Objective: To establish a highly sensitive and specific ELISA method for measurement of leptin and further to study the secretion of leptin during human preadipocytes differentiation and effects of troglitazone.
Methods: Rabbits Balb/c mice were immunized by recombinant human leptin and Balb/c mice were immunized by human leptin so as to produce rabbit anti-human leptin polyclonal antibodies (PAb) and mouse anti-human leptin monoclonal antibodies (MAb). Combination of the PAb as coating antibody, with a carefully paired biotinylated MAb as detector, and the avidin-horseradish peroxidase as the amplifier of detecting signals, a sandwich method, biotin-avidin ELISA (BA-ELISA) was established. Human omental preadipocytes were cultured, introduced to differentiate, and treated with 10 micromol/L troglitazone; the leptin secretion in the supernatant was detected by BA-ELISA. Peripheral blood samples were collected from 114 healthy persons and the serum leptin was detected by BA-ELISA.
Results: The sensitivity of BA-ELISA was 0.03 ng/ml with a working range of 0.05 - 5 ng/ml and a exogenous leptin recovery rate of 97.8%, and the intra- and interassay coefficients of variation (CVs) were less than 7.4% and 9.3% respectively. The assay detected only a single free leptin peak in gel chromatographic fractions from the mixed human sera or adipocytes culture media. The leptin secretion level detected by BA-ELISA showed that the leptin secretion of the preadipocytes increased strongly when the cells differentiated into mature adipocytes. The peak leptin secretion level of the troglitazone treated group was 2 times as that of the control group. The leptin concentration of women was than 7.6 ng/ml, significantly higher than that of the men (3.2 ng/ml, P < 0.001), and the serum leptin level was significantly correlated with body mass index both for men (r = 0.67, P < 0.001) and for women (r = 0.61, P < 0.001).
Conclusion: A highly sensitive BA-ELISA specific for free leptin has been developed that is especially suited for the accurate measurement of the rather low leptin levels of clinical blood specimens and for basic research use.