Quantitative ELISA for Secreted AGR2

For P3A5, each well of 96-well plates was coated with 0.2 μg P1G4 in 100 μL PBS overnight followed by rinsing with PBS-0.05% Tween, and blocking with 1% BSA-PBS. Cell-free prostate LuCaP xenograft tissue collagenase digestion media containing secreted AGR2 [42 (link), 43 (link)], diluted 1/10, was added. P3A5 (0.2 μg) or media (100 μL) of transfected 293F cells were used for detection, followed by goat anti-mouse IgG2a-HRP or goat anti-human IgG-HRP (Southern Biotech, Birmingham, AL, USA), where appropriate, diluted at 1:2000 in 1% BSA-PBS followed by 1-step ABTS (Thermo Fisher). Wells were scanned at λ = 415 nm after 5-30 min [13 (link)]. For P1G4, the wells were coated with P3A5. Goat anti-mouse IgG1-HRP for positive control and goat anti-human IgG-HRP were used for detection.