Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.
Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT-PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.
Coronella
J A
JA
Biotherapeutics Development Lab, Harvard Institute of Human Genetics, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Telleman
P
P
Truong
T D
TD
Ylera
F
F
Junghans
R P
RP
D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000276:immunology; D002469:Cell Separation; D017931:DNA Primers / Q000235:genetics; D004866:Equipment Contamination; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D009928:Organ Specificity; D014066:Palatine Tonsil / Q000276:immunology; D010950:Plasma Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013379:Substrate Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D012333:RNA, Messenger