Pi21134

Trop2 urine levels were determined by a Sandwich ELISA. Diluted anti-Trop2 capture antibody (SinoBiological, 10428-MM01, 1:100) was coated in a 96-well ELISA plate at 4 °C overnight. The wells were washed three times with PBST (PBS supplied with 0.01% Tween-20). 5% of BSA was used for blocking and incubated at 4 °C overnight. 20 µl of whole urine samples from cancer-free patients and clinically significant prostate cancer patients were incubated for 2 h at room temperature. The plates were washed three times with PBST and then incubated with the Goat-anti-Trop2-biotin detection antibody (R&D Systems; BAF650; 1:250) for 2 h at room temperature. The plates were washed three times with PBST and incubated with a streptavidin-HRP (Thermo Fisher Scientific, PI21134, 1:1000) at room temperature for 30 min. After washing with PBST three times followed by PBS two times, the signals were detected by ultra TMB-ELISA substrate (Thermo Scientific, #34028) for 20 min. After adding the stopping solution (Thermo Fisher Scientific, PIN600), plates were analyzed via plate reader (Promega) at 450 nm. The standard curve for Trop2 concentrations was determined using 0–500 pg/ml recombinant human Trop2 (SinoBiological, 10428-H08H-1).

30 µl of the whole urine samples from cancer-free patients and patients with clinically significant prostate cancer were denatured with sodium dodecyl sulfate (SDS) at 95 °C for 5 min. Samples were separated by SDS-PAGE (Invitrogen™ XP08165BOX). The proteins were transferred onto a nitrocellulose membrane (GVS Life Sciences, 1212632). 5% non-fat milk was used for blocking, and the membrane was incubated with a primary antibody (R&D Systems; BAF650; 1:1000) at 4 °C overnight. The HRP-conjugated secondary antibody was incubated (Thermo Fisher Scientific, PI21134, 1:2000) for one hour at room temperature and then developed with ECL Substrate (Thermo Fisher Scientific, 32106).