Derivation of cutoffs for the Elecsys® amyloid ß (1-42) assay in Alzheimer's disease.

INTRODUCTION: An Elecsys® Amyloid ß (Aß [1-42]) immunoassay cutoff for classification of patients with Alzheimer's disease was investigated. METHODS: Cerebrospinal fluid samples collected from patients with mild-to-moderate Alzheimer's disease were analyzed by Elecsys® immunoassays: (1) Aß (1-42), (2) total tau, and (3) phosphorylated tau. Cutoffs (Aß [1-42] and ratios with tau) were estimated by method comparison between AlzBio3 (n = 206), mixture modeling (n = 216), and concordance with florbetapir F 18 imaging-based classification (n = 75). RESULTS: A 1065-pg/mL (95% confidence interval: 985-1153) Elecsys® Aß (1-42) cutoff provided 94% overall percentage agreement with AlzBio3. Comparable cutoff estimates (95% confidence interval) were derived from mixture modeling (equally weighted: 1017 [949-1205] pg/mL; prevalence weighted: 1172 [1081-1344] pg/mL) and concordance with florbetapir F 18 imaging (visual read: 1198 [998-1591] pg/mL; automated: 1198 [1051-1638] pg/mL). DISCUSSION: Based on three approaches, a 1100-pg/mL Elecsys® Aß (1-42) cutoff is suitable for clinical trials with similar populations and preanalytical handling.

A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-ß 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

BACKGROUND: Amyloid-ß 1-42 peptide (Aß1-42) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aß1-42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents. METHODS: A sensitive digital ELISA for measurement of Aß1-42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aß1-42 in clinical samples from patients treated with the ß-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. RESULTS: The prototype assay measured Aß1-42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aß1-42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aß1-42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aß1-42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aß1-42 levels was also observed over the time course of sample collection. CONCLUSIONS: This digital ELISA has potential utility in clinical applications for quantification of Aß1-42 in plasma where high sensitivity and precision are required.

A validated LC-MS/MS method for neurotransmitter metabolite analysis in human cerebrospinal fluid using benzoyl chloride derivatization.

BACKGROUND: Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention. RESULTS: Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation. CONCLUSION: A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.

Validation and Clinical Utility of ELISA Methods for Quantification of Amyloid-ß of Peptides in Cerebrospinal Fluid Specimens from Alzheimer's Disease Studies.

The aim of this study was to validate assays for measurement of amyloid-ß (Aß) 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(®)ß-AMYLOID(1-42) and prototype INNOTEST(®)ß-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 Aß peptide spikes standard ranged from 77% to 93%. No interference was observed from the investigational drugs LY2811376, LY2886721, LY3002813, or semagacestat. Aß(1-40) and Aß(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, Aß(1-40) was mostly stable up to 3 years at ≤−70◦C, whereas stability of Aß(1-42) was limited to 221 days. Dose-dependent changes in measured CSF Aß 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.

Validation of assays for measurement of amyloid-ß peptides in cerebrospinal fluid and plasma specimens from patients with Alzheimer's disease treated with solanezumab.

The aim of this study was to validate new assays for measurement of amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF) and plasma specimens in clinical studies of solanezumab according to current regulatory recommendations. Four assays based on the INNOTEST® ß-AMYLOID(1-42) and prototype INNOTEST ß-AMYLOID(1-40) kits were developed and validated. To render these assays 'solanezumab-tolerant', excess drug was added to calibrators, quality control, and test samples via a 2-fold dilution with kit diluent. Validation parameters were evaluated by repeated testing of human CSF and EDTA-plasma pools containing solanezumab. Calibration curve correlation coefficients for the four assays were ≥0.9985. Intra- and inter-assay coefficients of variation for Aß1-40 and Aß1-42 were ≤13 and ≤15%, respectively for both matrices. Dilutional linearity, within and between assays, was demonstrated for both analytes in CSF and plasma at clinically relevant dilution factors. This dilution regimen was successfully applied during Phase 3 clinical sample analysis. Aß1-40 and Aß1-42 were stable in CSF and plasma containing solanezumab at 2-8°C and room temperature for up to 8 h and during 5 additional freeze-thaw cycles from ≤-20 and ≤-70°C. Results of parallel tests on stored clinical samples using INNOTEST methods and proprietary ELISA methods were closely correlated (r2 > 0.9), although bias in reported concentrations was observed between assays. In conclusion, the modified INNOTEST assays provided (relatively) accurate and precise quantification of Aß1-40 and Aß1-42 in CSF and plasma containing solanezumab according to established consensus validation criteria. The clinical experience with these assays post validation has shown them to be robust and reliable.

Validation of a multiplex assay for simultaneous quantification of amyloid-ß peptide species in human plasma with utility for measurements in studies of Alzheimer's disease therapeutics.

The aim of this study was to validate the INNO-BIA plasma amyloid-ß (Aß) forms assay for quantification of Aß1-40 and Aß1-42 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 Aß1-42 by 53%. Specimens from a Phase I human volunteer study of the ß-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.

Validation of ELISA methods for quantification of total tau and phosporylated-tau181 in human cerebrospinal fluid with measurement in specimens from two Alzheimer's disease studies.

Tau measurements in cerebrospinal fluid (CSF) are gaining acceptance as aids to diagnosis of Alzheimer's disease (AD) and differentiation from other dementias. Two ELISA assays, the INNOTEST® hTAU Ag and the INNOTEST® PHOSPHO-TAU(181P) for quantification of t-tau and p-tau181 respectively, have been validated to regulatory standards. Validation parameters were determined by repeated testing of human CSF pools. Specimens from Phase 2 studies of the γ-secretase inhibitor semagacestat and the therapeutic antibody solanezumab at baseline and following 12-14 weeks of treatment were also tested. Estimated intra-assay CV for repeated testing of 3 CSF pools were ≤11.5% and RE varied between -14.1% and +6.4%. Inter-assay CV for t-tau was <5% and RE was within ±8%. For p-tau181, inter-assay CV was <9% and RE was within ±2.5%. Total CV (intra-assay plus inter-assay) were below 10% for both analytes. Up to 20-fold dilutional linearity was demonstrated for both analytes. Stability of t-tau and p-tau181 was demonstrated in CSF during five freeze-thaw cycles at ≤-20 °C and ≤-70 °C and at 18-22 °C for up to 24 h. Neither semagacestat nor solanezumab interfered with either assay. Inter-individual t-tau and p-tau181 concentrations were highly variable but intra-individual variations were small. These assays are suitable for analysis of CSF t-tau and p-tau181 in a single laboratory supporting multi-center AD clinical trials. No effect of treatment with semagacestat or solanezumab was observed in response to three months of drug administration.