There are two principal immunological test formats used to check for the presence of anti-SARS-CoV-2 antibodies in blood as evidence of a previous COVID-19 infection. One is enzyme-linked immunosorbent assay (ELISA), a laboratory-based test in which blood, plasma, or serum of a sample drawn from an arm vein is applied to wells of a microplate coated with viral protein, i.e., the antigen against which the antibodies were generated. There is much more to ELISAs than will be covered here; the various detection systems used by assay developers involve different conjugated secondary antibodies designed to provide either colorimetric, chemiluminescent, or fluorescent readouts. ELISAs can be quite sensitive and provide quantitative results down to the pg/ml range. Results can be obtained in a few hours, but because the test is usually conducted at a lab remote from the site of blood collection, the time to receive results is often a few days.

The other antibody test format is called a lateral flow assay (LFA). An example of this format is the typical home pregnancy test, although LFAs for SARS-CoV-2 use blood from a finger prick instead of urine. The LFA contains a strip of membrane within a plastic cassette across which the sample flows by capillary action. As it moves, viral protein molecules (antigen) immobilized in a “test line” perpendicular to the flow will be bound by antibodies in the sample that recognize the antigen. A secondary “control line” checks that the sample flow was unimpeded. Depending on a threshold that is part of the assay device design, if enough antibodies are present to bind to the proteins on the strip, a band will materialize. If the antibodies are present but at a concentration below the threshold, no band will be visible. This qualitative (yes/no) answer can be achieved in a half hour or less, and the assay can be performed at home if authorized by the FDA.

MarinBio scientists have decades of experience in the development of ELISAs and other immunological assays. MarinBio can design and deliver antibody tests with low rates of false negatives and false positives that not only meet but exceed current FDA COVID-19 standards for overall specificity (≥95%) and sensitivity (≥90%). Contact MarinBio today if you are planning a project involving ELISA testing, and not just for SARS-CoV-2 antibodies but for any antigen of interest. Multiplex ELISAs for measuring levels of multiple antigens in a single assay are also a specialty of MarinBio.

Consequences and significance of sensitivity and specificity

The quality of these immunological tests is characterized in terms of their sensitivity and specificity. Adequate sensitivity is essential to minimizing false negatives, i.e., when antibodies are present but are undetectable by that test. On the other hand, specificity is crucial to minimizing false positives in which the test detects antibodies that may be closely related to those against the SARS-CoV-2 virus but are actually not. There are four other coronaviruses that together are responsible for about 20% of common colds, meaning that antibodies to these viruses are readily found in any human population. Therefore, the ultimate goal of assay designers is to create tests that will detect 100% of the antibodies formed against SARS-CoV-2 while recognizing 0% of antibodies against antigens from closely related coronaviruses. This all has to be accomplished across a diversity of human subjects, and the combinatorial nature of the immune system’s approach to antibody generation leads to a staggering potential number of antibodies recognizing viral epitopes. In addition, false positives do occur in qPCR tests, an outcome that makes a subsequent negative result in a SARS-CoV-2 antibody test appear to be a false negative when it is a true negative.

Viral antigens used in ELISAs and LFAs

Different viral proteins of SARS-CoV-2 can be used as the antigens in these immunological tests, but its spike protein is most commonly utilized, and for good reason. The protuberances that give this class of viruses the distinctive corona (and hence its name) seen in electron micrographs are composed of spike protein. Because of its exposed placement on the virus particle, spike protein is the portion of the virus most likely to be encountered by cells of the immune system and is therefore the most likely to have antibodies made against it. Some tests have used the SARS-CoV-2 neocapsid protein, but because it shows a higher degree of sequence similarity to the neocapsid proteins of other coronaviruses, the more diverged spike protein is usually preferred as a means of minimizing specificity issues.

The receptor binding domain (RBD) at the distal end of spike protein is designed by nature to recognize the ligand binding domain of human angiotensin-converting enzyme 2 (ACE2) in order to gain entry to the cell. Spike protein is somewhat labile due to a need to undergo radical conformational changes to accomplish cell entry upon binding of the receptor binding domain to ACE2. Therefore, some researchers have designed altered versions of spike protein to make it more stable during expression.

Expression systems for viral antigens

Native viral proteins are produced by human intracellular protein synthesis and modification machinery (ribosomes, endoplasmic reticulum, and Golgi apparatus) that are co-opted by the virus for replication after it successfully infects a human cell. However, recombinant versions of viral proteins expressed in cell culture are required to produce the huge quantities of antigens needed for large-scale manufacturing of tests. Accurate glycosylation of these recombinant proteins is a key issue in assay design, as the surface of spike protein (a homotrimer, each “mer” composed of two subunits) is densely decorated with branched sugar moieties. In responding to a COVID-19 infection, the human immune system will produce many antibodies to native spike protein that recognize epitopes at least partially formed by these externally facing carbohydrate groups. Therefore, it is critical that recombinant viral antigens express human glycosylation patterns faithfully. Any change from the native composition of these patterns can potentially alter epitopes to which the antibodies the assay is trying to detect were generated. If such a conformational change in an epitope causes the recombinant antigen not to be recognized by a subject’s antibody, this gives a false negative result, i.e., reducing sensitivity. On the other hand, if the change results in an epitope resembling that of another coronavirus, this can reduce specificity and give a false positive result.

Bacterial expression systems, while convenient, are not up to the task of expressing recombinant spike protein, as prokaryotic cells are unable to provide the precise post-translational modifications (glycosylations) added in human cells. However, differences in characteristic glycosylation patterns can also exist not only between different eukaryotes, e.g., between yeast and humans, but even between different mammalian species. Therefore, the cell culture system for large-scale expression of recombinant viral antigens must be carefully selected.

MarinBio has decades of experience in the finer points of mammalian cell culture for their cell based assay services and in the gene expression of recombinant proteins for their molecular biology services including insuring that glycosylation and other post-translational modifications occur as they should on proteins of interest. If you have projects requiring such expertise, please contact MarinBio for a consultation with one of our knowledgeable scientists to see how we might be able to help.

Types and timing of immunoglobulins detected

Another source of variability in assay performance derives from the immunoglobulin types of the antibodies that tests are designed to detect. In the progression of the COVID-19 disease, IgM antibodies are produced first, often measurable by about 10 days post-infection. IgG antibodies appear at about 20 days, typically after the viral RNA is no longer detectable. IgG antibodies generate a more specific and durable response than do IgMs, the signal from which is less robust and more variable. Independent assessments of various commercially available tests have shown that their ability to detect antibodies generally increases over the first three weeks after infection. The significance of this is that an early negative result could well mean that the subject had still been infected but had not yet developed a level of antibodies sufficient for the sensitivity of the assay. The most successful assay designs appeared to involve checking for both IgM and IgG, as detection rates were highest when the two were combined.

Quality required for individual versus population-level testing

ELISAs from Roche and Abbott appear to be able to corroborate claimed high levels of sensitivity and specificity for their assays, displaying performance suitable to aid decision making for individual patients. However, there are issues with some LFAs not only concerning specifications of sensitivity and specificity, but with interpretation of results, e.g., encountering faint bands without clear guidance on whether these would constitute a positive or a negative. Nevertheless, because of their low cost and ease of use, LFAs have excellent utility for seroprevalence surveys to identify the proportion of a population who have been infected with the virus, such as the NIH is carrying out with 10,000 at-home LFAs.

MarinBio is staffed by scientists who are well-versed in all aspects of designing and conducting immunoassays, and who are familiar with the many technical details that must be considered in generating high levels of specificity and sensitivity. If you are developing or even considering developing such a test, MarinBio stands ready to assist in your projects. With a strong track record of successful collaborations with biopharma companies, MarinBio is an ideal partner.