Optimizing immunoassay for ultra-sensitivity detection for pTau-181 for early Alzheimer’s disease diagnosis using upconverting nanoparticles

Abstract

Alzheimer disease (AD) is a progressive neurodegenerative disease affecting, which is estimated to affect 139 million people by 2050. The growing prevalence and severity of AD is pushing the research for tests capable of detecting early AD, giving better quality of life to the patients and easing the healthcare system burden. Biochemically, AD is characterized by formation of amyloid plaques and synaptic loss, with the presence of tau pathology and neurofibrillary tangles. Tau pathology results from hyperphosphorylated tau protein (p-Tau), with isoforms 181, 217 and 231 more common than others. P-tau can be detected in the cerebrospinal fluid and, at lower concentration in blood, years before symptoms onset, making it a favourable biomarker for the early detection of AD. The aim of this project is the optimization of an ultra-sensitive sandwich immunoassay able to reliably detect the presence of p-tau 181 in plasma, using upconverting nanoparticles (UCNPs) as a label, which provides the ability of the test to be measured again, in time, without signal decay. The target limit of detection (LOD) is 1 ng/L, thus aligning the assay to the commercially available Quanterix Simoa. The sandwich immunoassay was performed using streptavidin coated plates, biotinylated antibody, blockers and spiked p-Tau 181 in healthy plasma samples. The UCNP label used was UPCON®540-B coated with either polyacrylic acid (PAA) or C4 (Uniogen’s surface) and then conjugated with Anti-Tau-mAb. Each surface has been individually optimized in the assay, requiring different amounts and steps to achieve low sensitivity. The two different surfaces have been optimized in plasma matrix with their own specific requirements. When PAA surface is used with analyte pH wash 11, commercial blockers and 2.5% free PAA in UCNP buffer solution, the LOD achieved in plasma was 1.97 ng/L. In C4 surface, the LOD in plasma was 5.22 ng/L when tested with analyte pH wash 11, commercial blockers and 95% plasma + 5% commercial diluent.