Objective: This study is intended to screen potential antigen for Behcet's disease (BD) by using human microvascular endothelial cells (HUVEC). Methods: Following cell-based indirect immunofluorescence assay with sera from BD patients, proteins extracted from HUVEC were separated and detected by Western blotting. Then the target protein was identified by LC-MALDI-TOF/TOF, the recombinant target protein was expressed, purified and then used as coating antigen to test the prevalence of autoantibodies in patient's sera. Results: The Western blotting result showed that some patients' sera could react with a protein band with about 30 kDa of molecular weight, which was further identified as prohibitin by mass spectrometry. The prevalence of serum antibodies against recombinant human prohibitin was detected in 16 of 58 BD patients (28%) but none in healthy controls. (C) 2014 Elsevier Inc. All rights reserved.