Tetramethylbenzidine peroxidasesubstrate

Micro titer plates were coated with lipopolysaccharide (Salmonella typhosa LPS, Sigma) (2μg/well) in PBS (phosphate buffered saline) overnight at 4°C. After washing the plated wells with PBST (phosphate buffered saline and 0.05% tween) for 3 times, wells were treated with a blocking buffer (1% BSA in PBS) for 1hr at RT. Normal human serum (NHS) diluted to 50% with GVB⁺⁺ buffer (Sigma) supplemented with Mg⁺⁺(5mM)-EGTA(20mM), with or without pre-incubation with anti-P mAbs, were then added to the plated wells (50 μl/well). NHS samples diluted in the same way but containing EDTA (20mM) were used as positive controls of complete inhibition of AP complement activation. Pre-incubation of NHS with anti-human P mAbs was performed at 4°C for 1hr. AP complement activation in plated wells was allowed to proceed for 1 hr at 37°C, and reaction was stopped by addition of cold 10 mM EDTA in PBS (100 μl/well). After washing for 3 times with PBS-T, plated wells were incubated with a HRP-conjugated goat anti-human C3 polyclonal antibody (MP Biomedicals, Cat # 0855237)(1:4000 diluted in blocking buffer) for 1hr at room temperature. Wells were washed 3 times with PBS-T and developed with HRP substrate (100 ml tetramethylbenzidine peroxidasesubstrate (BD Pharmingen, San Jose, CA). After 5 min, reaction was stopped with 2N H₂SO₄ and plated wells were read at 450 nm in a micro plate reader.