Single, antigen-specific B cells used to generate Fab fragments using CD40-mediated amplification or direct PCR cloning.
A simple procedure for the generation of human antibody fragments directly from single B cells or B-cell clones is described here. The procedure is based on antigen-specific selection of single human B cells, using antigen-coated magnetic beads and a cellular amplification step based on a culture system involving both EL-4 thymoma cells and anti-CD40 antibodies, presented by CD32-expressing fibroblasts. Nested PCR was applied to rescue V-regions from both single B cells and B-cell clones obtained using the cellular amplification step. This amplification step both increased the cell number as well as activated the cells that amplified mRNA levels, thereby facilitating immortalization by cloning. The V-regions were cloned and expressed as Fab fragments and characterized by biosensor analysis. This approach allowed us to bypass cumbersome hybridoma technology and to obtain human antibody fragments that retained the original VH/VL pairing, a feature of importance when studying, e.g., the V-gene usage in various human diseases and in normal B-cell repertoires.
Lagerkvist
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Lund University, Sweden.
Furebring
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Borrebaeck
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D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D015374:Biosensing Techniques; D019013:CD40 Antigens; D002455:Cell Division; D002469:Cell Separation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D018189:Immunomagnetic Separation; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; D019013:CD40 Antigens; D017931:DNA Primers; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger