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Review 2: "Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 antibodies in patient plasma"

This pre-print offers a FRET-based diagnostic platform that is faster and less labor-intensive than ELISA-based diagnostics. The claims should be considered reliable.

Published onNov 15, 2020
Review 2: "Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 antibodies in patient plasma"
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key-enterThis Pub is a Review of
Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 antibodies in patient plasma

The human beta coronavirus SARS-CoV-2, causative virus of COVID-19, has infected more than 15 million people globally and continues to spread. Widespread, population level testing to detect active and past infections is critical to curb the COVID-19 pandemic. Antibody (serological) testing is the only option for detecting past infections outside the narrow window accessible to nucleic acid-based tests. However, currently available serological assays commonly lack scalability. Here, we describe the development of a rapid homogenous serological assay for the detection of antibodies to SARS-CoV-2 in patient plasma. We show that the fluorescence-based assay accurately detects seroconversion in COVID-19 patients from less than 1 microliter of plasma. Using a cohort of samples from COVID-19 infected or healthy individuals, we demonstrate detection with 100% sensitivity and specificity. This assay addresses an important need for a robust, low barrier to implementation, and scalable serological assay with complementary strengths to currently available serological platforms.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.



The manuscript by Yue et al. documents the development of a TR-FRET assay for measuring concentration of spike/RBD binding antibodies in serum samples using an established patient cohort. Appropriately the authors tested the assay for optimal labeling (donor vs receptor labels), functionality with different antibody classes, specificity and background with RBD vs spike usage, the effect of serum on sensitivity and the degree of labeling of the spike protein. The assay performed excellently and comparable to the standard ELISA assay. The limit of detection is in the low ng/ml level though the presence of serum reduces sensitivity to some degree.

The assay is simple, rapid, and sensitive. A great advantage over ELISA is that there is no need for washing. ELISA give a stronger signal at the lowest concentrations, but the TR-FRET has lower background which allows clear discrimination.

TR-FRET seems to have a broader range in that there doesn’t seem to be a ceiling effect as occurs with ELISA at high antibody concentrations. There is really no discussion of range or quantitation as the point of the assay as described is simplicity, specificity, and speed. The one step preparation without a need for washing contributes to the reproducibility and low background of the assay. However, this is also responsible for the strong prozone effect observed as well as the reduction in sensitivity caused by serum. These are minor problems and, overall, the assay works equivalently to the ELISA but is simpler and more rapid.

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