MPO-ANCA tested by ELISA
MPO-ANCA titer in serum samples was test by routine ELISA. In brief, plates were coated with MPO (M6908, Sigma-Aldrich, USA) 2ug/well at 4℃ overnight, then blocked with 3% skim milk and the serum samples (1:100 dilution) were added. Horseradish-peroxidase–conjugated anti-human Ig (IgG, IgM and IgA) (A18847, Invitrogen, USA) was used as secondary antibody (1:5,000 dilution) and reacted with substrate chromogenic agent for ten minutes. OD of 450nm wavelength was obtained by the same steps as ELISA above. OD value, which was more than mean plus 2 times standard deviation(SD) obtained in healthy control group, were taken as lower threshold for MPO-ANCA positivity in both groups.