Enhancing Sensitivity of Alzheimer’s Biomarker Detection Using BOLD (Binding Oligo Ladder Detection) : Development and Optimization of the BOLD-amplified Standard ELISA Assay

Sammanfattning

ELISA (Enzyme-Linked Immunosorbent Assay) is an analytical tool frequently used for the detection and quantification of biomolecules. It is frequently used within diagnostics for measurement of biomarkers such as hormones, antibodies or proteins related to different diseases. Levels of biomarkers often vary inhealthy and diseased patients; it can therefore be a very helpful approach for assessing patient status. Amyloid beta (Aβ) proteins are one of the most prominent biomarkers associated with Alzheimer's disease. They stem from incorrect cleavage of the amyloid precursor protein (APP) creating toxic peptides with high tendencies to aggregate and form large, insoluable structures called plaques. Measurement of Aβ is often done in cerebrospinal fluid (CSF) or through positron emission tomography (PET), methods that poses many challenges. As an alternative, researchers have begun investigating possible measurements of Aβ in blood, often requiring special signal amplification techniques due to low concentration levels. This project aimed to develop and optimize a signal amplified sandwich ELISA assay that can detect and quantify Aβ in blood. The center of this assay is the add-on reagent Exazym® containing Cavidi’s Binding Oligo Ladder Detection (BOLD) technology that uses primer-conjugated secondary antibodies, DNA-synthesis, and DNA-specific tertiary antibodies to amplify the signal and increase the sensitivity of the assay. By optimizing assay components and protocol parameters such as incubation time, blocking buffers and antibody concentrations, the background noise was reduced from 0.6 OD to 0.1 OD. Furthermore, in comparison to the standard ELISA assay, the increase of signals with BOLD-amplification ranged from 2-fold to 10-fold with increasing Aβ concentration. Lastly, the initial estimation of assay performance illustrated great potential regarding assay sensitivity with an LLOQ of 1.42 pg/mL, a value in the range of similar products available on the market.