A SARS-CoV-2 antibody magnetic immuno-detection test

A new report, released on the preprint server bio Rxiv* by researchers at the Fraunhofer Institute for Molecular Biology and Applied Ecology, describes the development of an innovative serologic test that could provide a fast, easy, and inexpensive method of detecting high titers of antibody in the serum of SARS-CoV-2 infected people.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing pandemic of COVID-19, which has affected over 6.59 million people worldwide and caused almost 400,000 deaths in just over five months. Asymptomatic infection is thought to be extremely common with this virus, making containment measures extremely difficult.

Why serologic testing?

The identification of infected individuals before they become symptomatic, if indeed they do, is therefore of the highest importance to public health measures. Another huge benefit is the ability to identify those who are immune, which could facilitate selective lockdown application in many regions while still allowing the resumption of economic activity, without unduly endangering public health.

Thirdly, the identification of infected individuals in an accurate manner can help to understand the actual disease potential, risk factors, and transmission characteristics of the virus. Finally, serologic testing will be useful to determine how strong and how lasting natural immunity is. For all such testing, it is essential to develop rapid, inexpensive, and conveniently applicable methods that can be used at the site of care. At present, there are few such available.

Current assays and their limitations

One currently used point-of-care testing system is the lateral flow assay, which offers fast and easy testing on-site but is not sensitive or specific enough, besides lacking the ability to measure antibody concentrations. These are therefore inferior to laboratory-based test formats that use enzyme-linked immunosorbent assay (ELISA).

Currently, the weak link in the serologic assays now available is the test antigen used. The strongest antigen is the spike (S) glycoprotein, which is targeted by neutralizing antibodies and is essential for virus entry into the host cell. The nucleocapsid (N) antigen is also immunogenic but cross-reacts with antibodies against other coronaviruses.

The S1 subunit of the S protein is typically recommended for specific detection of an immune response to SARS-CoV-2. Suitable tests, according to the above criteria, using this antigen, are not presently available.

The MInD Procedure

The use of Magnetic Immuno-Detection (MInD) could overcome this issue. This is a procedure based on immunofiltration, where an antigen-coated matrix is used to trap specific antibodies in the sample applied to the matrix by gravity flow. These antibodies are then detected using secondary antibodies.
Finally, specially designed magnetic nanoparticles (MNPs) are added to label the secondary antibodies, the unbound antibodies are washed out, and the magnetic read-out is then obtained with a portable device that uses frequency magnetic mixing detection technology (FMMD).

How the Study was Performed

The current paper reports a proof-of-concept study of the use of 
MInD vs. standard ELISA, in which  the former performs 
significantly better at specifically detecting SARS-CoV-2 antibodies
 in human serum.

The researchers used a peptide derived from the S protein of SARS-
CoV-2 to coat the immunofiltration columns. Next, reactive 
antibodies to this peptide were introduced at varying concentrations 
 into the columns and flushed through them. The specific antibodies
 bound to the coated antigen and were thus enriched in the column 
matrix.

They were then labeled using an isotype-specific biotinylated 
antibody. Then superparamagnetic streptavidin-functionalized 
magnetic particles were applied and flushed through the columns 
to label the secondary antibodies. After incubation, the excess 
MNPs were washed out, and the bound MNPs were detected using 
the FMMD-based portable magnetic reader.

The measuring signals correspond to the amount of bound antibody
 in the assayed sample.

Comparing the new method with laboratory-based ELISA, which 
was performed on the same sample as a reference point., threw up 
several observations.

Time-saving

The whole experiment took 161 minutes and can detect specific 
anti-SARS-CoV-2 antibodies at a range of 3.4 to 477 ng/mL. This 
is extremely sensitive with respect to the typical IgG levels in human 
serum, at about 10 mg/mL, and increasing when the individual is 
exposed to the antigen.

Higher range, broader sensitivity

The commercially available test kits for specific anti-S1-subunit 
antibodies can pick up IgG antibodies in three-fourths of samples
 within 10-20 days of infection. More sensitive assays might detect
 antibodies early in the course of infection.

However, when compared to the ELISA assay, the MInD assay 
was capable of detecting anti-SARS-CoV-2 antibodies in a range of 
2.95 to 2040 ng/mL, which is five times broader. This means the test 
is far more sensitive than ELISA, with a lower detection limit, and a 
wider range of detection. It also allows quantitative measurements 
due to the broad dynamic range.
The procedure took only 42 minutes, which is only a quarter of the 
 time required for the ELISA assay, which shows its potential for 
rapid testing. This can be further reduced by optimizing selected 
steps, such as reducing incubation time to 5 minutes or even less
 and using MNPs to which antigen-specific  antibodies are already 
bound. This would eliminate the need for incubation with secondary 
antibodies.With these adaptations, the total assay time would be 
below 20 minutes, which is comparable to that of a lateral flow
 assay.

Future Improvements and Implications

Other important innovations would be the use of antigens derived
 from the S1 subunit of the S protein,or multiple SARS-CoV-2 protein
 antigens in a mixture. Control antigens from other human 
coronaviruses should be tested to confirm the specificity of the 
enriched antibodies.

Multiplex testing using a range of MNPs could facilitate the 
detection of multiple subclasses of  antibodies in a single assay. 
This could be useful in that it not only demonstrates seroconversion 
but also the phase of the infection, and the course of antibody 
formation.Finally, optimizing the magnetic reader to a medical-
diagnostics instrument will make the test suitable  for such 
applications – such as including a barcode scanner and labeling 
the columns with patient-specific barcodes. The cost of the 
procedure is about a tenth or less of a typical ELISA,
 making it ideal for use in doctors’ offices.

The potential for the use of a single pipette for the assay procedure 
and the (perhaps battery-powered)  magnetic reader device makes 
the device appropriate for various medical applications. For 
instance, it could be used in nursing and elderly care homes, or at 
airports, for fast and convenient point-of-care testing.

Once a vaccine is available, the same method can be used to 
monitor the emergence of antibodies and  how long they last. Thus,
this technique can be used in multiple ways and at different time
 points, as a fast and convenient point-of-care measurement 
without extra cost or sophisticated equipment.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer
-reviewed and, therefore, should not be regarded as conclusive, 
 guide clinical practice/health-related behavior, or treated as 
established  information.

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Mount Sinai to Begin the Transfer of COVID-19 Antibodies into Critically Ill Patients

The Mount Sinai Health System this week plans to initiate a procedure known as plasmapheresis, where the antibodies from patients who have recovered from COVID-19 will be transferred into critically ill patients with the disease, with the expectation that the antibodies will neutralize it.

The process of using antibody-rich plasma from COVID-19 patients to help others was used successfully in China, according to a state-owned organization, which reported that some patients improved within 24 hours, with reduced inflammation and viral loads, and better oxygen levels in the blood.

Mount Sinai is collaborating with the New York Blood Center and the New York State Department of Health’s Wadsworth Center laboratory in Albany, with guidance from the U.S. Food and Drug Administration, and expects to begin implementing the treatment later this week.

“We are hoping to identify patients who can provide the antibodies,” says Dennis S. Charney, MD, Anne and Joel Ehrenkranz Dean of the Icahn School of Medicine at Mount Sinai, and President for Academic Affairs, Mount Sinai Health System. “We are at the front lines in fighting this pandemic and making discoveries that will help our patients.”

Late last week, researchers at the Icahn School of Medicine, in collaboration with scientists in Australia and Finland, were among the first to create an antibody test that detects the disease’s antibodies in a person’s blood. Development of the enzyme-linked immunosorbent assay (ELISA) was led by Florian Krammer, PhD, Professor of Microbiology, in collaboration with Viviana A. Simon, MD, PhD, Professor of Microbiology and Medicine (Infectious Diseases). Dr. Krammer, a renowned influenza researcher, recently made this so-called recipe available to other laboratories around the world so they can replicate it during the pandemic. In January, his lab was quickly retooled to begin studying COVID-19.

In addition to its widespread use in plasmapheresis, the antibody test will provide experts with an accurate infection rate so they can track the trajectory of the disease. The test will help identify health care workers who are already immune to the disease, who can work directly with infectious patients, and it can also help scientists understand how the human immune system reacts to the virus.

The new assay uses recombinant or manufactured antigens from the spike protein on the surface of the SARS-CoV-2 virus. That protein helps the virus enter cells, and it is a key target in the immune reaction against the virus, as the body creates antibodies that recognize the protein and seek to destroy the virus. The researchers also isolated the short piece of the spike protein called the receptor-binding domain (RBD), which the virus uses to attach to cells it tries to invade. The scientists then used cell lines to produce large quantities of the altered spike proteins and RBDs.

According to Dr. Krammer and his co-authors, the assay is “sensitive and specific,” and allows for the screening and identification of COVID-19 in human plasma/serum as soon as three days after the onset of symptoms. The antibodies were derived from three patients who had the disease. The study’s control participants—who did not have COVID-19 but had other viruses, including the common cold—ranged in age from 20 to 70.

Dr. Krammer says his preliminary findings also show that humans have no natural immunity to the SARS-CoV-2 virus, which would help explain why it spreads so quickly. But once the antibody sets in humans do become protected. He also says that at this early stage in the research, there is no evidence that people can lose their immunity and become re-infected.

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A NEW TEST TO DETECT CANCER, HIV AT THE EARLIEST FOUND

Scientists have developed a new ten times cheaper ultra-sensitive sensor test to detect the early stages of several cancers and viruses, including HIV, with the naked eye.

Researchers from the Imperial College London claim that their visual sensor technology is ten times more sensitive than the current gold standard methods for measuring bio-markers.

These indicate the onset of diseases such as prostate cancer and infection by viruses including HIV. The colour of a liquid changes to give either a positive or negative result.

Researchers say their sensor would benefit countries where sophisticated detection equipment is scarce, enabling cheaper and simpler detection and treatments for patients.

The team tested the effectiveness of the sensor by detecting a biomarker called p24 in blood samples, which indicates HIV infection.

The new approach affords for improved sensitivity, does not require sophisticated instrumentation and it is ten times cheaper, which could allow more tests to be performed for better screening of many diseases.

Researchers also tested samples for the bio-marker called Prostate Specific Antigen (PSA), which is an early indicator for Prostate Cancer. The team say the sensor can also be reconfigured for other viruses and diseases where the specific bio-marker is known.

The sensor works by analysing serum, derived from blood, in a disposable container. If the result is positive for p24 or PSA, there is a reaction that generates irregular clumps of nanoparticles, which give off a distinctive blue hue in a solution inside the container.
If the results are negative the nanoparticles separate into ball-like shapes, creating a reddish hue. Both reactions can be easily seen by the naked eye.

The team also said that the sensor was so sensitive that it was able to detect minute levels of p24 in samples where patients had low viral loads, which could not be diagnosed using existing tests such as the Enzyme-linked Immunosorbent Assay (ELISA) test and the gold standard nucleic acid based test.

The researchers have developed a test that we hope will enable previously undetectable HIV infections and indicators of cancer to be picked up, which would mean people could be treated sooner.