Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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B-Lymphocytes / cytology
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B-Lymphocytes / immunology
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B-Lymphocytes / metabolism*
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Base Sequence
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Breast Neoplasms / immunology
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Cell Separation
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DNA Primers / genetics
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Equipment Contamination
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Female
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Flow Cytometry
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Humans
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Immunoglobulin Fab Fragments / genetics
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Immunoglobulin Fragments / genetics*
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Immunoglobulin G / genetics*
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Immunoglobulin Heavy Chains / genetics
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Immunoglobulin Variable Region / genetics
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Immunoglobulin kappa-Chains / genetics
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Immunoglobulin lambda-Chains / genetics
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Organ Specificity
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Palatine Tonsil / immunology
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Plasma Cells / cytology
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Plasma Cells / immunology
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Plasma Cells / metabolism*
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Polymerase Chain Reaction / methods*
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RNA, Messenger / analysis
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RNA, Messenger / genetics
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Reproducibility of Results
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Reverse Transcriptase Polymerase Chain Reaction
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Sensitivity and Specificity
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Sequence Alignment
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Substrate Specificity
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Time Factors
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Tumor Cells, Cultured
Substances
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DNA Primers
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Immunoglobulin Fab Fragments
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Immunoglobulin Fragments
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Immunoglobulin G
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Immunoglobulin Heavy Chains
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Immunoglobulin Variable Region
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Immunoglobulin kappa-Chains
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Immunoglobulin lambda-Chains
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RNA, Messenger