Development and optimization of Gyrolab immunoassay targeting biomarker neurofilament light chain : Implementation of BOLD signal amplification on the Gyrolab platform

Immunoassays (IAs) are a collection of analytical tools used to detect and measure the concentration of different macromolecules such as proteins or nucleic acids in solutions. Referred to as biomarkers, these macromolecules tell us about different conditions in our bodies which makes them useful in a vast majority of different medical fields involving diagnostics, prognostics and drug development. Neurofilament light chain (NfL) is one example of a general biomarker indicating neuronal degradation, which is a typical hallmark for neurodegenerative diseases such as Alzheimer’s, Parkinson disorder and Multiple sclerosis. The aim of this project was to develop and optimize a sensitive immunoassay for detection and quantification of neurofilament light protein in human serum. The work was conducted at Gyros Protein Technologies using their highly flexible and automated immunoassay platform Gyrolab. With Gyrolab, immunoassays are performed at nanoliter-scale on microfluidic discs that generates fast results while covering a broad dynamic range. In this project, a NfL-assay was designed by initially testing different anti-NfL antibodies-pairs in a sandwich assay-setup. To increase the sensitivity of the assay, the Binding Oligo Ladder Detection (BOLD) signal amplification technique was applied on promising assay-candidates. The BOLD technique is a set of add-on reagents that expands the binding site for detection antibodies, generating more signal per antibody-antigen interaction. It works by conjugating primers to the original detection antibody to which enzymes, RNA-template and DNA-monomers are added. A DNA/RNA hybrid strand is synthesized which act as a binding spot for tertiary detection antibodies. The implementation of BOLD increased the signal significantly. Lowering the template-concentration further increased the sensitivity of the assay. The optimized assay was tested in human serum were LOD- and LLOQ-values were estimated to 2.28 pg/mL and 20pg/mL respectively. Although this study successfully applied a signal amplification strategy to a NfL-immunoassay previously untested on the Gyrolab platform, additional refinement is necessary to ensure and enhance assay sensitivity and reliability in serum matrices.