title: Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments.
abstract: The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.
authors: Drug Disposition, Eli Lilly and Company, Indianapolis, IN, USA. hansenry@lilly.com
authors: Ryan J
authors: RJ
authors: Hansen
authors: Robin M
authors: RM
authors: Brown
authors: Jirong
authors: J
authors: Lu
authors: Victor J
authors: VJ
authors: Wroblewski
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D007140:Immunoglobulin Fab Fragments / Q000097:blood* / Q000276:immunology* / Q000627:therapeutic use; D007700:Kinetics; D008024:Ligands; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D007140:Immunoglobulin Fab Fragments; D008024:Ligands
keywords: ELISA; Fab; PK/PD; antibody; free ligand
references: AAPS J. 2008 Jun;10(2):425-30
references: 18686041
references: J Pharmacol Exp Ther. 2010 Apr;333(1):2-13
references: 20089807
references: Basic Clin Pharmacol Toxicol. 2010 Mar;106(3):195-209
references: 20050847
references: Br J Clin Pharmacol. 2007 May;63(5):548-61
references: 17096680
references: AAPS J. 2011 Jun;13(2):169-78
references: 21336535
references: Expert Opin Drug Metab Toxicol. 2012 Feb;8(2):141-60
references: 22248267
references: Retina. 2006 Oct;26(8):871-6
references: 17031285
references: AAPS J. 2011 Mar;13(1):99-110
references: 21240643
references: J Allergy Clin Immunol. 2000 Aug;106(2):253-9
references: 10932067
references: J Pharm Sci. 2004 Nov;93(11):2645-68
references: 15389672
references: MAbs. 2010 Sep-Oct;2(5):576-88
references: 20676036
references: Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9820-5
references: 19443683
references: Br J Clin Pharmacol. 2009 Jul;68(1):61-76
references: 19660004
references: J Lipid Res. 2011 Jan;52(1):78-86
references: 20959675
references: J Allergy Clin Immunol. 1997 Jul;100(1):110-21
references: 9257795
references: Clin Pharmacol Ther. 2008 Nov;84(5):548-58
references: 18784655