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.
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.
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.
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.
The EURORealTime SARS-CoV-2, the Anti-SARS-CoV-2 ELISA (IgA and IgG), the Anti-SARS-CoV-2 NCP ELISAs (IgG and IgM) and the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) 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 approved by the U.S. food and drug administration (FDA) through an emergency use authorization (EUA) for use by authorised laboratories. The Anti-SARS-CoV-2 ELISA (IgG) is also approved by the Brazilian Health Regulatory Agency ANVISA.
Our tests are exclusively performed in the laboratory. The required samples (blood, swabs...) are taken by a physician, e.g. your GP, 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) which performs the tests.
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. Our range of antibody tests for COVID-19 diagnostics has meanwhile been extended by the CE-marked Anti-SARS-CoV-2 NCP ELISAs (IgG, IgM) and the Anti-SARS-CoV-2 QuantiVac ELISA (IgG). For acute diagnostics, in addition to the EURORealTime SARS-CoV-2 for direct detection of the virus via 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 and the EURORealTime tests are designed for use in diagnostic laboratories and are only supplied to these. These tests are no rapid tests for at-home testing.
If you wish to receive a quote for our tests, please contact your local distributor.
Please note that our test systems are no rapid tests for at-home testing. They are designed for the investigation of large samples volumes and their processing requires laboratory equipment. Cassette-based rapid tests for at-home use may present technical limitations such as reduced sensitivity or specificity. We therefore do not offer rapid tests.
If you suspect to be infected with SARS-CoV-2, please ask your physician for a laboratory diagnostic test.
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, multiparametric 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, butno 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 and Anti-SARS-CoV-2 QuantiVac ELISA 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 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 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-CoC-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.
As long as the virus can be detected in secretions of the respiratory tract by means of RT-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. 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.
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.
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: 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).
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) and the CMG-2015 Prepito Viral DNA/RNA200 Kit (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).
Validation of the EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2 Influenza A/B was based on RNA preparations from throat swabs. The validation of saliva as sample material is currently in planning. Other sample materials as RNA sources can be used as well, but have to validated by the customer.
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, around 4 hours with the antigen ELISA and around 1.5 hours with the EURORealTime tests.
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 with which the virus binds to human cells. Especially IgG antibodies against the RBD/S1 domain might have a virus-neutralising and thus protective function since 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.
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.
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.
The Anti-SARS-CoV-2 QuantiVac ELISA (IgG) allows quantification of the anti-S1 IgG antibody concentration. An excellent correlation of the test system with the recently approved WHO reference material “First WHO International Standard Anti-SARS-CoV-2 Immunoglobulin” has already been shown. Therefore, it is now possible to give the results in standardised units. Due to the outstanding linearity of the test system the same calculation factor can be applied over the entire broad measurement range. The quantification of the anti-S1 antibody concentration plays an important role in the development of S1-based vaccines.
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 presented 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.
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 are not yet 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. EUROIMMUN had the opportunity to participate in the characterisation of these reference materials. At the end of December 2020, a new international standard serum, the “First WHO International Standard Anti-SARS-CoV-2 Immunoglobulin”, was officially approved. An excellent correlation of the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) with this reference material has already been shown. Therefore, it is now possible to give the results in standardised units (BAU/ml, numerically identical to IU/ml).
At the end of 2020, two new SARS-CoV-2 variants VOC-202012/01 (B.1.1.7 lineage) and 501.V2 (B.1.351lineage) have been described to lead to an increased number of COVID-19 cases in southern England and South Africa, respectively. About another variant, 501Y.V3 (P.1 lineage) has been reported in Brazil in January 2021. . All three variants are mainly characterised by several mutations in the S gene, which encodes the spike protein. Since there was a great increase in infections with these variants within a short period of time, it is assumed that it is more infectious than other SARS-CoV-2 variants.
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 gene of SARS-CoV-2, they are not affected by the mutations in the S gene. For the variants, however, some additional mutations in other genes of the SARS-CoV-2 genome were described, which may affect primer and probe binding. Based on comparisons of the primer and probe sequences with published sequences of VOC*202012/01 (B.1.1.7 lineage), 501.V2 (B.1.351 lineage) and 501Y.V3 (P.1 lineage), it is expected that the three variants can be reliably detected with the EURORealTime SARS-CoV-2 and EURORealTime SARS-CoV-2/Influenza A/B tests.
ELISA test systems:
Although the discovered SARS-CoV-2 variants 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) and Anti-SARS-CoV-2-QuantiVac ELISA (IgG), it is not expected that these mutations affect the performance of these test systems. Usually, individual amino acid exchanges of an antigen do not essentially affect the binding properties of antibodies against the overall protein, because only a single epitope is affected. Since proteins have a large number of epitopes, even the failure of a single binding point can be compensated. In addition, an individual amino acid exchange does not necessarily mean that antibodies against this variant cannot bind to the respective epitope in the original variant. In many cases, no deterioration of the binding behaviour is to be expected, since, for example, not every site within an epitope is an antibody target.
Also with respect to the SARS-CoV-2 Antigen ELISA and the 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 antibodies against this protein respectively, and are thus not affected by the mutation within the S gene.
EUROIMMUN meticulously follows reports on the new SARS-CoV-2 variants from sources such as ECDC, WHO or FDA. Possible influences of new variants on our tests systems are checked immediately. Within the risk management procedures, the situation is regularly reevaluated based on this and other analyses, e.g. of current literature, reports from health institutions, and feedback from laboratories in order to be able to react with short notice if necessary and take preventative or corrective measures where required.
EUROIMMUN develops and produces test systems for doctors and laboratories that cannot be performed by private individuals.