Analytical Chemistry | May 25, 2023
Zhang J, Wiener AD, Meyer RE, Kan CW, Rissin DM, Kolluru B, George C, Tobos CI, Shan D, Duffy DC.
Anal Chem. 2023
https://doi.org/10.1021/acs.analchem.3c00918
Abstract
We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aβ-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.
Related Posts
Neurology
Comparative Spatial Analyses of the Tumor Immune Landscape in Mouse Models of Glioblastoma Using the PhenoCode Discovery Mouse Neuro Panel and Customized Markers
Poster Background Developing effective therapies for glioblastoma (GBM) relies on robust preclinical animal models that enable detailed investigation of tumor biology, discovery of therapeutic targets, and evaluation of new treatment…
Oncology
Development of a Spatial Metabolic Map of Immunotherapy Sensitive and Resistant Cutaneous Skin Carcinoma
Poster Background Ultrahigh-Plex Single-cell Spatial Phenotyping has revealed valuable insights into the tumor immune microenvironment (TiME) for biomarker discovery and stratification of clinical responses. Metabolic reprogramming is a key hallmark…
Spatial Immunometabolic Profiling of Non-Small Cell Lung Cancer Reveals Distinct Immune Exclusion Patterns and Functional Niches
Analytical validation of a multiplex immunofluorescence 7-Biomarker Immunoprint® assay on the Akoya PhenoImager HT platform for stage I/II Melanoma
Neurology
Neurodegenerative biomarkers in different chambers of the eye relative to plasma: an agreement validation study
Alzheimer’s Research and Therapy | August 26, 2024 Sampani K, Ness S, Tuz-Zahra F, Aytan N, Spurlock EE, Alluri S, Chen X, Siegel NH, Alosco ML, Xia W, Tripodis Y,…
Neurology
Clinical validation of the LucentAD p-Tau 217 as a lab developed test (LDT) for clinical use
Poster The intersection of new therapeutic options for Alzheimer’s disease (AD), the emergence of accurate blood-based tests for AD, and the anticipated health system log jam for confirmatory diagnostic testing…
Neurology
Development of thresholds and a visualization tool for use of a blood test in routine clinical dementia practice
Alzheimer’s & Dementia | August 3, 2024 Verberk IMW, Jutte J, Kingma MY, Vigneswaran S, Gouda MMTEE, van Engelen MP, Alcolea D, Arranz J, Fortea J, Lleó A, Chevalier C,…
Neurology
Discovering novel plasma biomarkers for ischemic stroke: Lipidomic and metabolomic analyses in an aged mouse model
Journal of Lipid Research | August 2, 2024 Becktel DA, Frye JB, Le EH, Whitman SA, Schnellmann RG, Morrison HW, Doyle KP. J Lipid Res. 2024 https://doi.org/10.1016/j.jlr.2024.100614 Abstract Ischemic stroke…