Investigating the complex transfer immunoassay approaches for plasma phosphorylated Tau-181

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disease and leading cause of dementia. Phosphorylated Tau-181 protein (pTau-181) is a specific biomarker for Alzheimer’s disease, but its concentrations in the blood are extremely low, therefore an extremely sensitive and specific assay is needed. Non-specific binding from the detection antibodies and antibody-antibody interactions are sensitivity limiting factors in conventional immunoassays. The aim of this study is to overcome the limitations by using an immune complex transfer (ICT) immunoassay. The ICT immunoassay is a two-step capture assay in which immune complexes are transferred from a first plate to a second plate. Thus, aiming to leave all non-specific binding on the first plate and allow for extremely sensitive background-free detection of complete immune complexes on the recapture plate. The aim of this study was to develop a highly sensitive ICT immunoassay for p-Tau-181 and achieve a limit of detection (LoD) of <1 ng/L.

In the ICT immunoassay, pTau-181 specific oligonucleotide-antibody conjugates were immobilized with biotinylated oligonucleotides. Antibody coated upconverting nanoparticles (UCNPs) were utilized in the detection. The formed complexes were eluted by specific oligonucleotides. The recapture on the second plate was done with immobilized Tau-specific antibodies. To achieve the highest elution and recapture efficiency, parameters affecting assay performance were studied, including the amount of eluting component, incubation times, and elution solution composition.

The assay reached a limit of detection (LoD) of 0.6 ng/L. In the first capture plate the elution efficiency was 48 % and recapture efficiency of the eluted complexes was 26 % in a spiked sample buffer (100 ng/L). The assay background signals were decreased close to the instrumental background signals on the recapture plate.

In the study the results indicate that the used platform in the ICT immunoassay is a highly promising method for detecting pTau-181 with extremely high sensitivity.