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Quantitation of Interleukin-10 using a Novel Enzyme-linked Darkening Assay (ELDA)

Tian Yu, Ph.D. – Bioelectronica

Abstract:

Antibody-based immunoassays are powerful tools used throughout the life sciences including drug discovery, diagnostic tests, and academic research in the STEM fields. This paper introduces the enzyme- linked darkening assay (ELDA), a novel hydrogel particle-based suspension array immunoassay that offers ease of use, adaptability to ELISA antibodies, a large capture area, and does not require expensive instruments for detection. ELDA achieves this using a novel chemistry that encapsulates the immunoassay complex and the enzymatic product within porous hydrogel beads. The beads are prepared using a proprietary method in which the mesh size and rigidity can be controlled. In an ELDA assay, the capture antibodies are immobilized in the beads’ inner matrix by anchors. As these “capture” beads are incubated with antigen, the antigen molecules bind to the capture antibodies and accumulate within the beads, which are then incubated with AP-conjugated detection antibodies and detection reagent. The reporter enzyme converts the soluble substrate into an insoluble product that is trapped within the matrix of the beads with picoliter volumes, resulting in progressive darkening of the beads based on antigen concentration. Unlike most particle-based immunoassays that measure fluorescence as signal outputs, the optical darkness of ELDA beads is measured as a function of antigen concentration using brightfield microscopy and computer vision detection, and can be imaged using Bioelectronica’s proprietary lensless imaging system. The computer vision workflow includes image processing methods to locate each bead and determine the grayscale intensity of the bead centroid region. The rapid imaging of thousands of beads provides grayscale distributions and statistically rigorous confidence intervals. As an initial proof of concept experiment, we demonstrate here that the ELDA assay can detect IL-10, a major inflammation biomarker, using standards prepared at concentrations of 48, 480, and 4800 pg/mL with a total hands on time of < 2 hrs. The limits of detection for a variety of biomarkers are still under investigation.


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