Serum FGF15 and FGF19 detection

The serum FGF15 levels of mice were detected using a sandwich ELISA Kit (LifeSpanBioSciences, Inc., Seattle, WA) following the manufacturer’s instructions. Briefly, 100 μL of standards, blank or samples were added to wells which had been pre-coated with the target specific capture antibody of FGF15, which will bind to the target antigen (FGF15). Then 100 μL of biotin-conjugated detection antibody (Detection Reagent A) was added to each well which, in turn, binds to the capture antigen, and the total mixture was incubated for 1 h at 37 °C followed by a wash step with 350 μL wash buffer per well x3.A100 μL aliquot of avidin-horseradish peroxidase (HRP) conjugate (Detection Reagent B) was then added (binds to the biotin) and the plate was incubated for 1 hour at 37 °C and further washed 5 times. About 90 μL of TMB substrate was added to each well followed by incubation for 30 min at 37 °C to allow reaction with the HRP enzyme for detection. Subsequently, 50 μL of sulfuric acid stop solution were added to each well to terminate the color development reaction. The optical density (OD) of each well was measured at a wavelength of 450 nm using a SpextraMax i3 Multi-mode Microplate Reader (Molecular Devices, USA). The standard stock solution was diluted with sample diluent to prepare a standard dilution series from 78 to 5000 pg/mL in order to generate a standard curve. A four parameter logistic curve fit was selected to generate the standard curve and calculate the concentration of each sample.

The serum FGF19 levels of human samples were quantified using a sandwich ELISA Kit (AIS, HongKong, China) according to the manufacturer’s instructions. 100 μL of standards and serum samples were pipetted to wells which were pre-coated with a rabbit polyclonal antibody specific for human FGF-19 to bind human FGF-19 followed by incubation for 1 h at room temperature. After washing away unbounded substances, a 100 μL aliquot of biotin labeled polyclonal detection antibody specific for human FGF19 was added to the wells followed by incubation for 1 h at room temperature. The wells were washed and 100 μL of astreptavidin-HRP conjugate solution were pipetted to each well followed by incubation for 20 min at room temperature. Then 100 μL of HRP substrate solution were added to each well and color development occurred after 15 min of incubation at room temperature. The color development was stopped by adding stop solution and the optical density (OD) of the wells was measured using a SpextraMax i3 Multi-mode Microplate Reader (Molecular Devices, USA). The standard stock solution was diluted with sample diluent and a standard dilution series from 31.2 to 2,000 pg/mL was prepared to generate a standard curve. A four parameter logistic curve fit was selected to generate the standard curve and calculate the concentration of each sample.