Detecting Autoantibodies via ELISA

Autoantibodies were detected using ELISA method, as described by Józsi and Uzonyi (36 (link)). Briefly, FH, FB, C3b, FI in 5 µg/ml, MBL and C1q in 2 µg/ml and HSA or alpha-1 antitrypsin as negative controls were immobilized on microtiter plates. After blocking with 5% BSA in DPBS-0.1% Tween-20, serum samples were added in DPBS-0.1% Tween-20 at a dilution of 1:50 for 1 hour at RT. To detect autoantibodies against solid phase C3 convertase of the alternative pathway (C3bBbP), the convertase was built up (see below) and incubated with serum samples diluted 1:50. The bound autoantibodies were detected with HRP-conjugated goat anti-human IgG, anti-human IgA or anti-human IgM. Color reaction was developed by adding TMB solution (BioLegend). After stopping with H₂SO₄, absorbance was measured at 450 nm and at 620 nm as reference wavelength. To detect anti-MBL autoantibodies, serum samples were diluted in DPBS containing 10 mM EDTA. In the case of detecting anti-C1q autoantibodies, serum samples were diluted in DPBS containing 1 M NaCl to avoid interaction of C1q globular heads with IgG Fc parts. Samples were accepted as positive if the mean OD value for the investigated protein was at least two-fold greater than the mean OD value for the negative control protein alpha-1 antitrypsin. The isotypes of the autoantibodies were determined using monoclonal antibodies specific for IgG1, IgG2, IgG3, IgG4, IgGκ and IgGλ.