Validation of a Rapid Assay for Determination of Leptin Binding in Serum† 417

We have previously reported the measurement of leptin binding protein(s) in serum by subjecting incubates of serum and ¹²⁵I- leptin, neat and with excess cold leptin, to filtration on Ultragel ACA 44 columns (BBRC233:818-822, 1997). We have now developed a more rapid “spun-column” method. With the plunger removed, a tuberculin syringe suspended in a 15 mL centrifuge tube was packed with 1.0 mL of Sephacryl S-200 HR resin by centrifugation at 860 × g_av for 3 min at 4⁰C. After packing, the spun-column was equilibrated against 0.05 M Tris-HCl buffer, pH 7.5, 0.15 M NaCl by three successive washes of 100 μL of buffer and centrifugation. To collect the sample eluate, a 1.5 mL microcentrifuge tube was placed at the bottom of the 15 mL centrifuge tube into which the spun-column was inserted with the syringe tip inside the microcentrifuge tube. ¹²⁵I-Leptin was purchased from Linco Inc.(St. Charles, MO) and further purified by filtration on an Ultragel ACA 34 and 44 column. Serum (150μL) was incubated with 150μL purified¹²⁵ I-leptin (approximately 10-20,000 cpm) and 100μL of buffer, neat or with 100 ng of cold leptin for 2 hr at 4⁰C. An aliquot (300μL) of the incubate was then assayed as previously described using the Ultragel ACA 44 column. At the same time, an aliquot (100μL) of the incubate was loaded on a spun-column and centrifuged. After centrifugation, the microcentrifuge tube containing the spun-column eluate was removed and counted to determine “bound” counts while the resin was blown from the spun-column into a test tube with an air jet and counted to assess “unbound” leptin. Total binding was determined by dividing “bound” by total counts. Percent specific binding was calculated by subtracting percent total binding determined in the presence of excess competitor leptin from percent total binding in the absence of competitor. Serum was collected from 27 patients and assayed simultaneously by both methods. Specific binding was higher by the Ultragel method (mean=18.3% vs 14.0%, p<0.02) and the values were highly correlative (r=.89,p<.0001). Specific binding in both methods correlated inversely with serum leptin levels (Ultragel, r=-0.79, p<0.0001; and spun-column, r=-0.63, p<0.001). We conclude that the “spun-column” method offers a simple, rapid, and quantitative method for determination of leptin binding in serum.