Analytical validation of a chemiluminescent immunoassay for plasma Aβ1–40 and exploratory clinical associations in neurological disorders

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Background Alzheimer’s disease (AD) remains a major unmet medical need. Increasing evidence indicates that disease-modifying therapies are most likely to be effective when initiated early, underscoring the need for accessible biomarkers that enable detection at the earliest stages. Aβ1–40 is a clinically relevant amyloid-β species, but current methods for its measurement are often limited by cost, complexity, and turnaround time. Accordingly, there is a need for a simple, rapid, and scalable assay for Aβ1–40 detection. Methods We developed a chemiluminescent sandwich immunoassay for plasma Aβ1–40 using two highly specific monoclonal antibodies and magnetic-bead separation. Assay conditions were optimized by titrating bead concentration and the working concentrations of a biotinylated capture antibody and an acridine ester-labeled detection antibody. Analytical performance was validated for linearity, limit of blank (LoB), limit of detection (LoD), limit of quantification (LoQ), accuracy, precision, stability, specificity (cross-reactivity), and method comparison. Plasma Aβ1–40 was measured in 222 participants. Results The assay showed excellent analytical performance: linearity r > 0.99, LoB 0.58 pg/mL, LoD 1.45 pg/mL, LoQ 3.93 pg/mL, accuracy bias and precision (CV) < 5%, 12-month stability, and no detectable cross-reactivity. Method comparison demonstrated strong agreement with a commercial kit (r = 0.9939; Bland-Altman bias –0.04 pg/mL). In an exploratory clinical analysis, plasma Aβ1–40 was significantly elevated in AD and Parkinson’s disease (PD) compared with healthy controls. After 10-fold cross-validation, the mean area under the ROC curve (AUC) for AD versus controls was 0.94 (95% CI 0.89–0.98) and for PD versus controls was 0.92 (95% CI 0.87–0.96). In cerebrovascular disease, Aβ1–40 increased in acute and lacunar infarction but not in old infarction. Conclusion This CLIA enables sensitive and reliable plasma Aβ1–40 quantification. The clinical associations are promising but exploratory; independent external validation is required before any diagnostic application.

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