A rapid, point of care red blood cell agglutination assay for detecting antibodies against SARS-CoV-2
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Abstract
The COVID-19 pandemic has brought the world to a halt, with cases observed around the globe causing significant mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess the prevalence of infection, as well as ascertain individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which may lack scalability to meet the demand of hundreds of millions of antibody tests in the coming year. Herein, we present an alternative method of antibody testing that just depends on one protein reagent being added to patient serum/plasma or whole blood and a short five-minute assay time. A novel fusion protein was designed that binds red blood cells (RBC) via a single-chain variable fragment (scFv) against the H antigen and displays the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Upon mixing of the fusion protein, RBD-scFv with recovered COVID-19 patient serum and RBCs, we observed agglutination of RBCs, indicating the patient developed antibodies against SARS-CoV-2 RBD. Given that the test uses methods routinely used in hospital clinical labs across the world, we anticipate the test can be rapidly deployed with only the protein reagent required at projected manufacturing cost at U.S. cents per test. We anticipate our agglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.
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SciScore for 10.1101/2020.05.13.094490: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources For testing patient serum, a dilution series of RBD-scFv was performed to test for optimal levels of protein to induce agglutination in presence of patient anti-RBD antibodies. anti-RBDsuggested: NoneExperimental Models: Cell Lines Sentences Resources (hyperactive piggyBac transposase) were co-transfected using Lipofectamine 3000 into HEK 293T cells. HEK 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Software and Algorithms Sentences Resources One prep of 800 … SciScore for 10.1101/2020.05.13.094490: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources For testing patient serum, a dilution series of RBD-scFv was performed to test for optimal levels of protein to induce agglutination in presence of patient anti-RBD antibodies. anti-RBDsuggested: NoneExperimental Models: Cell Lines Sentences Resources (hyperactive piggyBac transposase) were co-transfected using Lipofectamine 3000 into HEK 293T cells. HEK 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Software and Algorithms Sentences Resources One prep of 800 μL supernatant through one column of the kit yielded 102 μg/mL of RBD-scFv measured by NanoDrop™ 2000/2000c Spectrophotometers (ThermoFisher), which was used in subsequent experiments. NanoDrop™suggested: NoneThermoFishersuggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:A key limitation of this assay is that it does not distinguish between IgG, IgA, or IgM against SARS-CoV-2, which may be desired in certain clinical scenarios. IgG subclasses can similarly not be distinguished. Also, the assay is highly dependent on the viral antigen deployed, such that clinicians should be aware if RBD antibody levels are negative, the patient could have still had COVID19. While the assay is simple and can be read with the naked eye, there is more subjectivity to it compared to interpreting lines on lateral flow assays or light detection in chemiluminescent ELISA’s. Depending on mixing technique and provider comfort, there could be variability, likely necessitating training and/or deployment of controls in order to ensure proper reading of the assay. A negative control test without fusion protein will be important to include during clinical implementation for patients with positive tests, given that rare patients may have IgM autoantibodies causing agglutination.31 Given the similarities of this test to currently used ABO typing32 and Monospot assays for EBV antibodies,33 we ultimately do not see this being a significant barrier. Lastly, the fusion protein stability is unknown, which is important for use in low-resource countries. Past studies with similar fusion proteins for hemagglutination assays found stability at least 30 days at 37°C and 6 months at 4°C with no loss of assay agglutination activity.14 Ultimately, similar investigations will be important...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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