title: Validation and Clinical Utility of ELISA Methods for Quantification of Amyloid-β of Peptides in Cerebrospinal Fluid Specimens from Alzheimer’s Disease Studies.
abstract: The aim of this study was to validate assays for measurement of amyloid-β (Aβ) peptides in cerebrospinal fluid (CSF)specimens according to regulatory guidance and demonstrate their utility with measurements in specimens from Alzheimer’s disease (AD) studies. Methods based on INNOTEST(®)β-AMYLOID(1-42) and prototype INNOTEST(®)β-AMYLOID(1-40) ELISAkits were developed involving pre-analytical sample treatment with Tween-20 for reliable analyte recovery.Validation parameters were evaluated by repeated testing of CSF pools collected and stored in the same manner as clinical specimens. Intra- and interassay coefficients of variation were ≤11% and relative accuracy was within ± 10% for both analytes. Dilutional linearity was demonstrated for both analytes from a spiked CSF pool, but not from a non-spiked native CSF pool. Recovery of standard Aβ peptide spikes standard ranged from 77% to 93%. No interference was observed from the investigational drugs LY2811376, LY2886721, LY3002813, or semagacestat. Aβ(1-40) and Aβ(1-42) were stable in CSF for up to 8 hours at room temperature and during 5 f reeze-thaw cycles from ≤−20◦C and ≤−70◦C. In frozen native CSF specimens, Aβ(1-40) was mostly stable up to 3 years at ≤−70◦C, whereas stability of Aβ(1-42) was limited to 221 days. Dose-dependent changes in measured CSF Aβ were observed in healthy volunteers up to 36 hours after treatment with the-site cleavage enzyme inhibitor LY2886721. In conclusion, rigorous validation tests have successfully demonstrated the strengths and operational limitations of these INNOTEST(®)-based assays.They have proved to be robust and reliable tools for pharmacodynamic evaluations of investigational AD therapeutics in clinical trials.
authors.affiliation: Lilly Research Centre, Windlesham, UK. drlachno@lilly.com
authors.forename: D Richard
authors.initials: DR
authors.lastname: Lachno
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Evert
authors.forename: Kaia
authors.initials: K
authors.lastname: Maloney
authors.forename: Brian A
authors.initials: BA
authors.lastname: Willis
authors.forename: Jayne A
authors.initials: JA
authors.lastname: Talbot
authors.forename: Manu
authors.initials: M
authors.lastname: Vandijck
authors.forename: Robert A
authors.initials: RA
authors.lastname: Dean
mesh_terms: D000544:Alzheimer Disease / Q000134:cerebrospinal fluid*; D016229:Amyloid beta-Peptides / Q000134:cerebrospinal fluid*; D002555:Cerebrospinal Fluid / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006574:Heterocyclic Compounds, 2-Ring / Q000494:pharmacology; D006801:Humans; D008297:Male; D010446:Peptide Fragments / Q000134:cerebrospinal fluid*; D010848:Picolinic Acids / Q000494:pharmacology; D011136:Polysorbates / Q000494:pharmacology; D011569:Psychiatric Status Rating Scales; D015203:Reproducibility of Results; D013501:Surface-Active Agents / Q000494:pharmacology; D013997:Time Factors
chemical_list: D016229:Amyloid beta-Peptides; D006574:Heterocyclic Compounds, 2-Ring; C000596181:N-(3-(2-amino-4a,5,7,7a-tetrahydro-4H-furo(3,4-d)(1,3)thiazin-7a-yl)-4-fluorophenyl)-5-fluoropicolinamide; D010446:Peptide Fragments; D010848:Picolinic Acids; D011136:Polysorbates; D013501:Surface-Active Agents; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42)