title: DNA immunization followed by a single boost with cells: a protein-free immunization protocol for production of monoclonal antibodies against the native form of membrane proteins.
abstract: Recent advancements in antibody-based therapies require the development of an efficient method for generation of monoclonal antibodies (MAbs) against the native form of membrane proteins. We examined DNA immunization followed by a single boost with cells as a protein-free immunization protocol for production of MAbs. Mice immunized with plasmid cDNAs encoding human CD30 or Ret tyrosine kinase were given a single boost with cells expressing the corresponding antigen prior to cell fusion. A total of nine cell fusion experiments revealed that the cell boost is necessary for efficient generation of hybridomas and the DNA-cell boost method gave good yields of specific MAbs (5-59 MAbs from one mouse). All IgG isotypes except IgG3 were generated, although IgG2a was the dominant isotype. All the MAbs reacted with native antigens expressed on cells in a fluorescence-activated cell sorter (FACS) analysis as well as with recombinant CD30 or Ret protein genetically fused with human Fc in an enzyme-linked immunosorbent assay (ELISA). The affinities of the anti-CD30 MAbs to CD30-Fc protein ranged from 0.9 to 12.4 nM Kds, which were comparable to existing MAbs to these proteins, which range from 3.0 to 13.0 nM. Western blot analysis and topographical epitope mapping experiments based on the mutual competition of pairs of the anti-CD30 MAbs revealed that about 40% of the epitopes were linear epitopes and that each epitope was topographically classified into one of six groups. The large number of MAbs that react with high affinities to a variety of epitopes on the native form of antigens indicates that the method presented in this paper could be generally useful for generating MAbs to other membrane proteins.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr, Rm 5106, Bethesda, MD 20892-4264, USA.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagata
authors.forename: Giuliana
authors.initials: G
authors.lastname: Salvatore
authors.forename: Ira
authors.initials: I
authors.lastname: Pastan
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization / Q000379:methods*; D007117:Immunization, Secondary; D017730:Ki-1 Antigen / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology; D019444:Vaccines, DNA / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D017730:Ki-1 Antigen; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019444:Vaccines, DNA; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C494999:Ret protein, mouse