Development and Advanced Validation of an Optimized Method for the Quantitation of Aβ42 in Human Cerebrospinal Fluid

Abstract

Abstract. Cerebrospinal fluid (CSF) biomarkers have been extensively utilized in the diagnosis of Alzheimer's disease (AD) and characterization of progression. One important CSF biomarker is the amyloid beta 42 (Aβ 42 ) peptide, a key player in AD pathogenesis. The INNOTEST® Aβ 42 ELISA kit has been widely used but an advanced level of method development and validation has not been reported. To support a clinical trial in AD, we successfully completed a Good Laboratory Practices (GLP)-level validation of the method to establish the parameters of precision, accuracy, parallelism, selectivity, specificity, and linearity of dilution of the assay in CSF matrix, as well as CSF storage stability. Several modifications were required to optimize the assay and ensure consistent results in a clinical-trial setting. These included the use of additional calibrators, an adjusted standard curve range, a minimum required dilution (MRD) of CSF by 6-fold to avoid matrix interference and mitigation of analyte adsorption to labware by the addition of Tween-20. The optimized method displayed a quantitative range of 375-4,500 pg/mL. The inter-assay precision was ≤12.1 % CV and the inter-assay relative accuracy was ≤10.9 % absolute bias, bringing the total error of the assay to ≤23 %. The intra-assay precision of the assay at the high validation standard and below was ≤5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTEST® Aβ 42 ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.

Impact of cerebrospinal fluid matrix on the detection of Alzheimer's disease with Aβ42 and influence of disease on the total‐Aβ42/Aβ40 ratio

“…Cullen et al . ). If a ratio could be less affected by APOE status than Aβ1‐42 alone, then a ratio may also provide a more agnostic biomarker when unknown or complex genetic pressures are influencing the appearance of disease.…”

“…One property of current commercial Aβ1‐42 assays, contributing to the problems, is that they likely measure a poorly defined fraction of Aβ1‐42 present in a sample (Cullen et al . ; Slemmon et al . ).…”

Diagnostic Values of Cerebrospinal Fluid T-Tau and Aβ42 using Meso Scale Discovery Assays for Alzheimer's Disease

“…Although these assays are widely used and have been shown to perform well diagnostically in clinical and research settings [7–9], their analytical performance is less robust. A significant matrix effect has been reported for both platforms [10, 11] and the assays show substantial inter-laboratory variability, possibly originating from pre-analytical, analytical, post-analytical and kit manufacturing sources [12–14]. This might be one of the reasons that diagnostic cut-off values vary considerably between studies [15] such that currently, with the absence of absolute reference standards, each laboratory is recommended to have their own validated cut-offs to ensure adequate sensitivity and specificity [16].…”

Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

“…An initiative was started at the Sahlgrenska University Hospital in Mölndal, Sweden, to address this issue (Mattsson et al, 2011). A full validation study may lead to similar analytical refinements as has been achieved after a systematic validation of a commercially and widely used ELISA assay for quantification of Ab42 (Cullen et al, 2012). Moreover, because ELISA assays are susceptible to variation in assay performance, training of laboratory technicians by experienced assay performers could minimize the interlaboratory variances.…”