Immunologic Testing for COVID-19

Introduction

With over 93 million confirmed infections and 2 million reported deaths by January 2021, the on-going COVID-19 pandemic has devastated the global community deeply. This severe up-respiratory illness is caused by a positive-sense single-stranded RNA virus, SARS-CoV-2, which contains a genome of about 30K bp and interacts with the host cell through ACE2 binding via the viral spike protein. So far, to combat this disease, several vaccine candidates have been approved for emergency uses while many experimental medications are been exploited. Besides these efforts, preventive measures, accurate disease diagnostics, and effective contact tracing are still considered fundamental in containing the SARS-CoV-2 pandemic.

One of the strategies to control the spread of SARS-CoV-2 is accurate disease diagnostics at the early stage of infection. Currently, several lab-tests are available for the detection of the SARS-CoV-2. Among the established tests, RT-qPCR and NGS-based tests (nucleic acid tests), are considered as the golden standard for virus identification. However, these tests are not only time consuming and laborious, but they also rely heavily on specialized instrumentation, well-trained personnel, as well as sophisticated laboratory facilities, resulting in their limited availability. On the other hand, the nucleic acid tests are not error-proof since false-negative results are often generated due to reasons such as improper sample handlings and storage conditions, sampling time…. Thus, the development of complementary assay formats is necessary to expand the testing capacities. Immunologic tests are viable options and it has been reported that a combination of nucleic acid and immunologic tests significantly improved the sensitivity of pathogenic diagnosis for COVID-19 as early as during the 1st week of contracting the disease. These tests either detect the antibodies against SARS-CoV-2 (serological tests) or look for pieces of virus proteins in the serum (antigen tests). Immunologic tests can be performed under common clinic settings and the results are available in the duration of hours, which significantly expedite the diagnostic practices.

Serological Tests

Serological tests (antibody tests) identify CoV-2 specific antibodies (mainly IgM and IgG classes) in a patient's serum. Since IgM and IgG antibodies are the front-line responses in combating the viral infection in situ, they are the most useful biomarkers of SARS-CoV-2 serological diagnostics. Figure 1 shows the timeline from SARS-CoV-2 infection to the generation of immunity by the presence of IgM and IgG antibodies in serum.


Figure 1. Timeline from SARS-CoV-2 infection to immunity generation (DOI: 10.1063/5.0021554)

Serological tests have far-reaching epidemiological and clinical significances. Using these tests, it is possible to evaluate the duration and influence of immunity caused by the virus, assess the risk of re-infection, predict the potency of the vaccine, as well as identify asymptomatic individuals. Meanwhile, serological tests can also be used to facilitate the identification of convalescent plasma donors for the treatment of serious COVID-19 patients.

As of September 2020, more than 20 serological tests have been approved by the NMPA in China, while more than 40 serological tests are approved by the FDA in US for emergency uses. Formats for serological tests include rapid tests and non-rapid diagnostic tests. Rapid tests, mainly lateral flow tests (LFA), can detect antigen-specific IgG, and IgM or antigen-specific total antibodies in serum samples within 30 minutes. These tests are highly advantageous due to the low sample volume requirements, quick turn-around time, ease of use, as well as low cost…. Combined with other advanced sensory technologies, the sensitivity of LAF methods have been improved substantially. Non-rapid tests can provide quantification of the antibodies in the samples and they are usually more sensitive comparing to the LFA methods. The most commonly used non-rapid serological testing methods are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassays (CLIA). A general depiction of the basic work-flow of the methods mentioned can be found in Figure 2, while Table 1 summarizes the pros and cons of each serological test format.


Figure 2. Serological test formats: rapid test, LFA (A); non-rapid tests ELISA (B) and CLIA (C).

Table 1 Comparison of serological testing methods

Method LFA ELISA CLIA
Read-out Semi-quantitative Quantitative Quantitative
High throughput No Yes Yes
Ease of use Easy Moderate Hard
Accuracy low Moderate high
Cost low Moderate high

Sensitivity and specificity are the focus of any serological tests and they are heavily influenced by the quality of the core material, i.e. the recombinant proteins used in the assay developments. Recombinant proteins that closely reassemble their naturally occurring counterparts will greatly improve the test accuracy. For SARS-CoV-2, the most abundant viral proteins in circulation are spike (S) and nucleocapsid protein (NP). These proteins are also the major elicitors of the body’s antibody responses and they can both serve as key ingredients for serological test developments. Using recombinant protein expression technology, Sino Biological has successfully generated biologically active and stable Spike (40591-V08H, 40592-V05H) and NP proteins (40588-V08B, 40588-V07E) of SARS-CoV-2 (Figure 3). In the meantime, a collection of the S and NP proteins from other coronavirus strains are also available as core components in several commercial Serological diagnostic kits.

Figure 3: Examples of high-quality recombinant SARS-CoV-2 sand NP. Left) The RBD domain of the Spike protein was expressed in HEK293 via transient transfection. The high purity proteins with confirmed ACE2 binding activity exerted minimum batch-to-batch variance and applicable to detect anti-CoV-2 antibodies from patient serum samples. Right) The full-length NP was expressed in E.coli (40588-V07E). The protein was stable after 3X freeze-thaw cycles and presented as an oligomer.

Antigen Tests

Upon infection, the presence of viral antigens (pieces of virus proteins), are observed prior to the host's antibody responses Therefore, antigen-based immunoassays would be valuable in the early diagnosis of COVID-19. NP is the main target for antigen test developments for SARS-CoV-2.

So far, 13 antigen diagnostic tests have been authorized by the FDA in US for emergency uses. Similar to serological tests, specificity and sensitivity are also the key elements in antigen tests and they are determined largely by the antibodies used in test developments. In terms of assay formats, assay configurations used in serologic tests are also applicable for antigen tests, including immunofluorescence assays (IFAs), LFA, and CLIA ..

Using advanced antibody generation platforms, Sino Biological has generated a large panel of antibodies against the NP of SARS-CoV-2. Upon extensive screening efforts, we have identified several antibody pairs with high specificities and sensitivities towards NP. These antibody pairs are used as material for ELISA-based antigen tests (Figure 4, left). Meanwhile, a few antibodies with phantom-molar sensitivities have also emerged from the panning process and they have been proven compatible in ultra-sensitive detection platforms such as Simo (Figure 4 right).

  Capture Ab (Cat#) Detection Ab (Cat#)
Pair1 40143-MM05 40588-R001
Pair 2 40588-MM123 40588-R001
Pair 3 40143-MM05 40143-R001
Pair 4 40143-MM08 40143-R004
Pair 5 40143-R004 40143-R040
Antigen Capture Ab (Cat#) Detection Ab (Cat#) Standard (cat#) LOD
N 40143-MM05 40143-R001 40588-V08B 15.7 fg/ml

Figure 4. Recommended antibody pairs for ELISA-based quantitative antigen test kits (left) and ultra-sensitive single molecular array (SIMOA, right).

Challenge and Solutions

The Impact of SARS-CoV-2 Variants

RNA viruses are notorious for their high mutation rates. The dynamic nature of the viral proteome creates significant impact on the accuracy of immunological assays. It is crucial to assess the viability of the current assay materials in adaptation to the virus mutagenesis.

Recently, a SARS-CoV-2 variant with multiple mutations has been recorded in the UK. This strain, categorized in the B.l.1.7 or 20B/501V.Vl lineage, has raised concerns since evidence indicated that it exerted potential to escape vaccine or immunological diagnosis. To validate the effectiveness of the current immunological diagnostic reagents, especially antibodies used for antigen tests, scientists at Sino Biological have generated the NP protein of the UK strain and tested the performance of the current anti-NP antibodies (Figure 6). Results showed that the current antibodies performed similarly against the main mutants of B.l.1.7 of NP.


Figure 6. Activities of anti-NP antibody (left) and antibody pairs (right) against NP from UK variant

Antibody Cross-Reactivity

The NP of SARS-CoV-2 showed moderate sequence similarities (60~65%) against CoV strains that cause seasonal flu while it has ~75% and ~97% sequence similarity against MERS and SARS CoV, respectively. Thus, it is challenging to develop a highly specific SARS-CoV-2 immunological test with low cross-reactivity against other CoV strains. To do so, Sino Biological has successfully developed NP for seven CoV strains and use these proteins as screening agents, we have identified several SARS-CoV-2 NP specific monoclonal antibodies with negligible cross-reactivity (Figure 7).


Figure 7. Cross-reactivity of SARS-CoV-2 specific Anti-N antibodies

Reference:

1. https://www.acpjournals.org/doi/full/10.7326/M20-1495
2. https://doi.org/10.3389/fimmu.2020.610688
3. https://doi.org/10.1093/cid/ciaa344
4. https://doi.org/10.1101/2020.03.24.006544
5. https://aip.scitation.org/doi/10.1063/5.0021554

By Molecule Initial