Apelin-12 ELISA detection

Serum was isolated by centrifugation within 1 hour at 2500 g for 10 min and stored at −80°C. Serum concentrations of apelin-12 were assayed using commercially available enzyme immunoassay kits (Phoenix Pharmaceuticals, Belmont, California, USA). The protocol was as follows: add 50 µL/well of standard, sample or positive control, 25 µL of primary antibody and 25 µL of biotinylated peptide; incubate at room temperature (20–23°C) for 2 hours; wash immunoplate four times with 350 µL/well of 1×assay buffer; add 100 µL/well of Streptavidin horse radish peroxidase-solution and incubate at room temperature for 1 hour. Wash immunoplate four times with 350 µL/well of 1×assay buffer. Add 100 µL/well of 3,3′, 5,5′-tetramethylbenzidine substrate solution and incubate at room temperature for 1 hour. Terminate reaction with 100 µL/well of 2N HCl. The detection limit was 0.1 mg/L, with 1.26% and 5.4% intra-assay and interassay coefficients of variation, respectively. The measurements were performed in triplicates.