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

Abstract

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.

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

“…so many months after the initial vaccination), or individually, based on the monitoring the levels of neutralizing antibodies. For such an individually-based approach, HAT would seem to be a particularly appropriate solution since it is a very simple and cheap test based on the binding of antibodies to the RBD domain (Joly and Maurel Ribes, 2022; Townsend et al, 2021), which are those with neutralizing activity. Furthermore, because the only reagent in HAT simply comprises small amounts of soluble IH4-RBD protein, the hemagglutination test can be very easily adapted to detect antibodies binding to variant forms of the virus (Jayathilaka et al, 2021).…”

“…In the initial version of HAT, however, such a quantification can only be performed in a laboratory environment because it requires separation of the plasma or serum from the red blood cells. Making use of the Jurkat-S&R-flow test as a reference, we have been able to develop an adapted version of HAT called HAT-field, which can be performed pretty much anywhere, with no specialized equipment, and can provide a quantitative evaluation of the levels of antibodies in a single step (Joly and Maurel Ribes, 2022).…”

“…To be able to perform HAT on whole blood with the sensitivity and simplicity which we set out the reach, and to attempt to make it quantitative, various modifications and improvements had to be tested, and in order to do this, we felt that we needed a simple quantitative test that would allow us to evaluate the amount of antibodies present in the whole-blood samples we were using more simply and cheaply than by using ELISA or CLIA. Thanks to the use of the Jurkat-S&R-flow test, we have now managed to derive an adapted protocol of the HAT test called HATfield, which can provide a serological quantitative evaluation of the antibodies directed against the SARS-CoV-2 RBD is a single step, starting from a few microliters of blood such as can be obtained by a finger-prick, and requires absolutely no specialized equipment (Joly and Maurel Ribes, 2022) For validating the adaptation of the HAT test, we had initially turned to the S-flow test (Grzelak et al, 2020), which uses flow cytometry (FACS) performed on human cells expressing the S protein. The S-flow test appeared as a very promising approach since it is very simple to run and its performances compared favorably with three other serological tests (two ELISA directed towards the S or N proteins, and a luciferase immuno-precipitation system (LIPS) combining both N and S detection).…”

“…To be able to perform HAT on whole blood with the sensitivity and simplicity which we set out the reach, and to attempt to make it quantitative, various modifications and improvements had to be tested, and in order to do this, we felt that we needed a simple quantitative test that would allow us to evaluate the amount of antibodies present in the whole-blood samples we were using more simply and cheaply than by using ELISA or CLIA. Thanks to the use of the Jurkat-S&R-flow test, we have now managed to derive an adapted protocol of the HAT test called HAT-field, which can provide a serological quantitative evaluation of the antibodies directed against the SARS-CoV-2 RBD is a single step, starting from a few microliters of blood such as can be obtained by a finger-prick, and requires absolutely no specialized equipment (Joly and Maurel Ribes, 2022)…”