Polysorp elisa plates

Polysorp ELISA plates (Nunc) were coated with ASFV recombinant proteins p30 and CP312R (50 μl per well) diluted (1–10 μg/ml) in coating buffer (50 mM sodium carbonate/bicarbonate buffer, pH 9.6) and incubated overnight at 4°C. The wells were then washed three times with PBS plus 0.05 % Tween 20 and blocked with PBS plus 5 % milk (200 μl per well) at 37°C for 1 h. After blocking, plates were washed five times as above and incubated for 1 h at 37°C with pig sera diluted 1:100 in PBS plus 5 % milk (50 μl per well). The plates were again washed five times and incubated with protein A–horseradish peroxidase (Pierce) diluted 1:5,000 (100 μl per well) for 1 h at 37 °C. Finally, plates were washed again and developed with 3-dimethylaminobenzoic acid/3-methyl-2-benzothiazolinone hydrazine hydrochloride/H₂O₂ dissolved in 0.1 M phosphate buffer. After stopping the reaction with 3 M H₂SO₄ (50 μl per well), the absorbance at 620 nm was read on a Cytation3 microplate reader (Biotek).

Experiments were performed as described (18 (link)). Briefly, recombinant mouse or human CR3 (1 μg/well, R&D Systems) were coated on 96-well MaxiSorp™ELISA plates (Nunc) overnight at 4°C. Purified, native PGL-I (BEI Resources, NIAID, NIH) was dissolved in ethanol After washing and blocking, specified concentrations of PGL-I diluted in binding buffer were added to the wells and incubated at 37°C for 1 h. Bound PGL-I was detected by an indirect method using an anti-PGL-I antibody (Ab SC-48, BEI Resources, NIAID, NIH), followed by addition of a secondary horseradish peroxidase (HRP)-coupled goat anti-mouse Ab (BioRad). HRP activity, corresponding to bound PGL-I, was determined by reading the absorbance at 450 nm. For competition assays, purified native PGL-I diluted in ethanol (500 ng/well) was added to 96-well PolySorp™ ELISA plates (Nunc). After evaporation, washing and blocking, recombinant mouse CR3 (500 ng/well) was incubated with 50 μM of synthetic oligosaccharide domains of PGL-b and PGL-I (18 (link)), for 1 h at 37°C. CR3 binding to PGL-I coated to the plates was performed as above and bound CR3 was detected by an indirect method using anti-CD11b Ab (2LPM19c, Santa Cruz Biotechnology).