ELISA for detecting anti-properdin autoantibodies
ELISA plate (Greiner bio-one®) were coated with either 20 µg/ml of test
antigens – human Properdin (Complement Technology, Ins) in sodium
carbonate buffer (35mM NaHCO3, 15mM
Na2CO3, pH 9.6) for overnight at
4◦C. Blocking of the plates was done by 1% BSA in PBS
for 1h at 37◦C and washed three times with PBS
containing 0.05% Tween-20. Plasmas were diluted 1/100 in PBS-0.05%
Tween 20. After washing, HRP-conjugated anti-human IgG (Southern
Biotech) was applied in 1/1000 dilution in PBS-0.05% Tween 20. After
washing three times, the color was developed with 0.5 mg/ml
o-phenylenediamine (OPD) (Thermo, Scientific). The reaction was stopped
with 2N H2SO4 and absorbance at 490 nm
was measured using an ELISA plate Reader – Synergy 2.
Alternatively, plasma samples were serially diluted starting from 1/50
and applied on coated and blocked plates to evaluate the dose-response
of the binding of the anti-properdin IgG to their antigen.
A sample was considered positive if its optical density exceeded the
average of the optical density of the samples of the healthy volunteers
+ 3SD.