Generation of polyclonal antibodies directed against G protein-coupled receptors using electroporation-aided DNA immunization.
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.
Kaptein
Suzanne J F
SJ
Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands.
Jungscheleger-Russell
Joanne
J
Martínez-Martínez
Pilar
P
Beisser
Patrick S
PS
Lavreysen
Hilde
H
Vanheel
Annelies
A
De Baets
Mark H
MH
Bruggeman
Cathrien A
CA
Vink
Cornelis
C
Losen
Mario
M
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
D000906:Antibodies; C495131:BILF1 protein, Epstein-Barr virus; C120868:R78 protein, cytomegalovirus; D043562:Receptors, G-Protein-Coupled; D019444:Vaccines, DNA; D014764:Viral Proteins