ELISA assay

Serum levels of XPNPEP2 were measured by using a human XPNPEP2 ELISA pair set (cat: {"type":"entrez-protein","attrs":{"text":"SEK11903","term_id":"1095264981","term_text":"SEK11903"}}SEK11903, Sino Biological, Beijing). The ELISA assay was performed as follows. A 96-well microplate was coated with 100 μL monoclonal antibody specific for XPNPEP2 (2 μg/mL) overnight at 4 °C. The wells were then washed three times and blocked, and 100 μL of standards or samples (diluted 1:200) were added to individual wells. Each well was aspirated and washed three times with at least 300 μl of buffer per wash. Subsequently, 100 μL of HRP-conjugated anti-XPNPEP2 monoclonal antibody (0.5 μg/mL) was added. The aspiration and wash cycle was repeated, and then 100 μL of TMB substrate solution was loaded. 50 μL of stop solution was added and the absorbance of each well at 450 nm was measured. XPNPEP2 concentrations were calculated based on the curve generated from the standards.