title: Design and characterization of a plasmid vector system capable of rapid generation of antibodies of multiple isotypes and specificities.
abstract: We present a detailed method for constructing a vector system composed of plasmids encoding immunoglobulin genes that have been constructed in such a fashion so as to allow for the generation and secretion of antibodies of multiple specificities and isotypes via a rapid and easy cloning-and-ligation scheme. Restriction sites within each plasmid allow for the removal of variable domains, constant domains, leader sequences, or the entire immunoglobulin gene. Degenerate primers are used to clone variable regions from hybridoma cDNAs, allowing for the creation of antibodies with varying binding specificities. Sequence-specific primers are used to clone antibody constant domains, allowing for the creation of antibodies of multiple isotypes from a variety of lineages. A high-efficiency leader sequence has been inserted into the start of each gene to improve secretion. Antibodies constructed through this system are efficiently secreted by transfected cells, and are fully functional in antigen-specific binding assays.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 422 Curie Blvd, 505 Stellar Chance Labs, Philadelphia, PA 19104, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Morrow
authors.forename: Panyupa
authors.initials: P
authors.lastname: Pankhong
authors.forename: David B
authors.initials: DB
authors.lastname: Weiner
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology; D001709:Biotechnology / Q000379:methods*; D060908:CTLA-4 Antigen; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007132:Immunoglobulin Isotypes / Q000276:immunology / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C556707:Ctla4 protein, mouse; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins