A spun-column assay for determination of leptin binding in serum

Serum collected from 27 patients was assayed simultaneously using a spun-co lumn assay (SPC) and a traditional exclusion gel-filtration assay (GFC) to determine specific leptin binding. The levels of serum leptin binding deter mined by either assay correlated inversely with serum leptin levels (SPC, r = 0.63, P < 0.001; GFC, r = 0.79, P < 0.0001). Although specific leptin bi nding as determined by the traditional exclusion gel-filtration assay was g enerally higher than that obtained by the spun-column assay (mean = 18.3% v s 14.0%, P < 0.02, respectively); the values obtained between the two assay methods were highly correlative (r = 0.89, P < 0.0001). By varying either the amount of I-125-leptin or the amount of competitor, analysis was carrie d out using the spun-column assay to determine the intrinsic properties of serum leptin binding. Results yielded a K-d = 0.3 nM, where each variable a mount of leptin or competitor was carried out in duplicate. The complete an alysis was carried out in the time that it typically takes for a single sam ple determination by the traditional exclusion gel-filtration assay. We con clude that the "spun-column" assay is a useful method for rapid and accurat e quantification of leptin binding in serum. (C) 1999 Academic Press.