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

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 (A beta(42)) peptide, a key player in AD pathogenesis. The INNOTESTA (R) A beta(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 a parts per thousand currency sign12.1 % CV and the inter-assay relative accuracy was a parts per thousand currency sign10.9 % absolute bias, bringing the total error of the assay to a parts per thousand currency sign23 %. The intra-assay precision of the assay at the high validation standard and below was a parts per thousand currency sign5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTESTA (R) A beta(42) ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.