Validation and Clinical Utility of ELISA Methods for Quantification of Amyloid-β 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) ELISA kits 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 inter-assay 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 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 freeze-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 β-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.