Quantikine rat mouse igf 1 immunoassay kit

The Quantikine Rat/Mouse IGF‐1 Immunoassay kit (R&D Systems Inc., Minneapolis, MN, USA) was used to determine IGF‐1 protein expression. ELISA was performed according to the manufacturer's instructions. Briefly, 96‐well plates were precoated with monoclonal antibody specific for IGF‐1. Assay diluents were added to each well (50 ml/well). Standards were diluted serially (1:2) from 6 to 0 ng/ml or with the respective calibrator diluents and plated. Both standards and samples were incubated together for 2 h at room temperature. The wells were then rinsed in wash buffer five times then incubated in polyclonal anti‐mouse IGF‐1 antibody overnight at 4°C. The plates were washed then incubated with anti‐mouse IgG horseradish peroxidase conjugate for 1 h in the dark at room temperature. Tetramethyl benzidine/peroxidase substrate solution was added to the wells to produce the colour reaction. Colour reaction was stopped with 1N HCl (100 μl) and reaction was read in a microplate reader (Bio‐Tek, Winooski, VT, USA) at a wavelength of 450 nm (650‐nm reference wavelength). Protein concentration was determined from the regression line for the IGF‐1 standard corrected for the total amount of protein in the sample and assay sensitivity ranged from 1.6 to 8.4 pg/ml. Assay was performed in triplicates and measurements were averaged and used as one individual data point for statistical analysis.