Abstract
The development of phage-display technology and the construction of huge libraries of antibody (Ab) fragments have provided an unlimited source of binders to virtually any antigen (Ag) (1). However, it is unlikely that the heavy (VH) and light (VL) chains of the Abs obtained from these libraries resemble original in vivo pairings. In certain autoimmune diseases and immunological processes, such as B-cell tolerance, these VH and VL combinations can be of crucial importance. To be able to determine the original VH and VL combinations of Abs, we have set up a single B-cell culture system. This comprises the sorting of individual lymphocytes into culture wells using flow cytometry, a culture system to expand these cells (2) and polymerase chain reaction (PCR) amplification of their variable-region genes, thereby immortalizing the VH and VL regions from individual human B cells. The method relies on the clonal expansion of single B cells in which cell-cell interactions (CD40-CD40L), as well as soluble factors, have been shown to be essential. One advantage beyond conventional hybridoma technology is that this method circumvents laborious plating and screening; the advantage compared to phage-display technology is that original VH and VL pairings can be isolated. This system has been used to analyze VH and VL pairings of human immunoglobulin G+(IgG+) B cells of unknown specificity (3) and, combined with a selection on the Ag U1A, a frequent autoantigenic protein target in patients with systemic lupus erythematosus, to analyze pairings in Ag-specific B cells (4). The efficiency of the system makes it possible to analyze large numbers of B cells and should therefore allow rare B-cell activities to be studied.
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de Wildt, R.M., Hoet, R.M.A. (2002). 8 The Recovery of Immunoglobulin Sequences from Single Human B Cells by Clonal Expansion. In: O’Brien, P.M., Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology™, vol 178. Humana Press. https://doi.org/10.1385/1-59259-240-6:121
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DOI: https://doi.org/10.1385/1-59259-240-6:121
Publisher Name: Humana Press
Print ISBN: 978-0-89603-906-3
Online ISBN: 978-1-59259-240-1
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