Biotinylated cyclic V2 peptides from CAP45 (JPT peptides) were captured onto Streptavidin coated ELISA plates at a concentration of 2 ng/mL at 37°C for one hour. Plates were blocked with 5% milk and washed in PBS (0.05% tween20) before being probed at 37°C for one hour with serial dilutions of sample antibodies, starting at 10 mg/mL. After a subsequent washing step, HRP-conjugated goat anti-human IgG (H+L) polyclonal antibodies (Sigma-Aldrich) diluted to 1:5,000 were added to the plate and incubated for one hour at 37°C. Bound antibodies were detected using TMB substrate, and the reactions were stopped by the addition of 1 M H2SO4. Absorbance was read at 450 nm.
The CAP228 V1V2–1FD6 scaffolds were coated at 5 mg/mL onto high protein-binding microplates overnight at 4°C. Plates were blocked and washed as above, and probed with plasma serially diluted from 1:100, or mAbs from 10 mg/mL, for one hour at 37°C. Following washing, bound antibodies were detected with HRP-conjugated anti-human antibodies (Sigma-Aldrich) at a concentration of 1:1000 for one hour at 37°C, followed by TMB substrate stopped with 1 M H2S04. Absorbance was read at 450 nm.
The CAP228 V1V2–1FD6 scaffolds were coated at 5 mg/mL onto high protein-binding microplates overnight at 4°C. Plates were blocked and washed as above, and probed with plasma serially diluted from 1:100, or mAbs from 10 mg/mL, for one hour at 37°C. Following washing, bound antibodies were detected with HRP-conjugated anti-human antibodies (Sigma-Aldrich) at a concentration of 1:1000 for one hour at 37°C, followed by TMB substrate stopped with 1 M H2S04. Absorbance was read at 450 nm.