title: Validation of a multiplex assay for simultaneous quantification of amyloid-β peptide species in human plasma with utility for measurements in studies of Alzheimer's disease therapeutics.
abstract: The aim of this study was to validate the INNO-BIA plasma amyloid-β (Aβ) forms assay for quantification of Aβ1-40 and Aβ1-42 according to regulatory guidance for bioanalysis and demonstrate its fitness for clinical trial applications. Validation parameters were evaluated by repeated testing of human EDTA-plasma pools. In 6 separate estimates, intra-assay coefficients of variation (CV) for repeated testing of 5 plasma pools were ≤9% and relative error (RE) varied between -35% and +22%. Inter-assay CV (n = 36) ranged from 5% to 17% and RE varied from -17% to +8%. Dilutional linearity was not demonstrated for either analyte using diluent buffer, but dilution with immuno-depleted plasma by 1.67-fold gave results within 20% of target. Analyte stability was demonstrated in plasma at 2-8 °C for up to 6 h. Stability during frozen storage up to 12 months and through 3 freeze-thaw cycles at ≤ -70 °C was also demonstrated in 5 of 6 individuals but deteriorated thereafter. Neither semagacestat nor LY2811376 interfered with the assay but solanezumab at 500 mg/L reduced recovery of Aβ1-42 by 53%. Specimens from a Phase I human volunteer study of the β-secretase inhibitor LY2811376 were tested at baseline and at intervals up to 12 h after single oral doses, demonstrating a clear treatment effect. During 1,041 clinical assay runs from semagacestat studies over 10 months, the CV for plasma quality control pools at three levels were ≤15% and RE were <10%. In conclusion, the INNO-BIA plasma assay was successfully validated and qualified for use in clinical research.
authors.affiliation: Eli Lilly and Company, Windlesham, UK. drlachno@lilly.com
authors.forename: D Richard
authors.initials: DR
authors.lastname: Lachno
authors.forename: Julie K
authors.initials: JK
authors.lastname: Emerson
authors.forename: Hugo
authors.initials: H
authors.lastname: Vanderstichele
authors.forename: Celedon
authors.initials: C
authors.lastname: Gonzales
authors.forename: Ferenc
authors.initials: F
authors.lastname: Martényi
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Konrad
authors.forename: Jayne A
authors.initials: JA
authors.lastname: Talbot
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Lowe
authors.forename: Paul E
authors.initials: PE
authors.lastname: Oefinger
authors.forename: Robert A
authors.initials: RA
authors.lastname: Dean
mesh_terms: D000544:Alzheimer Disease / Q000097:blood* / Q000188:drug therapy*; D016229:Amyloid beta-Peptides / Q000097:blood*; D016231:Fluorescence Polarization Immunoassay / Q000379:methods / Q000592:standards*; D006801:Humans; D010446:Peptide Fragments / Q000097:blood*; D010949:Plasma / Q000737:chemistry*; D011743:Pyrimidines / Q000097:blood* / Q000627:therapeutic use; D013843:Thiazines / Q000097:blood* / Q000627:therapeutic use
chemical_list: D016229:Amyloid beta-Peptides; C569204:LY2811376; D010446:Peptide Fragments; D011743:Pyrimidines; D013843:Thiazines; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)