title: A rapid, quantitative functional assay for measuring leptin.
abstract: At present, leptin is quantitated using immuno-assays that measure leptin mass. Leptin biological activity is determined using protocols that measure feed consumption and weight reduction. These in vivo protocols are semi-quantitative and require large quantities of leptin. We describe a rapid, sensitive and quantitative in vitro assay for leptin using HEK-293 cells stably co-transfected with the leptin receptor Ob-Rb isoform and a STAT-inducible promoter regulating the firefly luciferase cDNA. The assay, performed in a 96-well format, has an EC50 of 150 pM and is linear from 3 to 700 pM of leptin. We demonstrate that the assay is capable of measuring leptin in plasma samples. We demonstrate that bacterially-expressed, recombinant leptin and in vivo expressed leptin are equipotent. Furthermore, we demonstrate that a leptin-derived peptide, leptin fragment 22-56, previously shown to be capable of reducing feed intake following ICV injection does not act directly through the leptin receptor.
authors.affiliation: Department of Obesity Research, Merck Research Laboratories, Merck, Rahway, NJ 07065, USA. charles_rosenblum@merck.com
authors.forename: C I
authors.initials: CI
authors.lastname: Rosenblum
authors.forename: A
authors.initials: A
authors.lastname: Vongs
authors.forename: M R
authors.initials: MR
authors.lastname: Tota
authors.forename: J P
authors.initials: JP
authors.lastname: Varnerin
authors.forename: E
authors.initials: E
authors.lastname: Frazier
authors.forename: D F
authors.initials: DF
authors.lastname: Cully
authors.forename: M A
authors.initials: MA
authors.lastname: Morsy
authors.forename: L H
authors.initials: LH
authors.lastname: Van der Ploeg
mesh_terms: D000818:Animals; D001681:Biological Assay; D002352:Carrier Proteins; D002460:Cell Line; D020738:Leptin; D051379:Mice; D010446:Peptide Fragments; D011506:Proteins / Q000032:analysis*; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D020738:Leptin; D010446:Peptide Fragments; D011506:Proteins; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; C515447:leptin receptor, mouse