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Isolation and Characterization of Single Anti‐U1A‐specific B Cells from Autoimmune Patients
Author(s) -
WILDT RUUD M. T. DE,
HOOGEN FRANK H. J. VAN DEN,
VENROOIJ WALTHER J.,
HOET RENÉ M. A.
Publication year - 1997
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1997.tb52097.x
Subject(s) - annals , citation , medicine , philosophy , library science , classics , computer science , art
Patients with systemic lupus erythematosus (SLE) or SLE-overlap syndromes often produce autoantibodies directed to UlRNA-binding proteins.] Previously, we isolated and characterized autoantigen-binding Ab fragments directed to the U1 RNAassociated A protein (UlA) from several variable (V) gene combinatorial phage libraries.2 Although these libraries have proven to be very successful, heavy (VH) and light (V,) chains are randomly combined during construction of such libraries. Because we were interested in the utilization of the original VHNL pairings of the encoding autoantibodies, we developed a single cell culture system for B cells using mouse thymoma EL-4 B5 cells.3 In combination with a preselection via recombinant antigen and single cell sorting using fluorescence-activated cell sorting (FACS), we were able to enrich for autoantigen-specific B cells. VH and VL genes originating from single U1A-specific B cells were cloned in a phage display vector for expression of single-chain variable fragments (scFv). Human mononuclear cells were isolated from SLE patients and selected against U 1 A-coated plates or biotinylated U1 A coated to streptavidin-coupled superparamagnetic microbeads (MACS). Adhering cells were collected from the plates via trypsin treatment or were directly used in the case of U1A-bound MACS (FIGURE 1). The selected lymphocytes were plated as single CD 19/CDZO-positive cells using FACS with an automatic cell deposit unit. Single selected B cells were seeded on 96-well plates containing 20,000 irradiated EL4 B5 cells and 10% supernatant of phorbol myristate acetate (PMA)and phytohemagglutinin (PHA)-activated human T cells (FIGURE 2). Cultures were grown for 16-11 days and tested in ELISA for antigen (Ag)-specific antibodies and total Ig production. Cell cultures that were positive in the Ag-specific ELISA screening were used for RNA isolation. An RT reaction was performed with an oligo-dT primer and the cDNA was used in a first PCR with VH family-specific primers or a mixture of V-kappa or V-lambda primers (in the case of the light chains) as in Marks et aL4 These first PCR products were used in a second