title: Production of recombinant Ig molecules from antigen-selected single B cells and restricted usage of Ig-gene segments by anti-D antibodies.
abstract: The Ig-genes of the heavy chains in anti-D-specific hybridomas and Fab/scFv-fragments selected from phage-display libraries are restricted to a group of closely related genes (IGHV3s genes). We analyzed the Ig-gene repertoire in anti-D-specific B cells of two hyperimmunized donors using a completely different method. Single B cells were cultured for 10 days in an EL4.B5 culture system. mRNA from anti-D-producing B cells was reverse transcribed into cDNA. Heavy- and light-chain gene rearrangements were amplified by PCR reactions, sequenced and cloned into a pNUT-vector system, thereby allowing the production of complete IgG and IgM. Eleven anti-D-specific B-cell clones were isolated and analyzed. Eight of these clones (including IgM-producing clones) had IGHV3s genes. We demonstrated that functional anti-D-specific IgM (4 clones) and IgG (2 clones) was produced. Using a new method, we analyzed the IGHV gene repertoire of anti-D-specific B cells of hyperimmunized donors and showed that it is indeed restricted. Moreover, we found a high frequency (1:100 and 1:500) of anti-D-specific B cells in the peripheral B cells of hyperimmunized donors. We suggest that this approach could be applied for the selection of human mAbs from immunized donors and for the analysis of Ig-gene repertoires at the single-B cell level.
authors.affiliation: Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Serge E
authors.initials: SE
authors.lastname: Dohmen
authors.forename: Arend
authors.initials: A
authors.lastname: Mulder
authors.forename: Onno J H M
authors.initials: OJ
authors.lastname: Verhagen
authors.forename: Chantal
authors.initials: C
authors.lastname: Eijsink
authors.forename: Marry E I
authors.initials: ME
authors.lastname: Franke-van Dijk
authors.forename: C Ellen
authors.initials: CE
authors.lastname: van der Schoot
mesh_terms: D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D002999:Clone Cells; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D007118:Immunoassay / Q000379:methods*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007518:Isoantibodies / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018029:Rho(D) Immune Globulin
chemical_list: D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007518:Isoantibodies; C061961:RHO(D) antibody; D011994:Recombinant Proteins; D018029:Rho(D) Immune Globulin