) pR78.
  
  
  METHODS
  pBILF1 and pR78 expression plasmids were first injected into the tibialis anterior muscle of rats and rabbits, respectively. Subsequently, the uptake of plasmids by the muscle cells was facilitated through in vivo electroporation.
  
  
  RESULTS
  Potent antisera against both vGPCRs were obtained, as determined by immunoblot analysis and immunofluorescence. By using the antisera, we were able to show that the EBV BILF1 protein is expressed as a 45-kD, glycosylated protein, and that it is localized in the cytoplasmic membrane of EBV-infected cells. Interestingly, we found the R78-encoded vGPCRs to have unusual perinuclear localization in both R78-transfected and RCMV-infected cells.
  
  
  CONCLUSIONS
  The in vivo DNA electroporation method is a useful technique for generating antibodies against GPCRs.
authors.affiliation: Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands.
authors.forename: Suzanne J F
authors.initials: SJ
authors.lastname: Kaptein
authors.forename: Joanne
authors.initials: J
authors.lastname: Jungscheleger-Russell
authors.forename: Pilar
authors.initials: P
authors.lastname: Martínez-Martínez
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Beisser
authors.forename: Hilde
authors.initials: H
authors.lastname: Lavreysen
authors.forename: Annelies
authors.initials: A
authors.lastname: Vanheel
authors.forename: Mark H
authors.initials: MH
authors.lastname: De Baets
authors.forename: Cathrien A
authors.initials: CA
authors.lastname: Bruggeman
authors.forename: Cornelis
authors.initials: C
authors.lastname: Vink
authors.forename: Mario
authors.initials: M
authors.lastname: Losen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D010957:Plasmids / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011917:Rats, Inbred Lew; D043562:Receptors, G-Protein-Coupled / Q000276:immunology*; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology
chemical_list: D000906:Antibodies; C495131:BILF1 protein, Epstein-Barr virus; C120868:R78 protein, cytomegalovirus; D043562:Receptors, G-Protein-Coupled; D019444:Vaccines, DNA; D014764:Viral Proteins