title: An improved and robust DNA immunization method to develop antibodies against extracellular loops of multi-transmembrane proteins.
abstract: Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.
authors.forename: Meredith
authors.initials: M
authors.lastname: Hazen
authors.forename: Sunil
authors.initials: S
authors.lastname: Bhakta
authors.forename: Rajesh
authors.initials: R
authors.lastname: Vij
authors.forename: Steven
authors.initials: S
authors.lastname: Randle
authors.forename: Dara
authors.initials: D
authors.lastname: Kallop
authors.forename: Vicki
authors.initials: V
authors.lastname: Chiang
authors.forename: Isidro
authors.initials: I
authors.lastname: Hötzel
authors.forename: Bijay S
authors.initials: BS
authors.lastname: Jaiswal
authors.forename: Karen E
authors.initials: KE
authors.lastname: Ervin
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Robby M
authors.initials: RM
authors.lastname: Weimer
authors.forename: Paul
authors.initials: P
authors.lastname: Polakis
authors.forename: Richard H
authors.initials: RH
authors.lastname: Scheller
authors.forename: Jagath R
authors.initials: JR
authors.lastname: Junutula
authors.forename: Jo-Anne S
authors.initials: JA
authors.lastname: Hongo
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002460:Cell Line; D018076:DNA, Complementary / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D007114:Immunization; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D010957:Plasmids / Q000276:immunology / Q000494:pharmacology; D017433:Protein Structure, Secondary; D019444:Vaccines, DNA / Q000276:immunology / Q000494:pharmacology
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019444:Vaccines, DNA; C489540:Abcc4 protein, mouse