Elisa kit for leptin

The ¹²⁵I radioimmunoassay (RIA) kits of T3 and T4 (Institute of Atomic Energy, Beijing, China) that were previously validated for Mongolian gerbil were used to quantify serum T3 and T4 concentrations. The intra-assay coefficients of variation (CV) were 5.1% for T3 and 4.4% for T4 (44 (link)).
Serum ghrelin levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (catalog no. CEA991Ra; Cloud-Clone Corp.), and serum leptin levels were measured using an ELISA kit for leptin (catalog no. SEA084Ra; Cloud-Clone Corp.) according to the manufacturer’s instructions. Absorbance was measured at 450 nm against a blank using an ELISA reader (RayBiotech, Canada). The intra- and interassay CVs were <10% and <15% for both kits. The minimum detectable dose for ghrelin was 52.3 pg/ml, and that for leptin was 0.129 ng/ml.

The glucose concentration in the whole blood from the tail vein was measured using a glucometer (“Life Scan Johnson & Johnson”, Denmark) and the test strips “One Touch Ultra” (USA). The concentration of insulin in the serum of mice and in the hypothalamus samples was measured using the Mouse Insulin ELISA kits (“Mercodia AB”, Sweden). The leptin levels in the serum and in the hypothalamus samples were measured using the ELISA kit for Leptin (“Cloud-Clone Corp.”, Houston, USA). To determine the intrahypothalamic leptin and insulin content, the samples of the hypothalamus tissue were homogenized in the ratio 1:10 in the lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.25 M sucrose, 0.5% Triton X-100, 0.5% sodium deoxycholate, 15 mM NaF, 10 mM sodium glycerophosphate, 10 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% NaN₃, and the protease inhibitor cocktail (“Sigma-Aldrich”, USA). The obtained homogenate was centrifuged (10 000 g, 5 min), and the concentration of leptin and insulin in the supernatant fraction was measured according to the manufacturer's instructions. The concentrations of total triglycerides and total cholesterol in the serum and the lactic acid levels in the blood plasma were measured using the enzyme colorimetric kits obtained from “Olvex Diagnosticum” (Russia).