An Innovative Method to Identify Autoantigens Expressed on the Endothelial Cell Surface: Serological Identification System for Autoantigens Using a Retroviral Vector and Flow Cytometry (SARF)

Figure 2

Identification of intercellular adhesion molecule 1 (ICAM-1) as a target antigen of anti-endothelial cell antibodies (AECAs). (a) Nonpermeabilized HUVECs were stained with 0.5 mg/mL of IgG of control or X10-3 from a patient with rheumatoid arthritis followed by secondary antibody and analyzed by flow cytometry. (b) HUVEC cDNA-expressing cells were stained with 0.5 mg/mL of X10-3 IgG followed by secondary antibody, and cells in the positive fraction were sorted (black box). (c) Unsorted and 4th sorted cells (left) and unsorted and cloned cells from 4th sorted cells, C5 (right), were stained with 0.5 mg/mL of X10-3 IgG followed by secondary antibody and analyzed by flow cytometry. (d) ICAM-1 cDNA fragments inserted into the genomic DNA of C5 were amplified, and PCR products were electrophoresed on an 0.8% agarose gel. (e) Unsorted and C5 were stained with isotype control or anti-ICAM-1 antibody, followed by secondary antibody and analyzed by flow cytometry. (f) Expression vector, empty-IRES-GFP, or ICAM-1-IRES-GFP were transfected into YB 2/0 cells, and these cells were stained with 0.5 mg/mL of control IgG or X10-3 IgG, followed by secondary antibody and analyzed by flow cytometry.