Validation of Immunoassay-Based Tools for the Comprehensive Quantification of Aβ40 and Aβ42 Peptides in Plasma

Abstract

Recent advances in neuroimaging and cerebrospinal fluid (CSF) biomarker assays have provided evidence of a long preclinical stage of Alzheimer’s disease (AD). This period is being increasingly targeted for secondary prevention trials of new therapies. In this context, the interest of a noninvasive, cost-effective amyloid-β (Aβ) blood-based test does not need to be overstated. Nevertheless, a thorough validation of these bioanalytical methods should be performed as a prerequisite for confident interpretation of clinical results. The aim of this study was to validate ELISA sandwich colorimetric ABtest40 and ABtest42 for the quantification of Aβ40 and Aβ42 in human plasma. The validation parameters assessed included precision, accuracy, sensitivity, specificity, recovery, and dilution linearity. ABtest40 and ABtest42 proved to be specific for their target peptide using Aβ peptides with sequence similar to the target. Mean relative error in the quantification was found to be below 7.5% for both assays, with high intra-assay, inter-assay, and inter-batch precision (CV <9.0% on average). Sensitivity was assessed by determination of the limit of quantification fulfilling precision and accuracy criteria; it was established at 7.60 pg/ml and 3.60 pg/ml for ABtest40 and ABtest42, respectively. Plasma dilution linearity was demonstrated in PBS; however, dilution in a proprietary formulated buffer significantly increased the recovery of both Aβ40 and Aβ42 masked by matrix interactions, allowing a more comprehensive assessment of the free and total peptide levels in the plasma. In conclusion, both assays were successfully validated as tools for the quantification Aβ40 and Aβ42 in plasma.

Plasma amyloid β 42/40 ratios as biomarkers for amyloid β cerebral deposition in cognitively normal individuals

“…In line with this, recent studies in CSF have reported better diagnostic performance for the Aβ42/40 ratio than for Aβ42 alone [42]. Furthermore, our approach allows a deeper knowledge of Aβ behavior in blood [32], by the assessment of Aβ bound to plasma components [43], [44], [45]. It is becoming increasingly clearer that sensitivity to cortical Aβ burden varied substantially among individuals and that the rate of SUVR change over time could be more relevant to predict disease progression than single cross-sectional measurements [46].…”

“…Aβ40 and Aβ42 peptides were quantified using ABtest40 and ABtest42, respectively (Araclon Biotech Ltd. Zaragoza, Spain), being blinded to all participant characteristics at the time of analysis. Analytical procedures and performance characteristics of these tests are described elsewhere [32].…”

“…With this in mind, we focused on the CN group of the Australian Imaging, Biomarker and Lifestyle Flagship (AIBL) Study using Ab-PET as the gold standard, supported by previous results showing an association between Ab42/ Ab40 plasma ratio and brain Ab levels [28][29][30][31]. Moreover, we have taken a comprehensive approach for the evaluation of Ab42/40 plasma ratio, differentiating the peptide fractions that are found free in plasma (FP42/40) from the total Ab peptides in plasma (TP42/40) and the amount of Ab that is bound to other plasma components (BP42/40), by means of validated enzyme-linked immunosorbent assays (ELISAs) [32]. In this study, the cross-sectional and longitudinal association of these plasma markers with brain Ab-PET results was evaluated, together with an assessment of their diagnostic performance and ability to predict brain Ab deposition trajectories, evaluating the potential of plasma Ab ratios as enrichment tools for secondary prevention clinical trials.…”

Plasma amyloid beta levels are associated with cerebral amyloid and tau deposition

“…Mean percentage calibration error was 2.2% for ABtest40 and 3.0% for ABtest42. The lower limit of quantification of the assays, empirically tested for precision and accuracy, are 7.6 pg/mL for ABtest40 and 3.6 pg/mL for ABtest42 [43]. All samples in our study were above these limits.…”

Neurofibrillary Tangles of Aβx-40 in Alzheimer’s Disease Brains

“…The anti A␤ x-40 and anti A␤ x-42 antibodies were purified by antigenaffinity chromatography using the same A␤ fragment inoculated to the rabbits (A␤ [33][34][35][36][37][38][39][40] and A␤ [35][36][37][38][39][40][41][42] , respectively). As described elsewhere, they have practically no cross-reactivity (less than 1%) between their target A␤ species nor with A␤ 1-38 (<2%), A␤ 1-43 (<1%) [31].…”