Research paperEfficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning
Introduction
The diversity of the antibody repertoire is based on somatic recombination of variable (V), diversity (D) and joining (J) gene segments (Tonegawa, 1983). In humans, immunoglobulin (Ig) genes are randomly assembled from about 50 V, 25 D and 6 J gene segments for heavy (H) chains and over 30 potentially functional Vκ and Vλ light (L) chain genes and 5 Jκ and 4 Jλ genes, respectively (Ravetch et al., 1981, Hieter et al., 1982, Schable and Zachau, 1993, Corbett et al., 1997, Kawasaki et al., 1997, Matsuda et al., 1998). Basic understanding of the expressed human antibody repertoire at different stages during B cell development came from Ig gene analyses from cDNA libraries of bulk isolated human B cell subpopulations and single B cells of defined origin (Huang and Stollar, 1991, Huang et al., 1992, Shiokawa et al., 1999, Wang and Stollar, 2000). Other studies compared the Ig gene usage and Ig gene characteristics among distinct B cell subpopulations from healthy individuals and patients with autoimmune diseases and demonstrated that differences in the overall representation of individual Ig genes are part of the normal B cell repertoire (Stewart et al., 1993, Demaison et al., 1995, Pascual and Capra, 1995, Demaison et al., 1996, Dorner et al., 1998, Dorner et al., 1999, Hansen et al., 2000).
Analyses of Ig genes from single B cells on a genomic level gave further insight in the molecular basics of Ig gene recombination, allelic exclusion and selection events in the antibody repertoire (Ehlich et al., 1993, Kuppers et al., 1993, Brezinschek et al., 1995, Ghia et al., 1996, Brezinschek et al., 1997, Casellas et al., 2001). However, sequence analysis alone does not allow predictions on antibody reactivities. To date human antibodies of defined specificities were obtained in large by cloning Ig genes from hybridomas or Epstein–Barr virus (EBV) transformed human B cells. Obtaining these stable human B cell lines involves low efficiency and therefore highly selective procedures (Stahli et al., 1980, Aman et al., 1984, Redmond et al., 1986, Borrebaeck et al., 1988, Crain et al., 1989, Laffly and Sodoyer, 2005). Thus, they cannot be used to document the frequency of B cells with defined specificities or Ig gene usage in individual B cell populations. To overcome these limitations we developed a strategy to clone and express antibodies from single human B cells of defined origin (Wardemann et al., 2003). The recombinant antibodies can be generated in large amounts to provide sufficient material for testing in various assays (Wardemann et al., 2003, Meffre et al., 2004, Ng et al., 2004, Herve et al., 2005, Samuels et al., 2005, Yurasov et al., 2005, Tsuiji et al., 2006, Yurasov et al., 2006, Herve et al., 2007, Tiller et al., 2007).
Section snippets
Isolation of single human B cells by fluorescence activated cell sorting
Human samples were collected after signed informed consent in accordance with Institutional Review Board (IRB)-reviewed protocols. Mononuclear cells were isolated from peripheral venous blood or bone marrow after enrichment with RosetteSep® human B cell enrichment cocktail (Stemcell Technologies Inc.) and purified by Ficoll-Paque® (GE Healthcare) density gradient centrifugation according to the manufacturer's instructions. The RosetteSep® human B cell enrichment antibody cocktail (Stemcell
Results
To be able to determine the frequency of B cells expressing autoreactive antibodies at different stages of their development we established an efficient method to produce recombinant monoclonal antibodies from single human B cells (Fig. 1). Isolation of individual cells is performed by single cell FACS sorting and allows the cloning of antibodies from defined B cell populations from various sources even if the cells are represented at low frequency or if the absolute samples size is small.
The
Discussion
Hybridomas and EBV immortalized B cell lines are the most commonly used technologies to generate human monoclonal antibodies.
However, overall transformation efficiencies are extremely low (about 1–3%), largely depend on the maturation status of the cell and are frequently performed after stimulation (Stahli et al., 1980, Aman et al., 1984, Redmond et al., 1986, James and Bell, 1987, Borrebaeck et al., 1988, Crain et al., 1989). Furthermore, detailed Ig gene information depends on additional
Acknowledgements
We thank all members of the Nussenzweig and Wardemann laboratory for discussion and S. Boscardin for helpful comments on the manuscript. We are indebted to Patrick Wilson and Anne Schaefer for their help and Aaron B. Kantor for advice on cDNA synthesis from single cells. This work was supported by grants from the National Institutes of Health (to M.C. Nussenzweig, E. Meffre) and the Dana Foundation Human Immunology Program (to E. Meffre, H. Wardemann). S. Yurasov is a Charles H. Revson Fellow
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