Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Export

@article{citeulike:2289784, 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.}, address = {Max Planck Institute for Infection Biology, D-10117 Berlin, Germany.}, author = {Tiller, T. and Meffre, E. and Yurasov, S. and Tsuiji, M. and Nussenzweig, M. C. and Wardemann, H.}, citeulike-article-id = {2289784}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.jim.2007.09.017}, citeulike-linkout-1 = {http://linkinghub.elsevier.com/retrieve/pii/S0022175907003122}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/17996249}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=17996249}, day = {1}, doi = {10.1016/j.jim.2007.09.017}, issn = {0022-1759}, journal = {J Immunol Methods}, keywords = {bcell, cell, cloning, mab, single}, month = {January}, number = {1-2}, pages = {112--124}, posted-at = {2008-05-17 20:39:56}, priority = {2}, title = {Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.}, url = {http://dx.doi.org/10.1016/j.jim.2007.09.017}, volume = {329}, year = {2008} }

TY - JOUR ID - citeulike:2289784 L3 - citeulike-article-id:2289784 N2 - 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. IS - 1-2 JF - J Immunol Methods SN - 0022-1759 AD - Max Planck Institute for Infection Biology, D-10117 Berlin, Germany. EP - 124 TI - Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. VL - 329 SP - 112 KW - bcell KW - cell KW - cloning KW - mab KW - single AU - Tiller, T AU - Meffre, E AU - Yurasov, S AU - Tsuiji, M AU - Nussenzweig, MC AU - Wardemann, H PY - 2008/01/01/ UR - http://dx.doi.org/10.1016/j.jim.2007.09.017 ER -