title: Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL.
abstract: To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.
authors.affiliation: Department of Assay & Automation Technology, Genentech, Inc., South San Francisco, CA 94080-4990, USA.
authors.forename: Laura E
authors.initials: LE
authors.lastname: DeForge
authors.forename: Danny H
authors.initials: DH
authors.lastname: Shih
authors.forename: Derek
authors.initials: D
authors.lastname: Kennedy
authors.forename: Klara
authors.initials: K
authors.lastname: Totpal
authors.forename: Anan
authors.initials: A
authors.lastname: Chuntharapai
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Bennett
authors.forename: Jason H
authors.initials: JH
authors.lastname: Drummond
authors.forename: Patricia
authors.initials: P
authors.lastname: Siguenza
authors.forename: Wai Lee T
authors.initials: WL
authors.lastname: Wong
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008252:Macaca fascicularis; D051379:Mice; D002679:Pan troglodytes; D051381:Rats; D017207:Rats, Sprague-Dawley; D044967:Serum / Q000276:immunology*; D013045:Species Specificity; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000097:blood / Q000276:immunology* / Q000494:pharmacology
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D053221:TNF-Related Apoptosis-Inducing Ligand