title: A tool kit for rapid cloning and expression of recombinant antibodies.
abstract: Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.
authors: 1] NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, London, UK [2] Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Tihomir S
authors: TS
authors: Dodev
authors: 1] NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, London, UK [2] St. John's Institute of Dermatology, Division of Genetics and Molecular Medicine, King's College London School of Medicine, King's College London, London SE1 9RT, UK.
authors: Panagiotis
authors: P
authors: Karagiannis
authors: 1] NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, London, UK [2] St. John's Institute of Dermatology, Division of Genetics and Molecular Medicine, King's College London School of Medicine, King's College London, London SE1 9RT, UK.
authors: Amy E
authors: AE
authors: Gilbert
authors: 1] NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, London, UK [2] St. John's Institute of Dermatology, Division of Genetics and Molecular Medicine, King's College London School of Medicine, King's College London, London SE1 9RT, UK [3] Division of Cancer Studies, King's College London, 3rd Floor Bermondsey Wing, Guy's Hospital, London SE1 9RT, UK.
authors: Debra H
authors: DH
authors: Josephs
authors: 1] NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, London, UK [2] Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Holly
authors: H
authors: Bowen
authors: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Louisa K
authors: LK
authors: James
authors: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Heather J
authors: HJ
authors: Bax
authors: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Rebecca
authors: R
authors: Beavil
authors: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Marie O
authors: MO
authors: Pang
authors: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Hannah J
authors: HJ
authors: Gould
authors: 1] NIHR Biomedical Research Centre at Guy's and St. Thomas's Hospitals and King's College London, London, UK [2] St. John's Institute of Dermatology, Division of Genetics and Molecular Medicine, King's College London School of Medicine, King's College London, London SE1 9RT, UK.
authors: Sophia N
authors: SN
authors: Karagiannis
authors: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors: Andrew J
authors: AJ
authors: Beavil
mesh_terms: D000818:Animals; D052179:Antigens, Plant / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000138:chemical synthesis; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E / Q000096:biosynthesis* / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D052179:Antigens, Plant; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D000939:Epitopes; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; C468525:Phl p 7 allergen; D010940:Plant Proteins; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E