title: A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells.
abstract: The phage display approach has proven to be a major step forward in studies on the human autoimmune repertoire. However, it remains doubtful whether the heavy and light chains of the antibodies obtained from these libraries resemble original in vivo pairings. Here we describe a novel, simple method for the immortalization of the variable heavy and light chain regions originating from individual, nonboosted, autoantigen-specific human B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD40L) as well as soluble factors were shown to be essential. This B cell culture system combined with a selection on antigen (the U1A protein, a frequent autoantigenic target in patients with systemic lupus erythematosus) and single cell sorting resulted in the isolation of U1A-specific human B cells and the subsequent expression of an U1A-specific single chain variable fragment (scFv). Our method circumvents laborious plating and screening and has the advantage that original heavy/light chain pairings can be isolated. Due to the high growth efficiency of single cultured B cells (50-70% outgrowth) even rare B cell activities can be studied using this system.
authors.affiliation: Department of Immunology, Organon International B.V., Oss, The Netherlands.
authors.forename: R M
authors.initials: RM
authors.lastname: de Wildt
authors.forename: P G
authors.initials: PG
authors.lastname: Steenbakkers
authors.forename: A H
authors.initials: AH
authors.lastname: Pennings
authors.forename: F H
authors.initials: FH
authors.lastname: van den Hoogen
authors.forename: W J
authors.initials: WJ
authors.lastname: van Venrooij
authors.forename: R M
authors.initials: RM
authors.lastname: Hoet
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000096:biosynthesis*; D001323:Autoantibodies / Q000032:analysis / Q000096:biosynthesis; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology; D003001:Cloning, Molecular; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein