A high-throughput test for neutralizing antibodies to SARS-CoV-2

The need for a reliable supply of accurate serology tests is paramount as countries globally have started opening their economies since the onset of the COVID-19 pandemic. Most ELISA-based serological tests for SARS-CoV-2 utilize either protein (typically receptor-binding domain (RBD) or truncations of the spike protein and/or nucleocapsid) or antibody (anti-IgG or anti-IgM) coated plates for capture and detection of IgG and/or IgM. Even more important than just detecting antibodies is the ability to detect the antibodies blocking the virus entry into the cell.

The newly developed cPass^™ Neutralization Antibody Detection kit utilizes the interaction between RBD and the human ACE2 receptor to provide greatly improved speed, scalability and ease of use compared to the gold standard virus neutralization assay. Since it uses soluble RBD for detection, the assay is also isotype and species independent for highly specific and sensitive immunoglobulin detection across animal models and human samples.

In a SelectScience webinar now available on demand, Sean Taylor, Field Applications Group manager, GenScript USA , gives the lowdown on this new alternative to live virus neutralization and immunoglobulin detection.

Read on for highlights from the Q&A discussion at the end of the live webinar or register to watch the full webinar on demand >>

Q: How does the sVNT kit complement a vaccine or drug development pipeline for SARS-CoV-2?

ST: If we use the virus neutralization assay, it can be used as a screen. This means we can use it as a screening tool to quickly screen the candidates for a vaccine or drug development program. Because it is so easy to use, it doesn't require a BSL 3 lab, it only requires one to two hours and it's very comparable to a traditional VNT test. This would be the obvious choice to use to quickly screen many, many drug candidates.

For the next tool, which would be a pseudovirus neutralization assay, still a little easier to use than a VNT assay, which requires a BSL 3 lab, we could use the pseudovirus test to further hone in on the candidates from the initial screen. We could then use this test as a further screen or ‘targeted screen’. The first test is used as a broad screening tool. This is then followed by use of the next test as a targeted screen from the results of the broad screen. This means we can narrow down our candidates.

Finally, the VNT test could be used for the final candidates from the pseudovirus test. Because this is the most representative screen to the actual infectious state. We work our way up through these tests from the surrogate virus test to the pseudovirus test and then up to the VNT test in a very logical order. This would make things go much faster and quicker to a final candidate drug or vaccine candidate.

Q: Can the sVNT kit replace the traditional IgG or IgM serology test?

ST: That's a very good question. The data that we have collected so far from various studies definitely supports this as an option. It really depends on what you are looking for in your tests though, because an IgG or IgM test will specifically detect IgG or IgM. And again, we have plates coded with the receptor-binding domain here, you would go in with the sample and, of course, the antibodies that bind to the RBD, the immune response antibodies will bind and, from this, it's the secondary antibodies that give you the specificity. You will use a secondary antibody to either IgG, IgM, or IgA, or whatever class of immunoglobulin to know how much of that immunoglobulin is bound to the receptor-binding domain.

For the neutralization test, same question just describing the neutralization test. Remember that the RBD is in solution and we are not specifically detecting it with any secondary antibody. This means any immunoglobulin that binds to the receptor-binding domain and blocks its ability to bind to the ACE2 receptor, which is coded on the bottom of the plate, is a candidate in this assay. This permits a much broader analysis of whether the individual, or if you are doing drug screening or vaccine development, whether an antibody binds and blocks ACE2. However, if you are doing screening of individual patients, it will tell you that that patient did once have COVID-19, and has recovered from COVID-19, and is generating antibodies to COVID-19.

You just won't know which specific immune response antibodies are being generated because we're not detecting the different classes of antibodies, we're detecting the ability of any antibodies, any immune response, the antibodies' ability to block the binding to ACE2 receptor. If it doesn't matter to know the IgG or IgM or IGA levels, and just to know if a patient has or has not been infected with COVID-19, then the sVNT kit could potentially be a good replacement to an IgG/IgM kit. I say this because of the ability of any antibody to bind to RBD. It could make this kit more sensitive and it could augment the specificity of the kit because the RBD is in solution, so it's in a more native state. I mentioned in the presentation permitting antibodies or requiring the immune response antibodies to bind more specifically to the antigens that are available on the native state of the receptor-binding domain.

Q: What does percent neutralization mean?

ST: It is either the amount of immune response antibodies that are circulating and inhibiting the binding of RBD to the ACE2 receptor. It could also mean an affinity of the antibodies, or it can mean a combination of both of those things. Remember, as I mentioned in the previous question, any immunoglobulins can bind to the receptor-binding domain and are in effect in this assay and measured in this assay. You could have a combination of several neutralizing antibodies bound to a given RBD which would potentially increase the percent of neutralization.

If a patient or an individual is making neutralization antibodies from either different classes of immunoglobulins, or even within the same class, if they are making different immune response antibodies that bind and neutralize the ability of RBD to bind to the ACE2 receptor, then the percent neutralization will increase. The same thing with affinity. They could be generating fewer neutralizing antibodies but the neutralizing antibodies they are producing have a very high affinity to the receptor-binding domain and block the ability of that receptor binding domain to bind to the ACE2 receptor.

There is a lot going on in an assumed sample from a recovered individual in terms of affinity, number, and the classes of antibodies that are binding to RBD. All these factors culminate to this percent neutralization.

Q: How many tests can be done in a given kit, and the number of tests if you include duplicate wells for the positive and negative control?

ST: That's four wells; it is a 96-well assay, so that leaves 92 wells to run individual samples. We recommend, if the kit is being used to assess the generation of antibodies from recovered individuals to know if they had or did not have COVID-19, then probably it's best to do each sample in duplicate. However, if it's a larger study where you are looking at study sets of people infected versus non-infected, and you want to look at trends or averages with a lot of samples, potentially those samples could be analyzed with iasingle kit.But, in total, there are 92 wells for running individual samples.

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