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HAT – accessible COVID-19 antibody testing for the global population

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Serodiagnostic tests detect antibodies after infection with, or vaccination against, SARS-CoV-2, the virus that causes COVID-19. This helps monitor the progress of the disease in the community and identify patients that need booster vaccinations. These tests have not been available all over the world, however, mostly because of their high cost and requirement for sophisticated equipment. Dr Etienne Joly at the Institute of Pharmacology and Structural Biology, University of Toulouse, France, has developed a quantitative serodiagnostic test for COVID-19 that is both affordable and easy to use, even in the most unlikely and challenging of settings.

The COVID-19 pandemic so far counts almost 400 million cases and over five million deaths globally. Scientists have been working on novel methods to tackle the disease, including diagnostic tests and vaccines. Diagnostic tools commonly used to detect the presence of virus in the human body are the polymerase chain reaction (PCR) test (a highly accurate test that detects even tiny amounts of virus RNA) and the rapid, lateral flow tests (that detect virus proteins). Serodiagnostic tests, by contrast, detect antibodies in the blood, which are proteins produced by the immune system after the patient has been infected with or vaccinated against the virus. Serodiagnostic tests not only give an indication of the body’s ability to fight off the virus, but also allow scientists and doctors to track the disease’s progress in the population and identify patients that need booster vaccinations.

Despite their benefits, however, these tests are not available to everyone who needs them. They are usually performed in a laboratory with specialised equipment, which is readily available in wealthy countries but not so easily accessed in remote locations or countries with poor medical provision. Dr Etienne Joly at the Institute of Pharmacology and Structural Biology (IPBS) of the University of Toulouse, France, has been developing a cost-effective and easy-to-apply method of detecting patients’ levels of antibodies against the severe acute respiratory syndrome virus, SARS-CoV-2 – the causative agent of COVID-19.

The haemagglutination test – HAT

Haemagglutination is the phenomenon in which red blood cells clump together to form aggregates. In their initial 2021 study, based on work carried out mostly at Oxford University, UK, the research team described HAT (short for haemagglutination test) to detect antibodies directed against the receptor binding domain (RBD) of SARS-CoV-2. The RBD is an area on the tip of the SARS-CoV-2 spike protein that is responsible for binding the virus to the host cell it is going to infect. Most neutralising antibodies are directed against this domain.

In HAT, haemagglutination is triggered by mixing a sample of the patient’s blood with a synthetic protein reagent that coats the red blood cells with the RBD domain of the virus spike protein, so ‘disguising’ the cells as virus. If the patient’s blood contains antibodies against the RBD, these will bind to the RBD domain presented by the red cells and cause them to clump, or agglutinate. In this case, the agglutination can be seen by the naked eye as a red ‘button’ of aggregated cells formed in the conical wells of the plastic plates used for the assay. If the patient’s blood does not contain antibodies against the RBD domain of the virus, the red cells would not agglutinate and will form red ‘teardrops’ when the wells are tilted. Thus, formation of a button rather than a teardrop reveals the presence of antibodies in the patient’s blood. This simple assay presented the opportunity to develop an antibody detection test that is both accurate and inexpensive to make.

Developing the HAT-field protocol

Based on the findings of the initial study, Joly decided to develop a testing protocol that could be used without specialised laboratory equipment such as adjustable pipettes. The goal was to produce a test that would be quantitative, cheap to use all around the world as a point-of-care (POC) test, and simple enough to be performed without specialist training.

For HAT to be performed in the field, Joly had to make a number of changes to the materials used. To simplify the procedure the reagent would have to be pre-distributed in the polystyrene wells used for the assay. To prevent the polystyrene wells from interacting with the IH4-RBD, the test’s sole reagent, and altering the results, Joly found that a mixture of BSA (bovine serum albumin) and sodium azide (NaN3) could be added to the dilution buffer, which also helped the reagent remain stable at a wide range of temperatures, eliminating the need for refrigeration. A further refinement using serial dilutions of the IH4-RBD reagent made the test quantitative. The method was tested on 60 blood samples from patients of Toulouse University Hospital and validated by comparing the results to those obtained by another quantitative, instrumentation-based serological test, recently described by the same research group (Ribes et al).

“If the patient’s blood contains antibodies against the RBD, these will bind to the RBD domain presented by the red cells and cause them to clump, or agglutinate.”

Performing the HAT-field test

The equipment needed for one test includes a single-use lancet (like that used daily by diabetics), a plastic disposable dropper pipette, a plastic tube to dilute a droplet of the patient’s blood in a solution of anti-coagulant, and eight conical wells containing a range of concentrations of the IH4-RBD reagent. The procedure starts with puncturing the skin on a clean fingertip, collecting the blood droplet using the pipette, and diluting it in the solution contained in the plastic tube. The solution is then collected with the pipette and a drop of it is placed into each of the eight wells. These wells contain seven different concentrations of the reagent IH4-RBD; the eighth well is a negative control. The plate is then incubated at room temperature for 60 minutes and is then tilted and photographed. The final step is to score the tests, which involves counting the number of wells that are fully agglutinated; the score ranges from zero to seven and corresponds to specific levels of antibodies against the RBD of SARS-CoV-2.

The team’s homemade lightbox for photographing HAT plates. youtu.be/e5zBYd19nIA

Fine-tuning HAT for widespread use

To make the results easier to see and interpret, Joly found that the plate translucency and contrast could be increased by using a simple home-made light box; he created a tutorial for constructing this on YouTube. Since the main disadvantage of this test is the long incubation period, he also found that centrifuging the plates briefly can shorten this phase to 15 minutes while increasing sensitivity. In remote locations with no access to lab equipment, creative methods can be used to centrifuge the wells, such as using adapted salad-spinners.

With these modifications, the HAT-field test offers a cost-effective and simple procedure that can be used even in the most deprived countries by people with no specialist medical training. Since it requires only cheap, disposable instruments and a very stable reagent, it can be used in almost any setting and at any temperature, making it available to people in the most remote locations. Such a widely available test would be a valuable tool for the quantitative detection of antibodies against the SARS-CoV2 and its variants, helping us trace and defend against the virus around the world.

What is the next step your team will take towards making the HAT test available to the world?
Identifying private and institutional partners that could help to further improve, validate, and distribute this test. This could involve the manufacture of strips of wells suited for individual tests rather than having to use plates with 96 wells. A further advantage would be that those would fit easily in hand-driven centrifuges of the salad-spinner type. Another avenue to explore will be to use HAT for other infectious diseases such as AIDS, tuberculosis, and malaria.

 

References

  • Joly, E, Ribes, AM, (2022) HAT-field: a very cheap, robust and quantitative point-of-care serological test for Covid-19. MedRxiv [online]. doi.org/10.1101/2022.01.14.22268980
  • Ribes, AM, Bessière, P, Guéry, JC, et al, (2021) A simple, sensitive and quantitative FACS-based test for SARS-CoV-2 serology in humans and animals. MedRxiv [preprint, online]. doi.org/10.1101/2021.09.06.21262027
  • Townsend, A, Rijal, P, Xiao, J, et al, (2021) A haemagglutination test for rapid detection of antibodies to SARS-CoV-2. Nature Communications [online], 12, 1951. doi.org/10.1038/s41467-021-22045-y
  • Lightbox instructions: youtu.be/e5zBYd19nIA
DOI
10.26904/RF-140-2387954619

Research Objectives

Developing a cost-effective and easy-to-use method of detecting patients’ antibodies against SARS-CoV-2, which could be used in the most remote locations.

Funding

ANR (HAT-field project)

Collaborators

Professor Alain Townsend (Oxford), with whom we devised the HAT approach and designed the IH4-RBD reagent.

Bio

Etienne trained as an MD in Paris and gained a BSc in immunology, virology, and genetics. After training as a postdoc at Scripps in La Jolla, USA, and as a junior group leader at the Babraham Institute in Cambridge, UK, he moved to Toulouse in 2000. His broad interests include immunogenetics, trogocytosis (which he co-discovered with Professor Denis Hudrisier), membrane biophysics, the mechanisms of speciation, and the development of a novel class of bio-inspired cytotoxic drugs. Over the years, Etienne has published more than 80 scientific papers, as well as contributing over 130 article evaluations on the Faculty Opinions platform. He has a particular interest in making science accessible and has actively supported the transition of scientific publishing towards open access for many years.

Dr Etienne Joly Photo Credit: Laura Petenatti

Contact
Institute of Pharmacology and Structural Biology (IPBS)
University of Toulouse, CNRS, Toulouse, 31000, France

E: [email protected]
T: +33 561175870
W: orcid.org/0000-0002-7264-2681
W: facultyopinions.com/Etienne Joly

Creative Commons Licence

(CC BY-NC-ND 4.0) This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Creative Commons License

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