Professor Matthias Wolf and his team have set up a blood antibody test that can be used to determine who has previously contracted COVID-19. The test could be used to screen residents in Okinawa to determine the true extent of the spread of the coronavirus.
Updated October 15, 2020
OIST Antibody test results released
In August 2020, all members of the OIST community were invited to submit a blood sample that was analyzed for COVID-19 antibodies. In total, 635 out of around 1200 people took part. The results found that none of these samples have the antibodies for COVID-19 and have been summarized in a manuscript submitted to the medRxiv pre-print server.
“This indicates, with a great level of certainty, that no one who submitted a sample had been infected with the virus,” said OIST provost and virologist, Dr Mary Collins. “My message is to keep up adherence to the COVID-19 guidelines on and off campus. “
Dr Collins emphasized that the antibody test established at OIST was very sensitive yet specific. It had reliably shown positive results when used on samples from Okinawa residents that were confirmed COVID-19 positive through PCR tests in April. It involved a two-step process, which meant that it was far more accurate than many other antibody tests conducted around the world.
“Testing volunteers has been a really useful exercise and has given us an idea of the challenges,” said Dr Collins. “The plan now is to test 6000 people in the Okinawa community over the next few months. This will give an indication of how many people in Okinawa have been infected.”
Dr. Hiroyuki Tahara, OIST’s Occupational Health and Safety Doctor, says that the results of this antibody test should not affect anyone’s behavior. “Keep adhering to the guidelines around the new normal – wear masks, wash your hands frequently, and social distance. This won’t change until a vaccine is widely available.”
Professor Matthias Wolf, from the Molecular Cryo-Electron Microscopy Unit, who supervised the project, added to this. “These results should tell everyone that we had no antibodies against this disease,” he said. “Having this knowledge is important because it emphasizes the need to follow health guidelines.”
Updated October 15, 2020
The OIST antibody test required an interdisciplinary approach
When a person is infected with a virus, their immune system makes antibodies, which help the body to fight the virus. The piece of the virus that the antibody attaches to is called an antigen. For SARS-CoV-2, the spike protein S, which is on the surface of the virus, is one of the main antigens. Antibodies can remain in the blood even after the virus has been killed off, and they can be detected using a method called Enzyme-Linked Immuno Sorbent Assay or ELISA. Thus, it is possible to test healthy individuals to determine if they have been infected and recovered from SARS-CoV-2.
Two-step process ensures accuracy
In early March, when the first ELISA assay for SARS-CoV-2 was developed by the Krammer Lab in New York, Professor Matthias Wolf had the shipped to Okinawa. These plasmids that encoded the spike protein and its receptor-binding domain. Professor Wolf had previously done work on the Ebola virus and wanted to try something similar for detecting SARS-CoV-2. His Molecular Cryo-Electron Microscopy Unit set about creating the OIST antibody test.
When the ELISA reagents arrived, staff scientist Dr. Tae Gyun Kim started to express and purify the spike proteins. This was necessary to test the samples to see if antibodies were present.
Meanwhile, the researchers also thought about how they would safely inactivate any samples received. “We had to centrifuge to separate the serum and heat the samples,” said staff scientist Dr. Satoshi Shibata. “We used a laboratory with biosafety level 2 containment and a dedicated centrifuge. We inactivated all the samples before we thought about testing them.”
Not all antibody tests are created equal. To maximize the accuracy of the test, the assay used at OIST consists of a two-step process.
The first step used one part of the spike protein to see if there were any antibodies in the sample that would bind to it. This was highly sensitive but not very specific to this virus, meaning that it could detect low levels of antibodies, but they weren’t necessarily from SARS-CoV-2
“If a serum sample was positive in step one, it was tested again in step two,” said Dr. Melissa Matthews, post-doctoral scholar in the Unit, who, alongside research assistant Noriko Shibata, was in charge of setting up the test. “Step two used the whole spike protein complex and a dilution series of the serum sample. This was highly specific so only samples which had been infected with the virus in the past several months would be positive in this test.”
The researchers showed this accuracy on control samples that they received from outside of OIST. The positive controls were samples that had tested positive for SARS-CoV-2 in PCR tests in April. The negative controls were serum samples from last year that were known not to have SARS-CoV-2 antibodies. In both instances, these controls were included on every assay plate and the test results rang true.
Finally, Christian Butcher, a technician from the Fluid Mechanics Unit, was instrumental in automating the testing process so the capacity could be expanded. Without this automation, the testing would have had to be performed manually, which would have been much less efficient.
Testing the OIST community
One issue with testing the OIST community was that samples needed to be provided without doctors to draw the blood. So, the researchers decided to use finger pricking. They ordered several different kits for this and tested them on themselves. They wanted something that was easy-to-use, safe, and inexpensive. “We ended up making our own kit by ordering individual components,” said Dr. Shibata.
Once the team had settled on the composition of the kit, they thought about the practicalities of testing volunteers anonymously. A QR code system was created and, with the help of the IT section, it was linked to a website, which included an instructional sheet and video. Each sample had a corresponding barcode and QR code. The barcode was left with the sample, whereas the participant kept and scanned the QR code to get their result. No personal information was retained nor required.
The final hurdle involved getting ethics approval. With the help of OIST’s Human Subjects Research Ethics Committee this was granted, and the tests were ready to be used by the OIST community in August 2020 at the onset of the second wave of infections on Okinawa.
“Our team really came together,” said Professor Wolf. “We had to work on a lot of different things – protein expression, ELISA testing, creating a website, QR code, sample and plate barcodes, 3D-printing tube racks, automation. We had people helping from other units across the University. It was really energizing for me and for my unit as a whole.”
Sometimes it proved difficult to extract enough serum from the blood droplet. These results were uploaded to the website as ‘not determined’ and individuals could repeat the test. In total, the researchers looked at 635 samples that were voluntarily given by the OIST community. None of these tested positive for COVID-19 antibodies. Their results have been summarized in a manuscript submitted to the medRxiv pre-print server. In collaboration with medical professionals, they have also tested 300 samples from clinics and a small cluster of infections in Naha. They will now test 6,000 samples from the Okinawan community provided in the scope of a contract with the Okinawan Prefectural Government.
Updated May 8, 2020
A team of OIST scientists, led by Professor Matthias Wolf from the Molecular Cryo-Electron Microscopy Unit, have set up a blood test that can detect the presence or absence of specific antibodies against SARS-CoV-2, the coronavirus that causes COVID-19.
Unlike PCR tests that detect RNA from the virus in currently infected patients, the blood test is used to determine who has previously contracted COVID-19. The specific immune response against the disease can be detected for a longer time after the virus is gone, but it is not yet known if this results in lasting immunity.
The antibody test being used at OIST is an enzyme-linked immunosorbent assay (ELISA), which was originally developed and validated by the Krammer Lab at the Icahn School of Medicine at Mount Sinai, New York.
“The test works by using parts from the SARS-CoV-2 virus surface called spike proteins, which act as antigens,” said Prof. Wolf. “When an individual is infected with the novel coronavirus, the immune system reacts to these antigens and produces specific antibodies which can bind to them.
“In the test, we expose blood serum to bound antigens. The serum of infected individuals, containing these specific antibodies, then bind to the antigens. In a series of stages, we can detect the quantity of antibodies that bind and therefore determine whether an individual had COVID-19.”
To establish the test, staff scientist, Dr. Tae Gyun Kim, with help from staff scientist Dr. Jaekyung Hyun and PhD student, Keon Young Kim, produced and purified components of spike proteins from genetic constructs supplied by the Krammer Lab. Spike proteins, which form the characteristic “crown” shape of coronaviruses, are key to allowing the virus to enter host cells.
Postdoctoral researcher, Dr. Melissa Matthews, is in charge of setting up the test. Currently, the team are awaiting positive control samples in order to validate the test.
Other units at OIST are now collaborating to further improve the antibody test. Dr. Saacnicteh Toledo-Patino, a postdoctoral researcher from the Protein Engineering and Evolution Unit, led by Professor Paola Laurino, is aiming to optimize the production of antigens, by using E. coli bacterial cells.
Meanwhile, Christian Butcher, a technician from the Fluid Mechanics Unit, led by Professor Pinaki Chakraborty, is working on automating the testing process to expand the daily testing capacity. Presently, the scientists are able to manually test around 1,000 samples each day.
“Ultimately, we hope that the test will be used to screen communities in Okinawa to fully understand the spread of COVID-19, helping officials make more informed decisions,” said Prof. Wolf.
A dialogue with the medical community on Okinawa has been established and the Prefectural Government has requested a pilot screen of 6,000 residents.