Enzyme immunoassay kit

RV and lung supernatants were acidified with 0.6% trifluoroacetic acid (TFA) to obtain a 10% (w/v) homogenate. The samples were centrifuged at 2,000 × g for 15 min at 4°C, and dried under a steam of nitrogen at 60°C. Enzyme-immunoassay kits (Phoenix Pharmaceuticals, CA, USA) were used to measure the Ang II and Ang-(1-7) contents in the RV and lung.

Plasma (0.8-1.0 mL) and lung (~100 mg) supernatants were acidified with 0.6% trifluoroacetic acid (TFA) to obtain a 10% (w/v) homogenate. The samples were centrifuged at 2,000 × g for 15 min at 4°C, washed, and dried under a steam of nitrogen at 60°C. Enzyme-immunoassay kits (Phoenix Pharmaceuticals, CA, USA) were used to measure the Ang II and Ang-(1-7) contents of the lung.

Fasting blood samples of approximately 5 mL were collected from the patients and immediately sent for biochemical examination by an autoanalyzer (Siemens Advia 1800; Siemens Healthcare GmbH, Henkestr, Germany). Random spot urine samples were sent for measurement of the ratio of albumin to creatinine (UACR) [16 (link),22 (link)]. Values of FGF-21 were measured by enzyme immunoassay kits (Phoenix Pharmaceuticals, Inc. Burlingame, CA, USA) [22 (link)]. Renal function was manifested as estimated glomerular filtration rate (eGFR) calculated from the Chronic Kidney Disease Epidemiology Collaboration equation [23 (link)].

Blood samples were drawn immediately prior to primary PCI and serum was isolated by centrifugation within 1 h at 2500g for 10 min, and stored at −80°C. Serum concentrations of apelin (human apelin-12) were assayed using commercially available enzyme immunoassay kits (Phoenix Pharmaceuticals, Belmont, CA). 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°C–23°C) for 2 hours. Wash immunoplate 4 times with 350 μL/well of 1 × assay buffer. Add 100 μL/well of Streptavidin-HRP solution and incubate at room temperature for 1 h. Wash immunoplate 4 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 h. Terminate reaction with 100 μL/well of 2N HCl. The intra-assay coefficients of variation (CVs) were 5% for apelin, and <14% for ghrelin, whereas the inter-assay CVs were 14% and 7.5%, respectively. Measurement of high-sensitivity C-reactive protein (hs-CRP) was performed using a particle-enhanced immunoturbidimetric assay (Hitachi 917 analyzer; Boehringer Mannheim, Germany). The detection limit was 0.1 mg/L, with intra- and inter-assay CVs of 1.34% and 5.7%, respectively.

Blood samples were collected after a 12-h fast. Biochemical and lipid parameters were measured using an automatic enzymatic analyser (Beckman Coulter, AU5800, Japan). For apelin, 5-mL blood samples were collected in tubes containing EDTA-K2. Immediately following centrifugation at 3600 rpm for 10 min, the plasma samples were frozen at −80 °C until analyses were conducted. Apelin-12, apelin-13, and apelin-36 levels were assayed using commercially available enzyme immunoassay kits (Phoenix Pharmaceuticals, Burlingame, CA, USA). For NO, blood (5 mL) was collected and centrifuged at 3600 rpm. The serum samples were separated and stored at −80 °C. The serum NO level was determined using a nitrate/nitrite colorimetric assay kit (Cayman Chemical, Michigan, USA).

Plasma samples (1.0 mL) and homogenous right ventricle (RV) were expressed in v/v for TFA and homogenate in w/v and then centrifuged at 2,000 × g for 15 min at 4°C. Enzyme-immunoassay kits (Phoenix Pharmaceuticals, Burlingame, CA, USA) were used to measure plasma and RV BNP contents according to the manufactural protocol.