Diagnostic TR-FRET assays for detection of antibodies in patient samples

• Published in: Cell reports methods Vol. 3; no. 3; p. 100421
• Main Authors: Yue, Hong, Nowak, Radosław P., Overwijn, Daan, Payne, N. Connor, Fischinger, Stephanie, Atyeo, Caroline, Lam, Evan C., St. Denis, Kerri, Brais, Lauren K., Konishi, Yoshinobu, Sklavenitis-Pistofidis, Romanos, Baden, Lindsey R., Nilles, Eric J., Karlson, Elizabeth W., Yu, Xu G., Li, Jonathan Z., Woolley, Ann E., Ghobrial, Irene M., Meyerhardt, Jeffrey A., Balazs, Alejandro B., Alter, Galit, Mazitschek, Ralph, Fischer, Eric S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.03.2023; Elsevier
• Subjects: Antibodies, Viral; CoraFluor; COVID-19 - diagnosis; COVID-19 Testing; Fluorescence Resonance Energy Transfer; Humans; neutralization assay; Nucleocapsid; SARS-CoV-2; serological testing; TR-FRET assays; vaccine efficacy studies; CP: Immunology; neutralization assay; CoraFluor; TR-FRET assays; serological testing; vaccine efficacy studies
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2023.100421

Serological assays are important diagnostic tools for surveying exposure to the pathogen, monitoring immune response post vaccination, and managing spread of the infectious agent among the population. Current serological laboratory assays are often limited because they require the use of specialized laboratory technology and/or work with a limited number of sample types. Here, we evaluate an alternative by developing time-resolved Förster resonance energy transfer (TR-FRET) homogeneous assays that exhibited exceptional versatility, scalability, and sensitivity and outperformed or matched currently used strategies in terms of sensitivity, specificity, and precision. We validated the performance of the assays measuring total immunoglobulin G (IgG) levels; antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle Eastern respiratory syndrome (MERS)-CoV spike (S) protein; and SARS-CoV-2 S and nucleocapsid (N) proteins and applied it to several large sample sets and real-world applications. We further established a TR-FRET-based ACE2-S competition assay to assess the neutralization propensity of the antibodies. Overall, these TR-FRET-based serological assays can be rapidly extended to other antigens and are compatible with commonly used plate readers. [Display omitted] •Homogeneous TR-FRET assay can accurately detect IgG levels in patient serum samples•TR-FRET assay can rapidly be extended to survey antibodies against different antigens•TR-FRET assay is compatible with diverse serological sample types Monitoring immune response is a crucial component of management and tracking of the spread of infectious diseases. Current serological testing laboratory assays are limited by the need for specialized automation, sample types, and cost. Time-resolved Förster energy transfer (TR-FRET) homogeneous assays provide an alternative, but their scope and limitations have not been extensively tested in a large-scale study. To address this, we have developed a suite of TR-FRET-based serological assays used to detect antigen-specific antibodies as well as total IgG levels, validated them in a large cohort study (>1,500 samples), and showed that they maintain high reproducibility and repeatability. TR-FRET assays perform on par or better than alternative laboratory tests that have previously been evaluated on the same set of samples while reducing the time to result (<1 h) and the costs per sample. Yue et al. develop a homogeneous TR-FRET assay for the detection of antibodies for specific antigens in human serum, plasma, and blood samples. The assay requires only a 1 μL sample, spans 1 h from sample to readout, and is easily adaptable to new antigens.