Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning Journal Article
| Authors: | Tiller, T.; Meffre, E.; Yurasov, S.; Tsuiji, M.; Nussenzweig, M. C.; Wardemann, H. |
| Article Title: | Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning |
| Abstract: | We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire. © 2007 Elsevier B.V. All rights reserved. |
| Keywords: | controlled study; gene sequence; human cell; flow cytometry; cells, cultured; reverse transcription polymerase chain reaction; gene amplification; cell maturation; cell line; immunoglobulin; transfection; b lymphocyte; b-lymphocytes; genetic vectors; monoclonal antibody; molecular cloning; cloning, molecular; antibodies, monoclonal; immunoglobulin heavy chain; immunoglobulin heavy chains; molecular sequence data; immunoglobulin g; reverse transcriptase polymerase chain reaction; eukaryota; nucleotide sequence; recombinant proteins; cell isolation; base sequence; antibody specificity; immunoglobulin light chain; immunoglobulin light chains; fluorescence activated cell sorting; cell separation; antibody; antibody production; expression vector; cross reactions; polyreactivity; self-reactivity; single cell pcr |
| Journal Title: | Journal of Immunological Methods |
| Volume: | 329 |
| Issue: | 1-2 |
| ISSN: | 0022-1759 |
| Publisher: | Elsevier Science, Inc. |
| Date Published: | 2008-01-01 |
| Start Page: | 112 |
| End Page: | 124 |
| Language: | English |
| DOI: | 10.1016/j.jim.2007.09.017 |
| PUBMED: | 17996249 |
| PROVIDER: | scopus |
| PMCID: | PMC2243222 |
| DOI/URL: | |
| Notes: | Erratum/Corrigendum issued, see DOI: 10.1016/j.jim.2008.02.010 -- "Cited By (since 1996): 51" - "Export Date: 17 November 2011" - "CODEN: JIMMB" - "Molecular Sequence Numbers: GENBANK: DQ407610;" - "Source: Scopus" |
