Radioimmunoassay for the detection of leptin in human serum

Abstract

Human leptin, which is encoded by the obese (ob) gene, is secreted specifically from adipocytes and is involved in the regulation of satiety and energy consumption. We developed a radioimmunoassay for the determination of leptin in human serum using polyclonal antibodies generated in rabbits against a C-terminal fragment of leptin, leptin(126-140), coupled to hemocyanin. The sensitivity of the assay was app. 5 pmol/l leptin(126-140) equivalent to 0.5 fmol/tube. The intra-assay variation at 100 pmol/l was less than 4.8% and the interassay variation less than 8.3%. Dilution curves of serum samples containing high levels of leptin(126-140) were parallel to the standard curve. Following G-50 Sephadex chromatography a single specific peak was detected at app. 16 kd. The assay procedure compared well to a commercially available assay (Linco, St. Louis, USA) using polyclonal antibodies directed against the intact recombinant protein (R = 0.96; p < 0.0001). Serum levels were significantly higher than plasma levels (app.20%) over a wide range of the standard curve. Levels of serum leptin126-140 immunoreactivity were not altered by meals and no day-to-day variation was found. In a group of 148 healthy female and 108 healthy male subjects with a BMI between 18.2 and 40 kg/m2 there was a significant difference between sexes with higher circulating serum levels in female than in male subjects when tested for identical BMI (p < 0.001). Serum leptin levels in both male and female subjects were positively related to BMI (p < 0.001) when analysed for lean and obese subjects whereas in lean subjects this relation was not apparent. No relation of serum leptin levels and age was detectable in subjects with a BMI up to 30 kg/m2. These data support an important role of leptin in the regulation of body fat stores and BMI which is modulated by gender specific factors.