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Analytical Performance Characteristics of a Novel Immunoassay for the Quantification of BACE-1 in Human Cerebrospinal Fluid

Emeric Chassaing, BS1, Mélanie Bodnar-Wachtel, PhD1, Tanja Schubert, PhD1, Hugo Marcel Vanderstichele, PhD2, Erik Stoops, Eng2 and Philippe Vergnaud, MSc1, (1)Bioclinica Lab, Lyon, France, (2)ADx NeuroSciences, Gent, Belgium

 

BACKGROUND

Accumulation of amyloid-β (Aβ) into plaques in the brain is one of the hallmarks of Alzheimer’s Disease (AD). This neurotoxic peptide is produced by the consecutive cleavage of the amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Due to its key role in amyloid plaque formation, BACE-1 is a major therapeutic target for reducing the levels of amyloid in AD brain. The BACE1 ELISA quantifies specifically BACE-1, in contrast to BACE-1 enzyme activity assays, which suffer from a BACE-2 interference. This study documents the analytical performance characteristics of a novel ELISA, targeting BACE-1 protein in human Cerebrospinal Fluid (CSF).

METHODS

The BACE-1 ELISA was developed with two monoclonal antibodies. Lyophilized calibrators, as well as run-validation control samples were included in the final kit format. Total assay times is 4 hours. After having performed familiarization runs and adaptation of the test procedures to the needs of a service provider, the assay was challenged internally for its analytical performance characteristics (e.g., precision, parallelism, spike-recovery, working range, sample stability) by using commercially available CSF samples (n=34).

RESULTS

The assay was qualified for measurement of BACE-1 protein in undiluted CSF and EDTA-plasma. Full validation was done for the CSF assay format. The new colorimetric ELISA specifically quantifies BACE-1 in the absence of matrix interference (parallelism 93 % ± 10 %; dilution range: neat – 1/16). Spike/recovery testing revealed good recovery rates (103 % ± 6 %). The precision resulted in intra- and inter-assay variability (% CV) below 5 % and 11 %, respectively. The working range is confirmed between 272 pg/mL (Lower Limit of Quantitation) to 10,752 pg/mL (Upper Limit of Quantitation) and lowest concentration detectable (LOD) is 29.2 pg/mL. CSF stability at +4°C and room temperature is acceptable after 24 hours of storage; consecutive freezing/thawing of CSF up to 4 cycles did not affect BACE-1 concentrations.

CONCLUSIONS

The newly developed BACE-1 colorimetric ELISA assay meets all internal acceptance criteria for clinical trial sample testing and represents an additional tool for patient management.

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