Quantification of the soluble leptin receptor in human blood by ligand-mediated immunofunctional assay.
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Panels of monoclonal antibodies (mAbs) were raised against recombinant human leptin and the recombinant human soluble leptin receptor. Using these mAbs, we established a ligand-mediated immunofunctional assay (LIFA) to quantify concentrations of the soluble leptin receptor, which has been shown to be a major binding protein for leptin in human serum. In performing the assay, a monoclonal antibody (mAb 2H6) against the soluble leptin receptor, which binds an epitope outside the leptin-binding site and equally recognizes both, free and leptin-occupied soluble leptin receptor, is used to capture the soluble leptin receptor on a microtiter plate. Recombinant human leptin is added to saturate all binding sites, and a biotinylated anti-leptin mAb (4D3) detects the amount of leptin (endogenous and exogenous) bound to the soluble leptin receptor. The same procedure, but without adding exogenous leptin, allows for measurement of the circulating endogenous leptin/soluble leptin receptor complexes. The LIFA assay has a linear working range of 0.5-200 microg/liter, intra- and interassay coefficients of variation ranged from 3.2-6.3% and from 5.2-7.9%, respectively. The assay has a linearity of 102.2 +/- 5.2% (mean +/- SD) and a recovery of 100.7 +/- 6.9%. Size-exclusion chromatography revealed that the assay measures a protein with a main peak eluted at 340 kDa. The soluble leptin receptor concentration (63.3 +/- 22.8 microg/liter (mean +/- SD), range 17.9-129.2 microg/liter, n = 43) in normal subjects (body mass index = 22.3 +/- 2.3 kg/m(2)) was not different from the concentration (54.4 +/- 19.8 microg/liter, range 23.7-104.8 microg/liter, n = 34, P > 0.05) found in obese subjects (body mass index = 40.9 +/- 15.7 kg/m(2)). However, the percentage of the total soluble leptin receptor complexed with endogenous leptin was significantly higher in obese subjects, compared with normal subjects (74.9% +/- 23.5% vs. 33.1% +/- 19.5%, P < 0.001). Higher serum leptin levels in obese subjects (38.4 +/- 23.7 microg/liter vs. 7.8 +/- 5.5 microg/liter in normal subjects, P < 0.001) together with comparable soluble leptin receptor levels result in a lower proportion of leptin bound to the soluble leptin receptor in obese subjects (19.3% +/- 19.4%, range 4.9-97.2%) than in normal subjects (39.0% +/- 22.5%, range 15.3-96.5%, P < 0.001). The development of this LIFA for the rapid and accurate quantification of total soluble leptin receptor and circulating leptin/soluble leptin receptor complexes provides a valuable tool for the further understanding of the role of leptin and its soluble receptor in health and disease.
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