Subcloning of autoantigen-expressing cells by using IgG from a patient with lupus nephritis. (A) Nonpermeabilized HUVECs were stained with 1:10 diluted sera of control or E10-19 from a lupus nephritis patient (left), and 0.5 mg/ml of IgG of control or E10-19 collected before (pre) or after (post) the treatments (right) followed by secondary antibody and analyzed with flow cytometry. (B) HUVEC cDNA-expressing cells were stained with 0.5 mg/ml of E10-19 IgG followed by secondary antibody, and cells in the positive fraction were sorted (black dotted box). Left indicates first sorting, and right indicates second sorting. (C) Unsorted and second-sorted cells (left), and unsorted and two clones from second-sorted cells, C9 and C18, respectively (right), were stained with 0.5 mg/ml of E10-19 IgG followed by secondary antibody, and analyzed with flow cytometry. (D) HUVEC cDNA fragments inserted into the genomic DNA of C9 and C18 were amplified, and PCR products were electrophoresed on an 0.8% agarose gel.
Shirai et al. Arthritis Research & Therapy 2012 14:R157 doi:10.1186/ar3897