title: Production of monoclonal antibodies using recombinant baculovirus displaying gp64-fusion proteins.
abstract: Generation of protein immunogens is often a rate-limiting step in the production of monoclonal antibodies (Mabs). Expressing domains of proteins as fusions to the baculovirus surface glycoprotein gp64 displays foreign proteins on the surface of the virion. Antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of the gp64 nucleotide sequence. This method allows immunization with whole virus, eliminating the need for purification of target antigens. Affinity-matured Mabs to the human nuclear receptors LXRbeta and FXR have been produced using baculovirus particles displaying gp64/nuclear receptor fusion proteins as the immunizing agent. Immunizations were performed directly with pelleted virus using the Repetitive Immunization Multiple Sites (RIMMS) immunization strategy for rapid Mab production. All Mabs were identified using insect cells infected with the immunizing virus. Characterization of these antibodies shows them to be class-switched and specific for LXRbeta or FXR. Additionally, high affinity antibodies that recognize gp64 and neutralize baculovirus infection of insect cells were isolated. Use of the recombinant baculovirus gp64 display system makes possible the production of Mabs once a partial DNA sequence is known. This allows the generation of antibodies prior to the isolation of purified protein, in turn providing antibodies to facilitate purification, characterization and immunolocalization of proteins.
authors.affiliation: Department of Molecular Sciences, Glaxo Wellcome Research and Development, Research Triangle Park, NC 27709, USA. kml48731@glaxowellcome.com
authors.forename: K M
authors.initials: KM
authors.lastname: Lindley
authors.forename: J L
authors.initials: JL
authors.lastname: Su
authors.forename: P K
authors.initials: PK
authors.lastname: Hodges
authors.forename: G B
authors.initials: GB
authors.lastname: Wisely
authors.forename: R K
authors.initials: RK
authors.lastname: Bledsoe
authors.forename: J P
authors.initials: JP
authors.lastname: Condreay
authors.forename: D A
authors.initials: DA
authors.lastname: Winegar
authors.forename: J T
authors.initials: JT
authors.lastname: Hutchins
authors.forename: T A
authors.initials: TA
authors.lastname: Kost
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D000071518:Liver X Receptors; D051379:Mice; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology*; D014760:Viral Fusion Proteins / Q000235:genetics / Q000276:immunology*; D000099150:Receptor, Farnesoid X-Activated
chemical_list: D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014760:Viral Fusion Proteins; D000099150:Receptor, Farnesoid X-Activated