A simple, sensitive and quantitative FACS-based test for SARS-CoV-2 serology in humans and animals

Ribes, Agnès; Bessière, Pierre; Guéry, Jean-Charles; Featherstone, Eloïse Joly; Bruel, Timothée; Robinot, Rémy; Abravanel, Florence; Izopet, Jacques; Joly, Etienne

doi:10.1101/2021.09.06.21262027

Cited by 1 publication

(10 citation statements)

References 67 publications

(114 reference statements)

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“…One limitation of HAT is that it is not as sensitive as ELISA, CLIA (chemiluminescence immunoassay) or FACS (Lamikanra et al, 2021; Maurel Ribes et al, 2021). As we have seen above (Figure 4), increased sensitivity can be attained by the use of more IH4-RBD reagent, but we found that another way to increase HAT sensitivity was to perform prolonged incubations.…”

Section: Resultsmentioning

confidence: 99%

“…The results obtained by HAT-field on these 60 whole blood samples were compared with those obtained by testing the plasma from the same samples by using the original HAT protocol with donor RBCs (Townsend et al, 2021), and by using the FACS-based Jurkat-S&R-flow test (Maurel Ribes et al, 2021), which is very sensitive, quantitative, and allows isotyping of the antibodies reacting against the S protein (Figure 6).…”

Section: Resultsmentioning

confidence: 99%

“…For the tilting and the photographing, we find it convenient to use a very simple home-made light box (see tutorial) and a standard smart phone camera. Figure 5: Schematic description of the HAT-field protocol One limitation of HAT is that it is not as sensitive as ELISA, CLIA (chemiluminescence immunoassay) or FACS (Lamikanra et al, 2021;Maurel Ribes et al, 2021). As we have seen above (Figure 4), increased sensitivity can be attained by the use of more IH4-RBD reagent, but we found that another way to increase HAT sensitivity was to perform prolonged incubations.…”

Section: The Hat-field Protocolmentioning

confidence: 86%

“…At that time, over 85% of the adult population had been vaccinated in France, and we thus expected a large proportion of the samples to be seropositive against the S protein of SARS-CoV-2, albeit at various levels. The results obtained by HAT-field on these 60 whole blood samples were compared with those obtained by testing the plasma from the same samples by using the original HAT protocol with donor RBCs (Townsend et al, 2021), and by using the FACS-based Jurkat-S&Rflow test (Maurel Ribes et al, 2021), which is very sensitive, quantitative, and allows isotyping of the antibodies reacting against the S protein (Figure 6). positive samples in the Jurkat-S&R-flow.…”

Section: Validation Of the Hat-field Protocolmentioning

confidence: 99%

“…Briefly, Jurkat-S and Jurkat-R cell lines, obtained and grown as previously described (Maurel Ribes et al, 2021), were resuspended in their own tissue culture medium at a concentration of 2.2 10 6 cells/ mL before pooling equal volumes of the two. Plasmas to be tested were diluted 1/10 in PFN (PBS / 2% FCS / 200 mg/L sodium azide ).…”

Section: Jurkat-sandr-flow Testmentioning

confidence: 99%

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HAT-field: a cheap, robust and quantitative point-of-care serological test for Covid-19

Joly

Ribes

2022

Preprint

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We have recently described a very simple and cheap serological test called HAT to detect antibodies directed against the RBD of the SARS-Cov-2 virus. HAT is based on hemagglutination, triggered by a single reagent (IH4-RBD) comprised of the viral RBD domain fused to a nanobody specific for glycophorin, which is expressed at very high levels at the surface of human red blood cells (RBCs). One of the main initial goals of this study was to devise a test protocol that would be sensitive and reliable, yet require no specialized laboratory equipment such as adjustable pipets, so that it could be performed in the most remote corners of the world by people with minimal levels of training. Because antibody levels against the viral RBD have been found to correlate closely with sero-neutralisation titers, and thus with protection against reinfection, it has become obvious during the course of this study that making this test reliably quantitative would be a further significant advantage. We have found that, in PBN, a buffer which contains BSA and sodium azide, IH4-RBD is stable for over 6 months at room temperature, and that PBN also improves HAT performance compared to using straight PBS. We also show that performing HAT at either 4°C, room temperature or 37°C has minimal influence on the results, and that quantitative evaluation of the levels of antibodies directed against the SARS-CoV-2 RBD can be achieved in a single step using titration of the IH4-RBD reagent. The HAT-field protocol described here requires only very simple disposable equipment and a few microliters of whole blood, such as can be obtained by finger prick. Because it is based on a single soluble reagent, the test can be adapted very simply and rapidly to detect antibodies against variants of the SARS-CoV-2, or conceivably against different pathogens. HAT-field appears well suited to provide quantitative assessments of the serological protection of populations as well as individuals, and given its very low cost, the stability of the IH4-RBD reagent in the adapted buffer, and the simplicity of the procedure, could be deployed pretty much anywhere, including in the poorest countries and the most remote corners of the globe.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: The Hat-field Protocolmentioning

confidence: 86%

Section: Validation Of the Hat-field Protocolmentioning

confidence: 99%

Section: Jurkat-sandr-flow Testmentioning

confidence: 99%

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