Validation of a Commercial Chemiluminescence Immunoassay for the Simultaneous Measurement of Three Different Amyloid-β Peptides in Human Cerebrospinal Fluid and Application to a Clinical Cohort

Abstract

A comprehensive assay validation campaign of a commercially available chemiluminescence multiplex immunoassay for the simultaneous measurement of the amyloid-β peptides Aβ38, Aβ40, and Aβ42 in human cerebrospinal fluid (CSF) is presented. The assay quality parameters we addressed included impact of sample dilution, parallelism, lower limits of detection, lower limits of quantification, intra- and inter-assay repeatability, analytical spike recoveries, and between laboratory reproducibility of the measurements. The assay performed well in our hands and fulfilled a number of predefined acceptance criteria. The CSF levels of Aβ40 and Aβ42 determined in a clinical cohort (n = 203) were statistically significantly correlated with available ELISA data of Aβ1-40 (n = 158) and Aβ1-42 (n = 179) from a different laboratory. However, Bland-Altman method comparison indicated systematic differences between the assays. The data presented here furthermore indicate that the CSF concentration of Aβ40 can surrogate total CSF Aβ and support the hypothesis that the Aβ42/Aβ40 ratio outperforms CSF Aβ42 alone as a biomarker for Alzheimer's disease due to a normalization to total Aβ levels.

A two-step immunoassay for the simultaneous assessment of Aβ38, Aβ40 and Aβ42 in human blood plasma supports the Aβ42/Aβ40 ratio as a promising biomarker candidate of Alzheimer’s disease

“…The comparison of two independent runs of the novel two-step immunoassay with the same set of 40 plasma samples revealed interassay coefficients of variation ranging from 0.12 to 27.2% with mean CV values of 10.2% ± 6.1 (Aβ38), 6.4% ± 4.3 (Aβ40) and 8.1% ± 6.7 (Aβ42) (Table 2 ). The observed degree of interassay variation appears to be comparable with previous findings regarding the assessment of diluted CSF with the same chemiluminescence Aβ multiplex assay kit [ 15 ]. However, Bland–Altman plots suggested nonrandom errors between the two different experiments regarding the measurements of Aβ38 and Aβ42.…”

“…Thus, the IP eluates are compatible with subsequent immunoassays without the need for pH neutralization or removal of denaturing substances. For the simultaneous measurement of Aβ38, Aβ40 and Aβ42 in the IP eluates, we employed a commercially available chemiluminescence multiplex immunoassay, which we have previously validated for measuring all three Aβ variants in CSF [ 15 ].…”

“…The lower limits of quantification (LLOQs) according to the certificates of analysis provided with the assay kits were 50 pg/ml (Aβ40), 60 pg/ml (Aβ38) and 3.13 pg/ml (Aβ42). The lower limit of detection (LLOD) was calculated with the MSD Discovery Workbench software automatically for each assay plate as the lowest concentration producing a signal three standard deviations above the zero calibrator with the option “use minimum error estimates” activated [ 15 ].…”

Reproducibility of Alzheimer’s Disease Cerebrospinal Fluid-Biomarker Measurements under Clinical Routine Conditions

“…Aβ 40 is the most abundant C-terminal Aβ-variant in CSF, followed by Aβ 38 and Aβ 42 [ 5 ]. In CSF, Aβ 40 correlates well with the total Aβ concentration [ 6 ] and thus can serve as a surrogate-marker for total CSF Aβ. In patients with either unusually low or high overall Aβ, the Aβ 42 to Aβ 40 concentration ratio (Aβ 42/40 ) can be a helpful tool to normalize the Aβ 42 levels to Aβ 40 (as a proxy of total Aβ) and thereby improve the diagnostic accuracy [ 7–9 ].…”

“…CSF Aβ 42/40 was reported to correlate more closely with tracer retention on Amyloid-PET imaging than Aβ 42 alone [ 10 ] and appears to be less prone to variances resulting from pre-analytical sample handling [ 9 ]. Furthermore, in our hands, Aβ 42/40 turned out to provide higher inter-assay and between lot reproducibility than CSF Aβ 42 alone, when assessed with a multiplex chemiluminescence immunoassay (Aβ V-PLEX, Mesoscale) [ 6 ].…”