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  • Frequently asked questions on SARS-CoV-2 and the COVID-19 diagnostics

Below, you can find the answers to frequently asked questions about SARS-CoV-2 and the EUROIMMUN tests for the diagnostics of COVID-19


SARS-CoV-2

What are coronaviruses?

Coronavirus constitute a large family of viruses which can infect humans as well as animals, for example birds and mammals. Coronavirus infection can lead to a simple cold, but also to severe or even fatal disease. The two highly pathogenic viruses SARS-CoV and MERS-CoV cause severe respiratory syndromes in humans. Infections with the other four human pathogenic coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1) mainly only result in mild diseases of the upper respiratory tract. In babies, infants and elderly people, however, the infection can take a severe course.

How is SARS-CoV-2 transmitted?

SARS-CoV-2 is mainly transmitted via aerosols during coughing or sneezing or at close contact with an infected person. Health care personnel and family members are among the high-risk populations.

What are the symptoms of COVID-19?

The symptoms of SARS-CoV-2 infection are fever, coughing, breathing difficulties and fatigue. Most patients suffer from a mild febrile illness with irregular lung infiltrates. Some patients, particularly elderly or chronically ill persons, develop severe acute respiratory distress syndrome (ARDS), which is fatal in three percent of cases. In February 2020, the disease caused by SARS-CoV-2 was named COVID-19 (coronavirus 19) by the WHO.

How is a person tested for COVID-19?

Laboratory tests for the diagnosis of SARS-CoV-2 infection include polymerase chain reaction (RT-PCR) with smears from the upper and lower respiratory tract (bronchoalveolar lavage fluid, tracheal secretion, sputum, nasopharyngeal secretion, oropharyngeal secretion, etc.) for direct detection of the virus. The test is used for primary laboratory diagnostic examination of patients with suspected SARS-CoV-2 infection. Additionally, there are serological tests for the detection of antibodies against SARS-CoV-2 in the blood. The antibody test is the ideal supplement to direct detection. It supports the diagnosis of SARS-CoV-2 infection and helps to confirm the RT-PCR results. Moreover, the determination of antibodies is relevant for the clarification of suspected cases of SARS-CoV-2 without symptoms or with negative results in direct detection. In addition to their importance for diagnostics, serological tests can also be used to gather epidemiological data and for outbreak control. 

Where can I find further information on SARS-CoV-2 and COVID-19?

World Health Organisation (WHO) https://www.who.int/

European Centre for Disease Prevention and Control (ECDC) https://www.ecdc.europa.eu/en/home 


EUROIMMUN tests for COVID-19 diagnostics

Are the SARS-CoV-2 tests from EUROIMMUN approved?

EUROIMMUN SARS-CoV-2 tests are CE-marked and can be used for COVID-19 diagnostics. The Anti-SARS-CoV-2 ELISA (IgG) and the EURORealTime SARS-CoV-2 were also approved by the US American Food and Drug Administration (FDA) through an emergency use authorisation (EUA) for use by authorised laboratories (validity of the FDA EUA according to the respective US-specific instructions for use), and by the Brazilian Health Regulatory Agency ANVISA.

Where can you get tested?

Our tests are exclusively performed in the laboratory. The required samples (blood, swabs, etc.) are taken by a physician, e.g. your doctor, and then sent to the respective laboratory in charge.

If you are interested, please get in touch with the Clinical Immunological Laboratory Prof. Dr. med. Winfried Stöcker (Seekamp 31, D-23560 Lübeck).

Where can you buy our tests for COVID-19 diagnostics?

With the Anti-SARS-CoV-2 ELISAs (IgA and IgG), EUROIMMUN was one of the first companies to offer CE-marked test systems for the detection of antibodies against SARS-CoV-2 already from March 2020. By now, our range of antibody tests for COVID-19 diagnostics has been extended by the CE-marked Anti-SARS-CoV-2 NCP ELISAs (IgG, IgM), the Anti-SARS-CoV-2 QuantiVac ELISA (IgG), the EUROLINE Anti-SARS-CoV-2 Profile (IgG) and the surrogate virus neutralisation test SARS-CoV-2 NeutraLISA. For acute diagnostics, in addition to the EURORealTime SARS-CoV-2 for direct detection of the virus via reverse-transcription real-time PCR (RT-PCR), we also offer the combination test EURORealTime SARS-CoV-2/Influenza A/B for differential diagnostics of COVID-19 and influenza as well as the SARS-CoV-2 Antigen ELISA for detection of the viral nucleocapsid protein. The ELISAs, immunoblot and EURORealTime tests are designed for use in diagnostic laboratories and are exclusively supplied to these. These tests are not rapid tests for at-home testing.

If you wish to receive a quote for our tests, please contact your local distributor.

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Can private persons perform the tests?

Please note that our self-developed test systems are not rapid tests for at-home testing. They are designed for the investigation of large samples volumes and their processing requires laboratory equipment.

If you suspect to be infected with SARS-CoV-2, please ask your physician for a laboratory diagnostic test.

What are the application areas of the test systems for direct pathogen detection (reverse-transcription real-time PCR, Antigen ELISA) and antibody detection (ELISA, immunoblot) in COVID-19 diagnostics?

Suitable methods for the identification of an acute infection are the detection of viral RNA via RT-PCR or that of virus proteins in sample material of the upper respiratory tract. RT-PCR tests enable pathogen detection a few days after virus contact even in subclinical or asymptomatic infections. The virus can be detected up to 14 days after the onset of symptoms. Moreover, by the combination of different parameters, multiparameter RT-PCR tests allow the detection and differentiation of SARS-CoV-2 and other respiratory pathogens, such as influenza viruses, in one reaction. However, as soon as the immune reaction starts and the viral load declines, the sensitivity of this test method decreases as well. The pathogen can then no longer be detected in all patients.

Serology, i.e. the detection of antibodies, expands the diagnostic window over the first one to two weeks. Serology allows identification of persisting, but no longer acute, or past infections with SARS-CoV-2.

Moreover, the S1 domain of the spike protein used in the Anti-SARS-CoV-2 ELISA, Anti-SARS-CoV-2 QuantiVac ELISA, EUROLINE Anti-SARS-CoV-2 Profile and SARS-CoV-2 NeutraLISA contains the receptor binding site (RBD) of SARS-CoV-2, by which the virus binds to human cells. Especially IgG antibodies against the S1 domain/RBD might therefore have a virus-neutralising and thus protective function. The assays can therefore also be applied to support the assessment of the immune reaction after infection or vaccination with S1-based vaccine (further information here).

What are the areas of application of the SARS-CoV-2 interferon-gamma release assay Quan-T-Cell?

The assay is suitable for the detection of a past contact with the pathogen. It enables identification of an immune reaction, especially with an unclear antibody status or a missing antibody reaction. Currently, it is assumed that in 10% of symptomatic and 40% of asymptomatic COVID-19 patients, specific IgG antibodies disappear over the course of time and a past infection can only be detected based on the T-cell activity.

According to current knowledge, a T-cell response is also linked to a high immunoprotection even if specific antibodies are absent.

Moreover, the Quan-T-Cell can be used to evaluate the immune reaction following a COVID-19 vaccination. This may be relevant especially for those groups at risk in whom a reduced antibody reaction is to be expected, e.g. immunosuppressed patients.

At what time after infection can reverse-transcription real-time PCR tests, antigen tests and assays for antibody determination be applied?

The WHO estimates an incubation period of 1 to 14 days for SARS-CoV-2. In most cases, it is approximately 5 days. This is the time that passes between the contact with the virus and the onset of first symptoms.

It is assumed that the virus can be detected directly after symptom onset in swabs of the upper respiratory tract by means of RT-PCR or antigen tests.

In viral infections, antibodies are generally only produced at the earliest one week, frequently two weeks after the onset of symptoms and are detectable only then. Specific IgG antibodies can be detected with high sensitivity using the Anti-SARS-CoV-2 ELISA (IgG) or Anti-SARS-CoV-2 QuantiVac, respectively, or the Anti-SARS-CoV-2 NCP ELISA (IgG) from approximately day 10 after symptom onset. IgG antibodies against the nucleocapsid protein (NCP) often occur some days prior to the anti-S1 IgG antibodies. A positive test result confirms virus contact. The Anti-SARS-CoV-2 ELISA (IgA) and the Anti-SARS-CoV-2 NCP ELISA (IgM) are both suitable for early monitoring of an immune response after positive direct detection when specific IgG antibodies have not yet been produced. Neither IgA nor IgM determination are suitable for identification of an acute SARS-CoV-2 infection. The surrogate virus neutralisation test SARS-CoV-2 NeutraLISA is intended for the detection of neutralising antibodies in samples taken at least 15 days after the onset of symptoms.

The T-cell response to SARS-CoV-2 occurs much earlier than the antibody reaction. The activity of specific T-cells can be determined already shortly after onset of symptoms via the release of the signal molecule interferon gamma (IFN-γ).

Do the test results allow drawing conclusions on infectiousness or immunity?

As long as the virus can be detected in secretions of the respiratory tract by means of reverse-transcription real-time PCR, the patient is assumed to be infectious.

The ELISA technique cannot show whether the detected antibodies have a neutralising effect on the pathogen or not. Whether the IgG results obtained by ELISA provide information on the immunity of a patient is a subject of current scientific research. However, by selection of a suitable antigen that induces the formation of neutralising antibodies, it might be possible to deduce the respective functionality of the antibodies. It can generally be assumed that immunity is mainly associated with class IgG antibodies, which are detected by our Anti-SARS-CoV-2 ELISA (IgG) and Anti-SARS-CoV-2 QuantiVac ELISA (IgG). These tests are based on the S1 domain of the spike protein, including the immunologically relevant receptor binding domain (RBD) as the antigen. Please find further information here. This is also indicated by many published results as, for instance, from the so-called Heinsberg Study by the University of Bonn. The study underlined the high quality of the Anti-SARS-CoV-2 ELISA (IgG) and showed that the ELISA correlated very well with neutralisation assays.

Moreover, EUROIMMUN has developed a surrogate virus neutralisation test which provides semiquantitative determination of neutralising antibodies that inhibit the binding of SARS-CoV-2 S1/RBD to the ACE2 receptors of the human host cells. The results obtained with the NeutraLISA show a very high agreement in the comparison with a plaque reduction neutralisation test (PRNT50).

 

Can the tests distinguish SARS-CoV-2 infections from infections with other coronaviruses?

EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2 Influenza A/B: Yes. Due to the detection of two specific gene sequences of the virus, SARS-CoV-2 infections can be reliably identified and distinguished from other coronavirus or respiratory infections.

Antigen ELISA: During validation of the SARS-CoV-2 Antigen ELISA no cross reactions with antigens of other worldwide occurring coronaviruses were observed. However, due to the close relationship between SARS-CoV(-1) and SARS-CoV-2, cross reactions with the antigen of SARS-CoV(-1) cannot be excluded.

Anti-SARS-CoV-2 ELISA/Anti-SARS-CoV-2 QuantiVac ELISA/Anti-SARS-CoV-2 NCP ELISA: During validation of the ELISAs, no cross reactions with antibodies against other worldwide occurring coronaviruses were observed. However, due to the close relationship between SARS-CoV(-1) and SARS-CoV-2, cross reactions with the antibodies against SARS-CoV(-1) cannot be excluded.

SARS-CoV-2 NeutraLISA: During validation of the assay, no cross reactions with antibodies against other worldwide occurring coronaviruses were observed. However, due to the close relationship between SARS-CoV(-1) and SARS-CoV-2, cross reactions with the antibodies against SARS-CoV(-1) cannot be excluded.

Interferon-gamma release Assay Quan-T-Cell: During validation of the Quan-T-Cell-ELISA in combination with the Quan-T-Cell SARS-CoV-2, no cross reactions with antibodies against other worldwide occurring coronaviruses were observed. However, due to the close relationship between SARS-CoV(-1) and SARS-CoV-2, cross reactions with the antibodies against SARS-CoV(-1) cannot be excluded.

Which laboratory equipment is required for performance of the ELISAs and the EURORealTime tests?

EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2 Influenza A/B: The tests are compatible with the standard equipment (real-time PCR thermocycler) present in most of the molecular-diagnostic laboratories. The EURORealTime SARS-CoV-2 was validated on the following real-time-PCR cyclers: 7500 Fast Real-Time PCR Instrument (Applied Biosystems), LightCycler® 480 II (Roche), CFX 96 Touch (Bio-Rad), RotorGene Q (Qiagen) and qTower3 (Analytik Jena). If other cyclers are used, they must be validated by the laboratories themselves. The EURORealTime SARS-CoV-2 Influenza A/B was validated on the following real-time-PCR cyclers: 7500 Fast Real-Time PCR Instrument (Applied Biosystems), CFX 96 Touch (Bio-Rad) and qTower3 (Analytik Jena). If other cyclers are used, they must be validated by the laboratories themselves.

Antigen ELISA: Photometer, incubator or water bath for 37°C. Each test kit contains a 96-well ELISA microplate (individual break-off wells, enabling adjustment to the number of samples to be analysed) and all reagents and control materials required for the test performance. One packaging unit allows the analysis of 93 patient samples. Like other ELISAs, the tests can be performed manually. Automated processing on our EUROIMMUN Analyzer I and I-2P is of course also possible.

Anti-SARS-CoV-2 ELISA/Anti-SARS-CoV-2 QuantiVac ELISA/Anti-SARS-CoV-2 NCP ELISA/SARS-CoV-2 NeutraLISA: Photometer, incubator for 37°C. Each test kit contains a 96-well ELISA microplate (individual break-off wells, enabling adjustment to the number of samples to be analysed) and all reagents and control materials required for the test performance. Depending on the number of calibrators, up to 93 patient samples can be analysed with one packaging unit. Like other ELISAs, the tests can be performed manually. Automated processing on our EUROIMMUN Analyzer I and I-2P is of course also possible (Analyzer I: up to 7 microplates, up to 180 samples per test run) and the EUROLabWorkstation ELISA (up to 15 microplates, up to 748 samples per test run).

Quan-T-Cell ELISA in combination with Quan-T-Cell SARS-CoV-2: The determination of the SARS-CoV-2-specific T-cell activity requires the stimulation tube set Quan-T-Cell SARS-CoV-2, consisting of 30 x 3 tubes (CoV-2 IGRA STIM, CoV-2 IGRA BLANK and CoV-2 IGRA TUBE), and the Quan-T-Cell ELISA (96-well ELISA microplate) for the quantification of the released IFN-γ. Since three preparations are required for each patient sample and the quantitative ELISA includes 6 calibrators and 2 controls, 29 determinations can be performed per packing unit. Of course the ELISA can be processed completely automatically on our EUROIMMUN Analyzer I and I-2P in combination with the evaluation software EUROLab Quan-T-Cell and the EUROLabWorkstation ELISA.

Which sample materials can be used for the SARS-CoV-2 interferon-gamma release assay Quan-T-Cell?

The validation of the SARS-CoV-2 interferon-gamma release assay Quan-T-Cell was performed using heparinised whole-blood samples. The samples must be stimulated within 16 hours following sample collection. After stimulating the samples and obtaining the plasma, this can be used immediately for analysis or kept in cool storage (long term storage at -20 ° C or short-term storage at 2 – 8 °C).

Which RNA extraction methods can be used for the EURORealTime tests?

RNA extraction can be performed by any method (automated or manual) which is suited and validated for the sample materials. The EURORealTime SARS-CoV-2 test was validated with the QIAamp Viral RNA Mini Kit (Qiagen), the NucleoMag® VET Kit (Macherey-Nagel),  the CMG-2015 Prepito Viral DNA/RNA200 Kit (Chemagen) and the CMG-1033 chemagic Viral DNA/RNA 300 Kit H96 (Chemagen). The EURORealTime SARS-CoV-2 Influenza A/B was validated with the QIAamp Viral RNA Mini Kit (Qiagen) and the CMG-2017 Prepito Viral DNA/RNA300 Kit (Chemagen).

Which sample materials can be used with the EURORealTime tests?

Validation of the EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2 Influenza A/B was based on RNA preparations from throat swabs. The EURORealTime SARS-CoV-2 is also validated for the use of saliva as sample material. Other sample materials as RNA sources can be used as well, but have to validated by the customer.

How long does it take to get the results?

Here, the shipping time of the sample to the laboratory and the laboratory capacities must be taken into account. The sole test performance is around 2 hours with the antibody ELISA or the NeutraLISA, around 4 hours with the antigen ELISA and around 1.5 hours with the EURORealTime tests.

Stimulation of the T-cells during the performance of the SARS-CoV-2 interferon-gamma release assay Quan-T-Cell takes up 20 to 24 hours; the subsequent quantification of released IFN-γ by ELISA approximately 4 hours.

Which proteins are used to stimulate the T-cells in the SARS-CoV-2 interferon-gamma release assay?

The stimulation is based on the SARS-CoV-2 spike protein (see also antigen description of the antibody ELISAs) Both CD8+- and CD4+-T-cells are activated by this.

Which antigen is applied in the Anti-SARS-CoV-2 ELISAs (IgA and IgG), the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) and the Anti-SARS-CoV-2 NCP ELISAs (IgG and IgM)?

The Anti-SARS-CoV-2 ELISAs (IgA and IgG) and the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) are based on the S1 domain of the spike protein (S), which is produced in a sophisticated procedure with a human cell line. This procedure allows the presentation of complex three-dimensional structures and post-translational glycosylations. Therefore, antibodies that exclusively react with authentic epitopes of SARS-CoV-2 can also be detected. Due to the use of this antigen, the Anti-SARS-CoV-2 ELISAs (IgA and IgG) and the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) can detect antibodies with high specificity and sensitivity. Moreover, the S1 domain contains the receptor binding site (RBD) of SARS-CoV-2 via which the virus binds to human cells. Especially IgG antibodies against the RBD/S1 domain might have a virus-neutralising and thus protective function:It could be shown that antibodies against S1/RBD effectively prevent attachment of the virus to human cells. The question of immunity is still subject of intensive research.

Accordingly, the currently most promising vaccines, for which preliminary results have already been published, are based on the use of different presentations of the SARS-CoV-2-specific spike protein. Thus, the S1 antigen plays a crucial role in the detection of antibodies produced following vaccination (more information can be found here).

The Anti-SARS-CoV-2 NCP ELISAs (IgG and IgM) are based on a modified variant of the particularly immunogenic viral nucleocapsid protein (NCP). Antibodies against the nucleocapsid protein are typical markers of infections with SARS-CoV-2. The nucleocapsid protein in its full length, however, presents many homologies within the coronavirus family, which is why the occurrence of unspecific (false positive) reactions with antibodies against other worldwide circulating human pathogenic coronaviruses cannot be excluded. Therefore, instead of the complete protein, the EUROIMMUN Anti-SARS-CoV-2 NCP ELISAs (IgG and IgM) are based on a designer antigen in which unspecific conserved regions were eliminated.

What is the kinetics of the different antibodies against SARS-CoV-2?

Antibodies develop approximately one to two weeks after the onset of symptoms. The course of the immune response may vary significantly between patients. Usually, antibodies against the viral nucleocapsid protein (NCP) are produced earlier (approximately seven days after symptom onset) than antibodies against the spike protein (S/S1 domain) (approximately 10 days after symptom onset). Antibodies of immunoglobulin classes A (IgA) and M (IgM) often occur earlier than those of class G (IgG). IgA and IgM indicate the starting immune response, while IgG antibodies most probably play a role in the development of immunity. Especially IgG antibodies against the S1 domain of the spike protein may have a virus-neutralising and thus protective function. The question of immunity is still subject of intensive research.

Moreover, in the literature, examples of individual patients have been repeatedly described in whom the secretion of antibodies only started with a time delay of several weeks or failed to occur entirely. These patients are negative for anti-SARS-CoV-2 IgG and reduce the clinical sensitivity of serological tests.

Which is the underlying principle of the SARS-CoV-2 interferon-gamma release assay Quan-T-Cell?

The test principle is based on the immunological test method of the interferon-gamma release assay (IGRA), which is used to quantify IFN-γ released of immune cells following pathogen-specific stimulation. The Quan-T-Cell SARS-CoV-2 kit contains 30 stimulation tube sets each consisting of three stimulation tubes per whole-blood sample: (1) CoV-2 IGRA BLANK: no T-cell stimulation, for determination of the individual IFN-γ background; (2) CoV-2 IGRA TUBE: specific T-cell stimulation using antigens based on the SARS-CoV-2 spike protein; (3) CoV-2 IGRA STIM: unspecific T-cell stimulation by means of a mitogen, for control of the stimulation ability. Fresh human whole blood from a heparin blood collection tube is pipetted into the three stimulation tubes and incubated. If stimulable immune cells are present in the sample, these are activated during the incubation to release IFN-γ. After the incubation, the tubes are centrifuged to obtain stimulated heparinised plasma, which can be used immediately for the determination of the IFN-γ concentration by means of the Quan-T-Cell ELISA or stored for analysis at a later time point.

Which is the underlying principle of the SARS-CoV-2 NeutraLISA?

The NeutraLISA is modelled upon nature: Neutralising antibodies in the patient sample compete in the same incubation step (no preabsorption required!) with the recombinantly produced and biotinylated host cell receptor (ACE2) for the binding to the equally recombinantly produced SARS-CoV-2 S1 domain/RBD, which is coated onto the ELISA plate. Bound ACE2 receptor protein is then detected by incubation with peroxidase-labelled streptavidin and through a subsequent colour reaction. The more neutralising antibodies are present in the sample and inhibit the binding of the biotinylated ACE2, the weaker is the colour reaction. As opposed to the classic ELISA format, the NeutraLISA only detects such antibodies which have a virus-neutralising effect.

What is the advantage of using the Anti-SARS-CoV-2 QuantiVac ELISA (IgG)?

The Anti-SARS-CoV-2 QuantiVac ELISA (IgG) allows quantification of the anti-S1 IgG antibody concentration. The test system has been already calibrated using the NIBSC Working Standard (NIBSC code 20/162), which is the only currently available reference material for anti-SARS-CoV-2 antibodies. As soon as an international standard serum is officially available, it will be possible to issue the results of the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) in IU/ml. The quantification of the anti-S1 antibody concentration plays an important role in the development of S1-based vaccines. Please find further information here.

What are the advantages of the SARS-CoV-2 NeutraLISA?

The SARS-CoV-2 NeutraLISA is modelled upon nature and imitates the natural process of virus neutralisation. The test system exclusively detects antibodies against SARS-CoV-2 that actually inhibit the binding of the viral S1 domain/RBD to the human ACE2 and thus contribute to the neutralisation of the virus. The test results show a very high agreement with those from a plaque reduction neutralisation test (PRNT50). Unlike the classic virus neutralisation assay, however, this ELISA-based test does not require a BSL-3 laboratory, can be easily integrated in the laboratory routine and is automatable even for high throughputs. In this way, the SARS-CoV-2 NeutraLISA makes an important contribution to the evaluation of the immune reaction after infection or vaccination with an S1-based vaccine.

Which role does the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) play in the development of a vaccine and the worldwide vaccination programs?

By using the S1 subunit of the spike protein in the Anti-SARS-CoV-2 QuantiVac ELISA IgG, antibodies induced by the spike-protein-based vaccines currently applied can be detected and quantified to support the assessment of the immune protection achieved through vaccination.

For neutralisation of the virus, a sufficient amount of neutralising antibodies against S1/RBD must have been produced. In a comparison study, an excellent correlation of the Anti-SARS-CoV-2 QuantiVac ELISA with the Neutralization Antibody Test (GenScript cPass) was achieved. It can therefore be assumed that the EUROIMMUN ELISA also detects neutralising antibodies. Moreover, the SARS-CoV-2 NeutraLISA is a system which exclusively detects antibodies against neutralising antibodies. The results from the NeutraLISA also show a very high agreement with the results of plaque reduction neutralisation test (PRNT50).Well-characterised immunisations against other pathogens have shown that protection is only achieved at a certain threshold. This threshold must be able to be reliably and comparably used, independently of the vaccine (provided that a similar antigen is used) and test system. The definition of such a value is based on the results obtained from large-scale studies or long-time observations and is generally established based on evidence. The values must therefore be defined by independent institutions or the manufacturers of vaccines with reference to study results.

Since the clinical studies for the assessment of the vaccination success are still being evaluated, thresholds for the assessment of the antibody activity after vaccination have not yet been determined. However, it can be assumed that the determination of the antibody level will be one of the most important instruments in the assessment of the vaccination success. It is therefore very likely that one or several thresholds will be established. Since all of the most promising vaccines are based on the spike protein, these values will probably refer to the IgG response to the spike protein. Due to the very exact quantification of the antibody concentrations in the Anti-SARS-CoV-2 QuantiVac ELISA based on a 6-point calibration the assay can be applied with regard to every prospective threshold that might possibly be used for assessing the vaccination success.

Exact definition of such a threshold requires the availability of standardisation of serological methods. The necessity of a serological standard for the determination of SARS-CoV-2-specific antibodies was recognised early on by the WHO. For this reason, the development of reference materials has already started some time ago. An international standard serum and an independent reference sample panel will soon be available and provided by the NIBSC.

EUROIMMUN had the opportunity to participate in the characterisation of these reference materials. As soon as these are available, it will be possible to issue the results of the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) in IU/ml. However, the test system has already been calibrated based on the NIBSC Working Standard (NIBSC code 20/162), which is currently the only available reference material for anti-SARS-CoV-2 antibodies.

Do the EUROIMMUN test systems detect the new SARS-CoV-2 variants?

Since the end of 2020, several SARS-CoV-2 variants have been described, Alpha (B.1.1.7 lineage), Beta (b.1.351 lineage), Gamma (P.1 lineage), Delta (B1.617.2 lineage), Lambda (C.37 lineage), Mu (B.1.621 lineage) and Omicron (B.1.1.529 lineage), which are assumed to be more transmissible than other SARS-CoV-2 variants or are classified as variants of interest. All variants are mainly characterised by several mutations in the S gene, which codes for the spike protein.

EURORealTime test systems:

Since the EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2/Influenza A/B test systems are based on the ORF1ab and N genes of SARS-CoV-2, they are not affected by the mutations in the S gene. For the above mentioned variants, however, further mutations in other genes of the SARS-CoV-2 genome have been described, which may affect primer and probe binding.  Based on comparisons of the primer and probe sequences with published sequences of  the variants Alpha (B.1.1.7 lineage), Beta (b.1.351 lineage), Gamma (P.1 lineage), Delta (B1.617.2 lineage), Lambda (C.37 lineage), Mu (B.1.621 lineage) and Omicron (B.1.1.529 lineage), all of these variants are expected to be reliably detected with the EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2/Influenza A/B tests.

ELISAs, NeutraLISA, Immunoblot and Quan-T-Cell (IGRA):

Although the variants Alpha (lineage B.1.1.7), Beta (lineage B.1.351), Gamma (lineage P.1) and Delta (lineage B1.617.2) present mutations in the receptor-binding domain (RBD), which is an important component of the S1 domain of the spike protein used as an antigen in the Anti-SARS-CoV-2 ELISA (IgA/IgG), Anti-SARS-CoV-2 QuantiVac ELISA (IgG), SARS-CoV-2 NeutraLISA, EUROLINE Anti-SARS-CoV-2 Profile and Quan-T-Cell SARS-CoV-2, these mutations are not expected to affect the performance of these test systems. First internal studies using samples from patients who have been infected with the Alpha, Beta or Gamma variant, confirm this assumption concerning the antibody test systems. At this early stage it is not yet possible to make a statement about the possible effects of the Omicron variant.

Generally, individual amino acid substitutions in an antigen do not significantly alter the properties of the antibody or T-cell binding to the whole protein since only a single epitope is affected. Due to the fact that proteins have a large number of epitopes, the loss of a single binding site can be easily compensated. Furthermore, an individual amino acid substitution does not necessarily mean that antibodies or T cells against a variant can no longer bind to the respective epitope of the original variant. In many cases, no deterioration in the binding behavior is to be expected because, for instance, not every site in the epitope is targeted by the antibodies or T cells.

With respect to the SARS-CoV-2 Antigen ELISA and Anti-SARS-CoV-2 NCP ELISA (IgG, IgM) it is not expected that the new variants affect the performance of the test systems. These tests are based on the detection of the viral nucleocapsid protein and human antibodies (IgG, IgM) against this protein, respectively, and are thus not affected by the mutations in the S gene.

EUROIMMUN carefully follows all reports on new SARS-CoV-2 variants from sources such as ECDC, WHO or FDA. Possible influences of new variants on our tests systems are investigated immediately. Based on these and other analyses of, for instance, current literature, reports from health authorities and feedback from laboratories, a reassessment of the situation is carried out regularly as part of risk management in order to be able to react with short notice if necessary and take preventative or corrective measures where required.