Azino bis 3 ethylbenthiazoline 6 sulfonic acid abts liquid substrate

MaxiSorp 96-well flat-bottomed plates were coated with 50 μl of 125 nM recombinant human or mouse ACE2 protein in PBS at room temperature for 1 h. All washes were performed three times using PBS and 0.1% Tween-20 and all incubations were performed for 1 h at room temperature. Coated plates were washed and blocked by incubation with 4% skim milk solution. Plates were washed and then incubated with 50 μl of 125 nM of anti-ACE2 monoclonal antibodies. The plates were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG secondary antibodies (ab6858, Abcam, 1:5,000). After a final wash, 50 μl of azino-bis-3-ethylbenthiazoline-6-sulfonic acid (ABTS liquid substrate; Sigma-Aldrich) was added and incubated in the dark at room temperature for 20 min and 50 μl of 1% SDS was used to stop the reaction. Absorbance was read at 405 nm and all samples were done in duplicate.

96-well flat-bottomed plates (Maxisorp, Nunc) were coated with each of the 6 PvRBPs (65 nM/well) in individual wells and incubated for two hours. For 65 nM of protein in 100 μL, we added 0.8 μg, 0.9 μg, 0.7 μg, 1 μg, 0.6 μg and 0.4 μg for PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, PvRBP2cNB and PvRBP2_P2 respectively. Plates were blocked with 5% skim milk/0.1% Tween-20 for one hour. After washing, specific anti-PvRBP polyclonal antibodies (1 mg/mL stock) were added at halving serial dilutions (from 1:2000 to 1:64000) for one hour. Plates were washed three times before the addition of HRP-goat anti-rabbit secondary antibodies (1:2000 dilution) for one hour. Azino-bis-3-ethylbenthiazoline-6-sulfonic acid (ABTS liquid substrate; Sigma-Aldrich) was used to detect HRP activity. 1% SDS was used to stop the reaction and absorbance was measured at 405 nm. All experiments were performed at room temperature. All washes were done in PBS/0.1% Tween-20, and dilutions of antibodies in 0.5% skim milk/0.1% Tween-20. Samples were tested in duplicates.