Establishment of a highly sensitive leptin radioimmunoassay and detection of increased leptin levels in hyperlipidemia and pregnancy

The highly effective antibody has been obtained by immunizing rabbits with recombinant leptin many times. The leptin is iodinated with the chloramine-T method and purified with a Sephadex-G25 chromatography column. The reaction between antigen and antibody is carried out by a one-step balance method and cultured at 4 degrees C for 24 h; the binding and free antigen was then separated by PR reagent. The determining range of this method is about 0.5-24 ng/mL; limited detection level is 0.45 ng/mL, relative standard deviation in a group, and among groups, are less than 5.4% and 8%, respectively. The level of blood leptin in 277 samples of normal persons, in 112 samples of overweight persons (weight/hieght m2 > or = 25) and 224 samples of hyperlipidemic patients have been measured by this method. It is demonstrated that the level of blood leptin in males is much lower than that of the females, and becomes elevated with increased age. Serum leptin level in overweight persons and hyperlipidemic patients is also much higher than that of normal groups (P < 0.01). Serum leptin of 21 workers in our lab at 8:00 AM and 4:00 PM has been tested. It was found that there are no differences between the two time points. The same results are obtained within age groups. Leptin levels of pregnant women's serum is higher than those of the control group (P < 0.001). Leptin in newborn's serum is significantly lower than those of mothers (P < 0.01). There is no obvious correlation between leptin level of mother and newborns by correlation analysis (r = 0.19, P > 0.05). The body weight and body weight index of pregnant women are well correlated with their serum leptin levels (r = 0.33 and 0.35, P < 0.05). The body weight and body weight index of newborns are well correlated with their serum leptin levels (r = 0.54 and 0.49, P < 0.001). The serum leptin level of pregnant women is not correlated with newborn's body weight (r = 0.10). These results have shown that the proposed method is stable, simple, and specific, being sensitive enough to determine leptin levels in human serum or plasma.