Hemagglutination Test (HAT) to detect antibodies against the RBD domain of the SARS2-Covid19 virus

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. We describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specicity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sucient for ten thousand test wells free of charge to qualied research groups anywhere in the world.

Incubate 1 hour at RT for Red Cells to form a pellet in the "-ve" well Tilt plate against a well-lit white background for ~30 seconds to allow Tear drop to form in "-ve" well.
The presence of antibodies to RBD is shown by loss of Tear Drop formation in the "T" and "+ve" wells. Occasionally a partial tear drop forms -these wells are counted as Negative.
Photograph the plate to record the results with the date and time. Results can be reviewed and tabulated later. Taking picture from a distance and using the zoom function helps to take a clear picture of all wells in a 96-well plate.
The negative (PBS) control should be done on every sample for comparison. The Positive control induced by CR3022 is used to check that all the reagents are working, and that the glycophorin epitope recognised by VHH(IH4) is present on the red cells. Absence of the IH4 epitope should be very rare (Habib et al., 2013). For setting up cohorts a positive control on every sample is therefore not necessary but should be included in every batch of samples.
15. If a 25 µL sample of blood was taken from the nger prick there should be 850 µL of the 1:40 diluted blood left. The red cells can be removed and a preparation of 1:40 O-ve red cells used as above to titrate the sample. In principle the autologous red cells could be washed x3, resuspended in the same volume of PBS, and used as indicators for the titration, however we have not attempted to do this. The supernatant is 1:40 plasma that can be used in con rmatory ELISA or other tests.

Troubleshooting
Time Taken 15 minutes manipulation, 60 minutes incubation.
Anticipated Results