DE202020105116U1 - Reagents and uses in diagnosing SARS-CoV-2 infection - Google Patents

Abstract

Use of a polypeptide comprising SEQ ID NO1 or a variant thereof which can bind to an antibody against SED ID NO1 from a sample from a patient for the production of a diagnostic kit.

Description

The present invention relates to the use of a polypeptide comprising SEQ ID NO1 or a variant thereof, which can bind to an antibody against SED ID NO1 from a sample from a patient, for the production of a diagnostic kit or use of an antibody against SEQ ID N01 for diagnosing a SARS-CoV-2 infection or for differential diagnosis of a coronavirus infection or for screening donor blood, preferably for contamination with a coronavirus SARS-CoV-2 or use of an antibody, optionally a recombinant antibody that binds to SEQ ID NO1, which is preferred is recognized by a secondary antibody which binds to antibodies of the immunoglobulin class IgA, IgG or IgM, preferably IgA, as a calibrator for the early diagnosis of a SARS-CoV-2 infection.

Towards the end of 2019, an increasing number of patients with pneumonia caused by an unknown pathogen emerged in Wuhan, the capital of Hubei Province in China, and spanned almost the entire country of China. A new coronavirus has been isolated, and based on its phylogeny, taxonomy, and established practices, the Corona Study Group (CSG) identified it as a relative of the Severe Acute Respiratory Syndrome Corona Virus (SARS-CoV-1) and named it Severe Acute Respiratoy Syndrome Corona virus 2 (SARS-CoV-2).

Although SARS-CoV-2 is generally less pathogenic than SARS-CoV-1 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), it shows a comparatively high transferability. Since the symptoms can be mild and can be mistaken for a cold, there is a risk that patients will be unaware of their infection and will contribute to the spread of the virus.

SARS-CoV-2 is a coronavirus with four structural proteins, more precisely the spike (S), E (envelope), M (membrane) and N (nucleocapsid) proteins. The N protein carries the RNA genome, whereas the other three structural proteins are components of the viral envelope. The S-protein is responsible for enabling the virus to attach to the membrane of a host cell and fuse with it. It comprises an S1 domain that catalyzes attachment and an S2 domain that catalyzes fusion with the host cell. The S1 domain comprises the receptor binding domain (RBD), the binding site for the angiotensin converting enzyme 2 (ACE2) receptor on human host cells. Therefore, the RBD is the binding site of neutralizing antibodies that block the interaction between the virus and its host cells and thus confer immunity.

In contrast to the group of coronaviruses, which includes SARS-CoV-1 and SARS-CoV-2 and which are associated with high mortality and serious illness, there is another group of coronaviruses that are usually associated with mild and rapidly passing illness and it includes coronaviruses 229E, NL63, OC43 and HKU1.

Due to the high transferability, reliable methods for diagnosing SARS-CoV-2 infection and immunity to SARS-CoV-2 are of paramount importance. In the past, a combination of serological methods and RT-PCR (Real Time Polymerase Chain Reaction) was used to detect and confirm infections with coronaviruses.

RT-PCR methods are based on the detection of the nucleic acids of the coronavirus of interest, more precisely the RNA. They are quick and with the help of throat or nasal / throat swab samples, they can be used for the detection of SARS-CoV-1 during the first week of illness. However, their usefulness depends very much on how long the nucleic acid can be detected in samples, which differs from virus to virus. In particular, a negative result from a diagnostic test carried out on a sample obtained after the early stage of the disease lacks meaningfulness. Therefore, several samples should be checked, one from the first week of infection and a follow-up sample obtained three to four weeks later. A sample from the upper part of the patient's airway is also required. Incorrect extraction of such a sample can also lead to false negative results.

Corman et al. published an RT-PCR-based test for the detection of SARS-CoV-2 ( Corman, VM, Landt, O., Kaiser, M., et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25 (3): 2000045. doi: 10.2807 / 1560-7917. ES.2020.-25.3.2000045 ).

Therefore, serological tests are also widely used. The special feature of these tests is the detection of antibodies that are specific to a certain virus such as SARS-CoV-2 are associated. If the presence of such an antibody is detected in patient serum, this indicates that the patient may be or have been infected. In contrast, the absence of the antibody suggests that the patient was not previously infected. However, a negative result can also be obtained at a very early stage of the disease, ie before the patient's body was able to produce specific antibodies. Further serological monitoring, taking into account whether a detected antibody belongs to a particular immunoglobulin class, can help to monitor the course of the disease and to differentiate an acute from a previous infection or vaccination. For example, a low level of IgG that persists for months or years suggests that there was a past infection or that vaccination was present.

When a test is first developed and validated, the time at which an antibody will first be produced and detectable after infection with a new virus cannot be predicted because it depends on a variety of factors, including the virus itself, the extent of his immunogenicity, the patient and his immune system, e.g. B. suppressed or overstimulated, the immunoglobulin class of the antibody of interest and the target antigen selected for the development of the diagnostic test.

In addition, the specificity and sensitivity of such tests are crucial. For example, a group of viruses, e.g. B. Flaviviruses, antigens with an epitope with a conserved amino acid sequence have in common. The detection of an antibody against such an epitope then does not indicate which type of virus was detected. For example, it would be impossible to distinguish between langat flavivirus, which is not associated with any known disease in humans, and dengue flavivirus, which can cause severe dengue, a life-threatening disease. In terms of sensitivity, the concentration of an antibody that is considered as an indicator of infection may be too low to be detected in routine diagnostic operations.

Therefore, a problem underlying the present invention is to provide a test and reagents for the early serological detection of SARS-CoV-2.

Another problem on which the present invention is based is to provide a test which can be used to distinguish infections with known coronaviruses.

Another problem on which the present invention is based consists in providing a test with a high level of optimized reliability, in particular with regard to sensitivity and / or specificity, preferably sensitivity, over a long period of time.

WO14045254 discloses tests for diagnosing MERS infection.

US2005 / 0112559 discloses methods and agents relating to SARS-CoV. The nucleocapsid protein is considered the main antigen for diagnostics.

WO2005 / 118813 discloses a variant of the SARS-CoV-related S protein and the detection of its presence or the presence of antibodies binding against it in samples.

US2006 / 0188519 discloses peptides for the diagnosis of SARS-CoV infection.

Hsueh et al., Reported that antibodies of the immunoglobulin class IgG could already be detected four days after the onset of a SARS infection, at the same time or one day earlier than antibodies of the immunoglobulin classes IgM and IgA ( Hsue, PR, Huang, LM, Chen, PJ, Kao, CL, and Yang PC (2004) Chronological evolution of IgM, IgA, IgG and neutralization antibodies after infection with SARS-associated coronavirus, Clinical Microbiology and Infection, 10 (12) , 1062-1066 ).

The problems are solved by the subject matter of the independent and dependent claims.

In a first aspect, the problem is solved by a method for diagnosing a SARS-CoV-2 infection, comprising the step of detecting the presence or absence of an antibody against SEQ ID NO1, preferably an antibody of the immunoglobulin classes IgM, IgG and / or IgA, more preferably an antibody of the immunoglobulin class IgA, in a sample from a subject.

In a second aspect, the problem is solved by a method for differential diagnosis of a coronavirus infection, preferably for differentiating a SARS-CoV; preferably SARS-CoV-2-; MERS, NL63, 229E, OC43 and HKU1 infection, comprising the step of detecting the presence or absence of an antibody against SEQ ID NO1 in a sample from a subject, preferably an antibody of the immunoglobulin classes IgA and / or IgG, more preferably an antibody of the immunoglobulin class IgA.

In a preferred embodiment, the presence of an antibody of the immunoglobulin classes IgG and / or IgM against SEQ ID NO1 is detected in addition to that of an antibody of the immunoglobulin class A against SEQ ID NO1.

In a preferred embodiment, the sample is a blood sample, preferably from the group comprising whole blood, serum, capillary blood or plasma. The capillary blood is preferably in the form of a third blot spot, which can be prepared by the patient and sent to the laboratory, followed by the extraction of the blood.

In a preferred embodiment, the antibody is detected using a labeled secondary antibody which preferably binds to antibodies of the immunoglobulin classes IgA, IgG or IgM.

In a preferred embodiment, the antibody, preferably an antibody of the immunoglobulin classes IgA, IgG or IgM, is detected using a method selected from the group comprising colorimetry, immunofluorescence, detection of enzymatic activity, chemiluminescence and radioactivity.

In a preferred embodiment, the infection is detected in an early phase.

In a third aspect, the problem is solved by using an antibody against SEQ ID NO1, preferably an antibody of the immunoglobulin class IgA, for diagnosing a SARS-CoV-2 infection or for the differential diagnosis of a coronavirus infection, preferably to differentiate a SARS -CoV-2, MERS, NL63, 229E, OC43 and HKU1 infection.

In a preferred embodiment there is a use for the early diagnosis of the infection of a SARS-CoV-2 infection.

In a fourth aspect, the problem is solved by a kit which comprises a polypeptide comprising SEQ ID NO1 or a variant thereof, preferably coated on a diagnostically useful carrier, and one or more than one reagent, preferably all reagents from the group comprising one Antibodies against SEQ ID NO1, a washing buffer, a means for detecting the presence or absence of an antibody against SEQ ID NO1, preferably an antibody of the immunoglobulin classes IgA, IgG and / or IgM, preferably a secondary antibody that is specific against antibodies of the immunoglobulin -Class IgA, IgG and / or IgM binds, preferably comprising a detectable label, and a dilution buffer.

In a preferred embodiment, the diagnostically useful carrier is selected from the group comprising a bead, preferably a magnetic bead, a test strip, a microtiter plate, a membrane, preferably from the group comprising Western blot, line blot and dot blot, a lateral flow device, a glass surface, a microscope carrier, a microarray, a chromatography column and a biochip and is preferably a microtiter plate.

In a preferred embodiment, the kit comprises two or more than two, preferably three or more than three calibrators.

In a preferred embodiment, the calibrator is a recombinant antibody which binds against SEQ ID NO1 and which is preferably recognized by a secondary antibody which binds to antibodies of the immunoglobulin class IgA, IgG and / or IgM.

In a sixth aspect, the problem is solved by using a polypeptide comprising SEQ ID NO1 or a variant thereof or an antibody against SEQ ID NO1, preferably an antibody of the immunoglobulin classes IgA, IgG and / or IgM, for producing a diagnostic kit.

In a seventh aspect, the problem is solved by using a recombinant antibody that binds against SEQ ID NO1, which is preferably recognized by a secondary antibody that binds against antibodies of the immunoglobulin classes IgA, IgG and / or IgM, as a calibrator Early diagnosis of SARS-CoV-2 infection.

Various antigens and antibodies have been used as the basis for the immunological detection of coronaviruses, including the whole virus or any of the structural proteins or fragments thereof, as well as antibodies that bind against all of these antigens.

The present invention is based on the surprising finding of the inventors that antibodies against SEQ ID NO1, the S1 domain of the S protein, can be detected during an early phase of the infection. They can be used to determine the presence of an infection at an early stage of the infection, before or at least just before PCR-based tests begin, giving false negative results for many patients.

Furthermore, the present invention is based on the surprising finding that antibodies of the immunoglobulin class IgA against SARS-CoV-2 antigens such as S1 and N protein can be detected in many patients earlier than other antibodies such as antibodies of the immunoglobulin class IgG, also in a subpopulation of patients who lack detectable antibodies of the immunoglobulin class IgM.

Furthermore, the invention is based on the surprising finding that antibodies against SEQ ID NO1 persist longer and can be detected over a longer period of time than antibodies against the SARS-CoV-2-N protein.

Furthermore, there is a surprisingly low cross-reactivity with a number of antibodies in samples from patients who are infected with coronaviruses other than SARS-CoV-1 and SARS-CoV-2, which explains the surprising specificity of the detection of antibodies against SEQ ID NO1.

In a preferred embodiment, the term "diagnosis" as used herein is to be used in its broadest sense and can mean any type of procedure aimed at obtaining information useful for assessing whether a patient has a history of At the time of diagnosis or in the future, suffers or will suffer from a particular disease or disorder, or is likely or more likely than an average subject or comparative subject to suffer or will suffer from it, the comparative subject preferably having similar symptoms or being helpful to find out how the disease is progressing or is likely to progress in the future, or to evaluate the response of a patient, or patients in general, to a particular treatment, preferably vaccination, or to find out whether a sample is from such a patient . Such information can be used for clinical diagnosis, but can also be obtained from an experimental and / or research laboratory for the purpose of general research, e.g. B. to determine the proportion of subjects suffering from the disease in a patient cohort or in a population as part of epidemiological studies. In other words, the term “diagnosis” encompasses not only diagnosing, but also prognosticating and / or monitoring the course of a disease or disorder, including monitoring the response of one or more than one patient to the administration of an active substance or an active substance candidate , e.g. B. to determine its effectiveness. While the result may be assigned to a specific patient for clinical diagnostic applications and communicated to a physician or facility treating the patient, it may not necessarily be the case for other applications, e.g. B. in diagnostics for research purposes, where it may be sufficient to assign the results to a sample from an anonymized patient. Preferably the disease is a SARS infection comprising SARS-CoV-1 and SARS-CoV-2, more preferably a SARS-CoV-2 infection.

In a preferred embodiment, the methods and products according to the present invention can be used for interaction studies, comprising determining whether a drug candidate or other compound, including a vaccine candidate or a compound from a body such as a blocking antibody, is present and binding an antibody interfere with SARS-CoV-2 or influence any subsequent process. In a preferred embodiment, they can be used for monitoring the immune response, more preferably the appearance and / or concentration of antibodies to a polypeptide comprising, preferably consisting of SEQ ID NO1, after the administration of an immunogenic composition comprising SEQ ID NO1 or an immunogenic variant of which, e.g. B. to a mammal, which may be a mammal other than a human, such as an experimental animal.

The subject is likely or more likely to have a SARS-CoV-2 infection if an antibody of the immunoglobulin classes IgA and / or IgG and / or IgM against SEQ ID NO1 can be detected in a sample from the subject. The person skilled in the art is familiar with the interpretation of results that reflect the presence of antibodies (e.g. Doerr, HW, and Gerlich, W., Medical Virology: Basics, Diagnostics, Prevention and Therapy, Thieme 2010, e.g. .7). In short, the first detection of specific antibodies can be used for the initial diagnosis of infections. Immunoglobulin class IgM antibodies are usually the first antibodies to be detected, followed by antibodies of the immunoglobulin class IgG. Antibodies of the immunoglobulin class IgA are normally not diagnostically relevant except in specific cases such as enteroviruses (Gressner / Arndt, Lexikon der Medizinischen Laborordiagnostik, 2nd edition, Springer, 2013, page 1387). If they occur, it can happen after the appearance of IgG antibodies, and their concentration will be less than that of antibodies of the immunoglobulin classes IgM and IgG. A low concentration of antibodies of the immunoglobulin class IgG in the absence of antibodies of the immunoglobulin classes IgM and IgA indicates a previous infection. An increasing concentration or a high concentration in the early stages of the disease of antibodies of the immunoglobulin class IgG can indicate re-infection. A low concentration of antibodies of the immunoglobulin class IgG, which persists for a long time, may indicate an earlier immunization, but this does not represent an acute disease, especially in the absence of antibodies of the immunoglobulin classes IgM and / or IgA. If a patient has been vaccinated with a vaccine or vaccine candidate comprising SEQ ID NO1 or a variant thereof, the detection of an antibody against SEQ ID NO1 or an increasing concentration of such an antibody can indicate successful immunization.

The sample is preferably from a mammal, more preferably from a human.

Therefore, the term "diagnosis" preferably does not imply that the diagnostic methods or agents according to the present invention are definitive and sufficient to make the final diagnosis on the basis of a single test, let alone parameter, but means a contribution to a procedure known as “differential diagnosis”, that is, a systematic diagnostic approach that takes into account the likelihood of a number of possible diseases based on a number of diagnostic parameters. According to the invention, a distinction can be made as to whether a patient, preferably one who is already suspected of having a coronavirus infection, has a SARS, preferably SARS-CoV-1 and / or SARS-CoV-2 infection, or a another coronavirus infection, preferably with a coronavirus from the group comprising MERS, NL63, 229E, OC43 and HKU1. For example, a patient might initially be suspected of having a coronavirus infection based on possible exposure in a risk environment and / or based on common symptoms such as fever, cough, or shortness of breath. A PCR and / or an immunoassay can then be carried out. If the PCR is negative, e.g. B. because the sample was taken later than a week after infection, the method according to the invention can be used to detect the presence or absence of an antibody against SEQ ID NO1. In addition, the presence or absence of an antibody against an antigen from another coronavirus can be detected, preferably from the group comprising SARS-CoV-1, MERS, NL63, 229E, OC43 and HKU1, preferably MERS, NL63, 229E, OC43 and HKU1. Antibodies, preferably antibodies of the immunoglobulin class IgA, IgG and / or IgM against homologues of SEQ ID NO1 from SARS-CoV-2 can be detected. The diagnosis may be final based on specific clinical symptoms such as headache and body aches, which are more characteristic of SARS-CoV-1, or loss of taste, smell, or swollen throat, which are more characteristic of SARS-CoV-2 will. A distinction between SARS-CoV-1 and SARS-CoV-2 is possible based on the different time-resolved immunoglobulin class signature, in particular the later appearance of antibodies of the immunoglobulin class IgA in SARS-CoV-1 ( Hsue, PR, Huang, LM, Chen, PJ, Kao, CL, and Yang PC (2004) Chronological evolution of IgM, IgA, IgG and neutralization antibodies after infection with SARS-associated coronavirus, Clinical Microbiology and Infection, 10 (12) , 1062-1066 ). Antibody concentrations can be monitored, e.g. B. over several weeks, e.g. B. to detect the disappearance or appearance of an antibody of interest, which can help to distinguish a primary and a secondary infection or to detect an infection with more than one coronavirus.

In a preferred embodiment, the term “SARS-CoV-2”, as used herein, means a virus which is characterized by the genome, as it is in the GenBank database under the accession number MN908947 or SEQ ID NO13 is characterized, preferably as shown in SEQ ID NO13, as well as derivatives thereof that contain at least 80, preferably 85, preferably 88, preferably 90, preferably 91, preferably 92, preferably 93, preferably 94, preferably 95, preferably 96, preferably 97, preferably 98, preferably 99, preferably 99.5, preferably 99.8, preferably 99.9 or 99.99 percent sequence identity over the entire nucleotide sequence of the genome. All database entries used herein correspond to the version that was available on the Internet on the earliest priority date or the filing date of the application.

In a preferred embodiment, the term "diagnosis" means that the method or product or use can be used to aid in the diagnosis of a disease or the identification of a subject at risk of suffering from the disease. The term "diagnosis" can also mean a method or agent that is used to select the most promising treatment regimen for a patient. In other words, the method or agent may involve selecting a treatment regimen for a subject.

In a preferred embodiment, the method according to the present invention comprises the step of providing the diagnostically useful carrier and a sample from a patient suspected of being infected, preferably a mammalian patient, more preferably a human patient. The carrier is coated with the polypeptide comprising SEQ ID NO1 or a variant thereof. The carrier can then be contacted with the sample and under conditions which include binding of any antibodies to the polypeptide comprising SEQ ID NO1 or a variant thereof. The sample can then be removed and the support with the polypeptide washed to remove any residue of the sample. A secondary antibody or like reagent or agent which binds to the antibody and bears a detectable label can then be contacted with the support under conditions which permit the formation of a complex between any bound antibody and the secondary antibody. The support can then be washed to remove unbound secondary antibody. Finally, the presence of the antibody can be detected by checking that the secondary antibody can be detected.

In a preferred embodiment, the term "SARS-CoV-2 infection" or similar terms as used herein means infection of a subject, preferably a human, with SARS-CoV-2, i. the presence of the virus, at least temporarily, in the body of the subject, more preferably with detectable concentrations of the virus itself and / or of one or more than one biomarker from the group comprising a SARS-CoV-2 polypeptide from the group comprising the structural proteins, in particular S and N protein, more preferably S1 protein, and antibodies that bind to them, or a nucleic acid, the latter being detectable by PCR. The subject may have clinical symptoms, preferably one or more than one, more preferably all symptoms from the group comprising fever, fatigue, dry cough, nasal congestion, runny nose and swollen throat. More severe symptoms include difficulty breathing, chest pain or pressure, and sudden confusion. The disease can lead to complications such as pneumonia, acute respiratory distress syndrome, sepsis, septic shock, and kidney failure. However, many infected subjects have mild symptoms and may not even be aware of their infection.

In a preferred embodiment, the method is used more than once to examine samples from the same patient, preferably on different days. For example, the presence or absence of antibodies can be examined daily for a week or two. In a preferred embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 samples are examined on different days. In a preferred embodiment, samples are taken over a period of at least 2, 3, 4, 6, 8, 12 or 16 weeks.

The methods and reagents according to the present invention can also be used to study the efficacy and utility of an antiviral agent or vaccine or vaccine candidate. A vaccine can be useful when the presence, preferably the increase in concentration, of an antibody to SEQ ID NO1 is detected. An antiviral agent can be useful when the absence or a decrease in the concentration of antibodies against SEQ ID NO1, preferably of the immunoglobulin classes IgA, IgM or IgG, indicates that the patient is no longer suffering from an acute infection or is in the process of getting away from it recover from an acute infection.

The method and the reagents according to the present invention can also be used to screen whether donated blood is contaminated with a coronavirus or whether a blood donor produces antibodies against SARS-CoV-2, preferably SEQ ID NO1, from his donated blood can be taken, e.g. B. for therapeutic uses. After detecting the presence or absence of antibodies of the immunoglobulin classes IgG, IgM and / or IgA, preferably IgG and IgA, even more preferably IgA, against SEQ ID NO1, an antibody against SARS-CoV-2, preferably SEQ ID NO1, be purified from the purified blood of the blood donor, e.g. By affinity chromatography, by contacting the carrier according to the present invention with the donated blood under conditions compatible with the formation of a complex comprising the antibody to SEQ ID NO1 and the carrier, removing remaining donated blood and separating the antibody from the carrier. In a preferred embodiment, donated blood is selected from the group comprising red blood cells, plasma, platelets and whole blood, and can comprise an anticoagulant reagent. In addition, a compound can be present which is selected from the group comprising citrate, phosphate, dextrose and adenine, preferably all of these compounds.

An antibody to be detected preferably binds specifically to its target, even more preferably SEQ ID NO1. Specific binding preferably means that the binding reaction is stronger than a binding reaction, which is determined by a dissciation constant of 1 × 10 ^-5 M, more preferably 1 × 10 ^-7 M, more preferably 1 × 10 ^-8 M, more preferably 1 × 10 ^-9 M , more preferably 1 × 10 ^-10 M, more preferably 1 × 10 ^-11 M, more preferably 1 × 10 ^-12 M, is characterized as determined by Surface Plasmon Resonance using Biacore equipment at 25 ° C in PBS buffer at pH 7 becomes.

The teaching of the present invention can be carried out not only using polypeptides which have the exact sequences referred to in this application, such as, for example, SEQ ID NO1, for example by function, name, sequence or accession number, or implicitly, but also using variants of such polypeptides.

In a preferred embodiment, the term "variant" as used herein means at least one fragment of the full-length sequence referred to or a polypeptide comprising such a fragment, more specifically one or more than one amino acid or nucleic acid sequence, which is shortened by one or more than one amino acid at one or both ends relative to the full-length sequence. Such a fragment comprises or codes for a peptide which comprises at least 10, 15, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500 or 600 consecutive amino acids of the original sequence, or for a variant thereof . For example, SEQ ID NO12 is an exemplary fragment that can be used.

In a further preferred embodiment, the term “variant” relates not only to at least one fragment, but also to a polypeptide or fragment thereof that comprises amino acid sequences, preferably a fragment comprising at least 25, more preferably 50, more preferably 200 consecutive amino acids, at least 40, 50 , 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence referred to or to the fragment thereof with amino acids other than those for the biological activity, e.g. B. the ability to specifically bind to an antibody of interest, or the folding or structure of the polypeptide essential amino acid are deleted or substituted and / or one or more than one such essential amino acid is conservatively replaced and / or amino acids are added or deleted, so that the biological activity of the polypeptide is at least partially retained. The prior art encompasses numerous methods that can be used to align two given nucleic acid or amino acid sequences and to calculate the extent of identity, see e.g. B. Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition . In a preferred embodiment, the ClustalW software ( Larkin, MA, Blackshields, G., Brown, NP, Chenna, R., McGettigan, PA, McWilliam, H., Valentin, F., Wallace, IM, Wilm, A., Lopez, R., Thompson, JD, Gibson, TJ, Higgins, DG (2007): Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948 ) is used with the basic settings applied.

SARS-CoV-2 publications on specific amino acid sequences, such as Beal et al. can help the specialist to design suitable variants ( Beal, J., Mitechell, T., Wyschogrod, W., Manthey, J, and Clore, A. (2020) Highly Distinguished Amino Acid Sequences of 2019-nCoV (Wuhan Coronavirus) doi: https://doi.org/ 10.1101 / 2020.01.31.929497 ), as well as publications that concern SARS-CoV, e.g. B. Hua, R., Zhou, Y., Wang, Y., Hua, Y and Tong, T. (2004). Identification of two antigenic epitopes on SARS-CoV spike protein, BBR 319, 929-935 , whereby homologous epitopes can be found and SARS-CoV-2 epitopes can be identified on the basis of their homology. For example, possible epitopes can be derived from SEQ ID NO5. Dahlke et al. show an epitope mapping based on a microarray comprising overlapping peptides with a length of 15 amino acids, which are derived from the S1 polypeptide ( Dahlke, C., Heidepriem, J., Kobbe, R., Santer R., Koch, T., Fathi, A., Ly, M. L, Schmiedel, S., Seeberger, PH, ID-UKE COVID-19 study group, Addo, MM, and Loeffler, F. F. (2020) https://doi.org/10.1101/2020.04.14.20059733doi ). Specifically, peptides have been identified comprising the amino acid sequences SSVLHSTQDLFLPFF (SEQ ID NO14, which is 30-44 of SEQ ID NO1), TWFHAIHVSGTNGTKRFDNPV (SEQ ID NO15, which is 48-68), NVKMQQ16 (SEQ ID NOKS is 110-166), DLPQGFSALEPLVDL (SEQ ID NO17, which is 200-214), LLALHRSYLTPGDSSSGWTAGAAAY (SEQ ID NO18, which is 226-250), QPRTFLLKYNENGTITDAVDCALDP (SEQ ID NO.19, which is .19) 256-277), NATRFASVYAWNRKR (SEQ ID NO20, which is 328-342), TGKIADYNYKLPDDF (SEQ ID NO21, which is 399-414), YNYLYRLFRKSNLKP (SEQ ID NO22, which is 434- 448), FGRDIADTTDAVRDPQTLEILDI (SEQ ID NO23, which is 550-572), SNQVAVLYQDVNCTE (SEQ ID NO24, which is 590-604), AGCLIGAEHVNNSYECDIP (SEQ ID NO25, which is 632-650) of the S1 protein were identified as IgA reactive epitopes. Peptides comprising the amino acid sequences YNSASFSTFKCYGVS (SEQ ID NO26, which is 354-368), STGSNVFQTRAGCLI (SEQ ID NO27, which is 622-636) of the S1 protein have been identified as IgG-reactive epitopes. Peptides comprising the amino acid sequences SSVLHSTQDLFLPFF (SEQ ID NO28, which is 30-44) and LLALHRSYLTPGDSSSGWTAGA AAY (SEQ ID NO29, which is 226-250) of the S1 protein were identified as IgM-reactive epitopes. In addition, the inventors have shown that peptides with the sequences RTQLPPAYTNS (SEQ ID NO34), LTPGDSSSGWTAG (SEQ ID NO35), YQAGSTPCNGV (SEQ ID NO36) and YGFQPTNGVGYQ (SEQ ID NO37) are reactive with antibodies from SARS patient CoV-2 are.

In a preferred embodiment, variants may include additional chemical modifications, e.g. B. labels such as isotope labels or detectable labels or covalent labels such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation and the like. Those skilled in the art are familiar with methods of modifying polypeptides. In addition, variants can also be created by fusion with other known polypeptides or variants thereof, e.g. B. artificial linkers, affinity tags, other antigens and the like. For example, SEQ ID NO3 is a fusion protein according to the present invention.

According to the present invention, a medical or diagnostic device such as the diagnostically useful carrier by expressing a recombinant variant of SEQ ID NO1 comprising an affinity tag, optionally with an artificial linker, which may comprise a protease cleavage site, in a cell such as a eukaryotic or prokaryotic cell, by contacting the expressed variant with a ligand that specifically binds to the affinity tag, the ligand being immobilized on a solid phase, by washing the solid phase in such a way that non-specifically bound material is removed from the cell, and by Elute the expressed variant from the solid phase, preferably by adding an excess of non-immobilized ligand. The variant can then be immobilized on the device. Optionally, the affinity tag can be removed by contacting the variant with a protease, preferably a protease that recognizes the protease cleavage site, before the immobilization. The affinity tag can be selected from the group of tags that include 18A, ACP, aldehyde, Avi, BCCP, calmodulin, chitin-binding protein, E-tag, ELK16, FLAG, Flash, polyglutamate, polyaspartate, GST, GFP, HA , Isope, maltose-binding protein, myc, nus, NE, ProtA, ProtC, Tho1d4, S-Tag, SnoopTag, SpyTag, SofTag, Streptavidin, Strep-tag II, T7-Epitope-Tag, TAP, TC, Thioredoxin, Ty , V5, VSV and Xpress-Tag includes. Useful proteases include, but are not limited to, TEV, thrombin, factor Xa, or enteropeptidase. Suitable linkers are part of vectors, e.g. B. from the pET vector series (Novagen).

The variant of the polypeptide has biological activity. In a preferred embodiment, such biological activity is the ability to bind to the corresponding antibody. In a preferred embodiment, it comprises an epitope which has the ability to bind to an antibody against SEQ ID NO1, or the variant itself has the ability to bind to an antibody against SEQ ID NO1, preferably an antibody of the immunogobulin class IgA , IgG or IgM against SEQ ID NO1, preferably from a sample from a patient suffering from SARS-CoV-2, the epitope more preferably comprising a sequence comprising at least 5, 6, 7, 8 or 10 amino acid residues. More preferably it does not bind specifically to homologues of SEQ ID NO1 from other coronaviruses, preferably from the group comprising MERS (SEQ ID NO6), NL63 (SEQ ID NO10), 229E (SEQ ID NO7), OC43 (SEQ ID NO8) and HKU1 ( SEQ ID NO9), more preferably from the group comprising SARS-CoV-1 (SEQ ID NO11), MERS, NL63, 229E, OC43 and HKU1, with specific binding preferably being defined and determined as set out above using Biacore equipment .

Those skilled in the art are familiar with the preparation of diagnostically useful reagents based on suitable amino acid sequences and are able to use both linear peptides, e.g. B. by chemical synthesis, and To provide polypeptides, e.g. B. by recombinant expression. In particular, those skilled in the art are aware that both conformational and sequential epitopes exist and that a suitable strategy for expression and purification must be chosen in order to obtain a diagnostically useful polypeptide that can be used to produce an antibody such as the antibody against SEQ Evidence of ID NO1 (e.g. Reischl, U., Molecular Diagnosis of Infectious Diseases, Humana Press, 1998, e.g. B. Chapters 12, 15, 16, 18 and 19 ). Those skilled in the art are also familiar with procedures for evaluating the usefulness of recombinant proteins for test systems (e.g. Reischl, U., Molecular Diaignosis of Infectious Diseases, Humana Press, 1998, e.g. B. Chapters 21-26 ).

The detection of the antibody or complex for the prognosis, diagnosis, method or the kit according to the present invention comprises the use of a method selected from the group comprising immunodiffusion techniques, immunoelectrophoretic techniques, light scattering immunoassays, agglutination techniques, immunoassays with labeling, such as from the group comprising radiolabeled immunoassays, enzyme immunoassays such as colorimetric tests, chemiluminescent immunoassays, and immunofluorescence techniques. In a preferred embodiment, the complex is detected using a method selected from the group comprising immunodiffusion techniques, immunoelectrophoretic techniques, light scattering immunoassays, agglutination techniques, labeled immunoassays from the group including radiolabeled immunoassays, chemiluminescence immunoassays and immunofluorescence techniques. Those skilled in the art are familiar with this method and are also described in the prior art, e.g. B. in Zane, HD (2001): Immunology - Theoretical & Practical Concepts in Laboratory Medicine, WB Saunders Company, especially in Chapter 14 . Preferably the test format is an ELISA and a microtiter plate comprising wells is used as a diagnostically useful carrier.

A polypeptide comprising SEQ ID NO1 or a variant thereof is preferably immobilized on a solid phase of the carrier. It can be immobilized directly on the solid phase when it is contacted with the sample, but a competitive test, a capture bridge test, an immunometric test, a class-specific secondary antibody on the solid phase, a class capture test, directly or indirectly, can also be used. The principle of each of these formats, edition in The Immunoassay Handbook 3 ^rd, edited by David Wild, Elsevier, described in detail of 2005. More preferably, the solid phase is a test strip or a well of a microtiter plate for ELISA, preferably a well of a microtiter plate for ELISA.

Any polypeptide, e.g. B. a polypeptide comprising SEQ ID NO1 or a secondary antibody, can be provided in any form and in any degree of purity, from tissues, fluids and cells comprising the polypeptide in an endogenous form, preferably cells expressing the polypeptide, crude or enriched lysates such cells, to purified and / or isolated polypeptide, which may be essentially pure. In a preferred embodiment, the polypeptide is a native polypeptide, the term “native polypeptide”, as used herein, meaning a folded polypeptide, more preferably a folded polypeptide purified from cells, even more preferably from prokaryotic or eukaryotic, preferably mammalian cells. A glycosylated form of the polypeptide can be used. Secondary antibodies are preferably pure, e.g. B. after purification based on protein A or protein G as affinity ligand. In a preferred embodiment, any polypeptide used or provided in accordance with the present invention is used or provided in a folded state, in the absence of significant concentrations of denaturing reagents such as thiol containing compounds such as DTT.

In a preferred embodiment, the presence or absence of an antibody of the immunoglobulin classes IgA, IgM and / or IgG against SEQ ID NO1 is detected. In a further preferred embodiment, only the presence or absence of IgA is detected. In a further embodiment, only the presence or absence of IgM is detected. In a further preferred embodiment, only the presence or absence of IgG is detected. In a preferred embodiment, the presence or absence of IgA and the presence or absence of IgG is detected. More preferably, an immunoglobulin class IgA secondary antibody and an immunoglobulin class IgG secondary antibody are used or provided to do so.

In a preferred embodiment, the presence or absence of an antibody, e.g. B. the immunoglobulin classes IgA, IgM and IgG, against SEQ ID NO1, using a polypeptide comprising SEQ ID NO1 or a variant thereof, detected, but the polypeptide can comprise additional sequences, preferably artificial sequences or, for example, linkers and / or affinity -Tags. However, such additional sequences are selected such that the ability of SEQ ID NO1 or a variant thereof to specifically bind to the antibody to be detected, or the diagnostic reliability, in particular sensitivity and / or specificity, is not changed significantly. For example, a domain that binds to SEQ ID NO1 or a fragment thereof should not be fused or present. According to the present invention, a secondary antibody which detects antibodies of the immunoglobulin class IgA against a SARS-CoV-2 antigen, preferably from the group comprising an antigen comprising SEQ ID NO1, an antigen comprising SEQ ID NO30, an antigen comprising SEQ ID NO31 and an antigen comprising SEQ ID NO33 can be used for the diagnosis, preferably early diagnosis, of a SARS-CoV-2 infection.

In a preferred embodiment, the products, methods and uses according to the present invention are configured in such a way that the presence of an antibody against SEQ ID NO1 is preferably based on the presence of an antibody against another SARS-CoV-2 antigen or one or a further antigen the group comprising SARS-CoV-2 N protein, more preferably from the group comprising SARS-CoV 2-N protein, SARS-CoV 2-M protein and SARS-CoV-2 E protein, most preferably from the group comprising SARS-CoV-2 N-protein, SARS-CoV-2 M-protein and SARS-CoV-2 E-protein and SARS-CoV-2 S-protein epitopes, if not present in SEQ ID NO1, can be distinguished. In a more preferred embodiment, a polypeptide comprising SEQ ID NO1 or a variant thereof is spatially separated from such other SARS-CoV-2 antigens or other coronavirus antigens if it is used to detect the presence or absence of an antibody against SEQ ID NO1 . For example, the polypeptide comprising SEQ ID NO1 or a variant thereof can be present in pure form and / or isolated on a carrier. For example, it can be spatially separated from other antigens on a blot or microtiter well or bead.

In a preferred embodiment, the absence of an antibody against SEQ ID NO1 is detected using an isolated, pure and / or recombinant polypeptide comprising SEQ ID NO1 or a variant thereof.

In a preferred embodiment, the presence or absence of an antibody against SARS-CoV-2 N protein, defined by SEQ ID NO30, is additionally determined, preferably an antibody of the immunoglobulin classes IgA, IgG and / or IgM, more preferably IgG or IgM, most preferably IgG. For this purpose, the carrier according to the invention can comprise a polypeptide comprising SEQ ID NO30 or a variant thereof.

In a preferred embodiment, the presence or absence of an antibody against the receptor binding domain, defined by SEQ ID NO31, is detected. For this purpose, the carrier according to the invention can comprise a polypeptide comprising SEQ ID NO31 or a variant thereof.

In a preferred embodiment, the presence or absence of an antibody against the S2 domain, defined by SEQ ID NO32, of SARS-CoV-2 spike protein is detected. For this purpose, the carrier according to the invention can comprise a polypeptide comprising SEQ ID NO32 or a variant thereof.

In a preferred embodiment, a secondary antibody is an antibody that binds against all antibodies of an antibody or immunoglobulin class, preferably a human antibody class, preferably one of the immunoglobulin classes IgA and / or IgG and / or IgM, preferably IgA antibodies . Secondary antibodies typically recognize the constant domain of such a class or have polyvalent binding sites against various epitopes over the sequence or three-dimensional structure common to antibodies of the immunoglobulin class. Secondary antibodies are typically derived from a mammal that is not a human or from a bird, preferably from a chicken, rabbit, mouse, rat, horse, pig, donkey, goat, cow, or primate. A variety of them are commercially available.

According to the present invention, the SARS-CoV-2 infection can be detected in an early phase with increased sensitivity, preferably 5 or fewer days after the onset of the disease symptoms.

According to the invention, a diagnostically useful carrier is provided, which is preferably selected from the group comprising a glass carrier, preferably for microscopy, a biochip, a microarray, a microtiter plate, a lateral flow device, a test strip, a membrane, preferably a line blot, comprises a chromatography column and a bead, preferably a microtiter plate.

In a preferred embodiment, the diagnostically useful carrier is a line blot ( Raoult, D., and Dasch, GA (1989), The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram negative bacteria. J. Immunol. Methods, 125 (1-2), 57-65 ; WO2013041540 ). In a preferred embodiment, the term “line blot” as used herein means a test strip, more preferably membrane-based, that has been coated with one or more than one agent for specifically capturing an antibody, each of which is preferably a polypeptide. If two or more agents are used, they are preferably spatially separated on the carrier. The width of such bands is preferably at least 30, more preferably 40, 50, 60, 70 or 80% of the width of the test strip. The line blot can comprise one or more than one control band to confirm that it has been contacted with a sample for a sufficiently long time and under suitable conditions, in particular in the presence of human serum or with a secondary antibody.

In a further preferred embodiment, the diagnostically useful carrier is a bead. Various beads for numerous applications are commercially available, mainly based on carbohydrate, e.g. B. Sepharose or agarose, or plastic. They comprise active or activatable chemical groups such as a carboxyl or tosyl or ester group which can be used for the immobilization of an agent for specifically capturing an antibody. The beads preferably have an average diameter of 0.1 μm to 10 μm, 0.5 μm to 8 μm, 0.75 μm to 7 μm or 1 μm to 6 μm. The beads can be coated with the agent for specifically capturing an antibody directly or via affinity ligands, e.g. B. biotin or glutathione and streptavidin and GST. For example, the bead can be coated with biotin or glutathione and the antigen can be fused with streptavidin or glutathione-S-transferase or a variant thereof. The bead is preferably provided in the form of an aqueous solution which has a bead content of 10 to 90%, preferably 20 to 80%, preferably 30 to 70%, more preferably 40 to 60% (w / w).

In a particularly preferred embodiment, the beads are magnetic beads which can easily be concentrated on a surface with the aid of a magnet. For this purpose, commercially available magnetic beads usually contain a magnetic mineral, e.g. B. iron oxide. A variety of suitable magnetic beads are commercially available. A bead can be marked with a detectable mark.

In a preferred embodiment, a magnetic bead is used and washed or incubated in a buffer by application of a magnetic field to concentrate and immobilize the beads, followed by removing the existing buffer and adding new buffer. The magnetic field can then be turned off to make the suspension of the beads in the new buffer more efficient. A buffer can be any solution used in accordance with the present invention comprising a diluted patient sample, an incubation buffer, or a buffer comprising a secondary antibody.

In a preferred embodiment, the antibody is detected using a chemiluminescent label. In a preferred embodiment, this is an enzyme capable of chemiluminescence, preferably selected from the group comprising luciferase, peroxidase, alkaline phosphatase and β-galactosidase or a variant thereof, which can convert a substrate capable of chemiluminescence without being consumed itself ( Kricka, LJ (2003). Clinical applications of chemiluminescence. Analytica chimica acta, 500 (1): 279-286 ). In a further preferred embodiment, the chemiluminescent label is a small organic compound that has no enzymatic activity with regard to catalyzing a chemiluminescent reaction that emits a chemiluminescent signal and is degraded when it is contacted with a chemiluminescent substrate solution that contains inorganic and / or non-enzymatic organic compounds which are necessary for the transmission of the signal. The small organic compound without enzymatic activity is preferably selected from the group comprising acridinium esters ( Weeks, I., Beheshti, I., McCapra, F., Campbell, AK, Woodhead, JS (1983) Acridinium esters as high specific activity labels in immunoassay. Clin Chem 29: 1474-1479 ) and luminol or a chemiluminescent derivative thereof, such as isoluminol. Such small organic compounds can be linked to a secondary antibody. In the case of luminol, the substrate solution comprises H ₂ O ₂ at high pH. In the case of an acridinium ester, a mixture of H ₂ O ₂ and sodium hydroxide is often used. The small organic compound is consumed when the chemiluminescent signal is emitted. In a preferred embodiment, the chemiluminescence of the chemiluminescent label is detected for 1 to 60 seconds, preferably 2 to 20 seconds, even more preferably 3 to 15 seconds after the chemiluminescence detection reaction has been triggered. In a further preferred embodiment, the chemiluminescence of the chemiluminescent label is detected for at least 0.5, 1, 1.5, 2, 2.5 or 3 seconds, preferably 3 to 15, more preferably 5 to 10 seconds.

In a further preferred embodiment, the carrier is a microtiter plate comprising at least 8 wells which can be used for an ELISA. At least one of the wells is coated with the agent for specifically capturing an antibody, either directly or indirectly, preferably a polypeptide comprising SEQ ID NO1 or a variant thereof. At least 3, preferably 4, even more preferably 5 calibrators with defined concentrations can be used to set up a calibration curve for a semi-quantitative analysis. When the method according to the invention is carried out, the calibrators can be processed and developed in parallel with the samples. A secondary antibody comprising a detectable label, such as an enzymatically active label, can be provided, e.g. B. a label with horseradish peroxidase activity or alkaline phosphatase activity or an enzyme that is chemiluminescent.

In a further preferred embodiment, the carrier is a microarray. In a preferred embodiment, the term “microarray” as used herein means a chip that is coated with a plurality of spatially separated antigens, preferably at least 20, preferably 30, 40, 50, 80 or 100. Each antigen is preferably a peptide comprising or consisting of 5 to 25, preferably 7 to 15 consecutive amino acids of the antigen, preferably SEQ ID NO1. Preferably a library is used with overlapping peptides which comprise a fragment or the full sequence of SEQ ID NO1, more preferably comprising the RBD. The peptides are typically chemically synthesized and can include a blocking group. A secondary antibody comprising a label, preferably a fluorescent label, can be used for detection.

In a further preferred embodiment, a glass slide is used which is on one or a part of a support for microscopic immunofluorescence analysis. A cell, preferably a eukaryotic cell such as a HEK293 cell, is located on the carrier. It can be covered with a cover medium. Various compositions and methods are described in the prior art, e.g. B. in "Mountants and Antifades", published by Wright Cell Imaging Facility, Toronto Western Research Institute University Health Network (https: // de. Scribd. Com / document / 47879592 / Mountants-Antifades) , Krenek et al. (1989) Comparison of antifading agents used in immunofluorescence, J. Immunol. Meth 117, 91-97 and Nairn et al. (1969) Microphotometry in Immunofluorescence, Clin. Exp. Immunol. 4, 697-705 . The cell expresses, preferably overexpresses, a polypeptide comprising SEQ ID NO1 or a variant thereof. The carrier may comprise a mock transfected cell which is transfected with the same vector as the cell which overexpresses a polypeptide comprising SEQ ID NO1 or a variant thereof, but without the nucleic acid which codes for the polypeptide. Such a mock transfected cell can serve as a negative control. Another cell can comprise another coronavirus, preferably SARS, more preferably SARS-CoV-2 antigen, e.g. B. N-protein, S2-protein or RBD to detect an antibody.

According to the present invention, a polypeptide provided, preferably the polypeptide comprising SEQ ID NO1 or a variant thereof, be a recombinant protein, the term “recombinant”, as used herein, meaning a polypeptide that is produced at any stage of the production process using genetic engineering methods, e.g. B. by fusing a nucleic acid that codes for the polypeptide to a strong promoter for overexpression in cells or tissues or by changing the sequence of the polypeptide itself. The person skilled in the art is familiar with methods for genetic engineering of nucleic acids and polypeptides for which they code, familiar (e.g. described in Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown TA (1986), Gene Cloning - an introduction, Chapman & Hall ) and for the production and purification of native or recombinant polypeptides (e.g. Handbooks "Strategies for Protein Purification", "Antibody Purification", published by GE Healthcare Life Sciences, and in Burgess, RR, Deutscher, MP (2009): Guide to Protein Purification ). In a further preferred embodiment, the polypeptide is an isolated polypeptide, the term "isolated" meaning that the polypeptide is enriched in comparison to its state during production using a biotechnological or synthetic approach and is preferably pure, i.e. at least 60, 70, 80, 90, 95 or 99 percent of the polypeptide in the particular fluid consists of the polypeptide as judged by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection. Preferably, any polypeptide on a carrier used as a means of capturing an antibody is pure.

A secondary antibody comprising a detectable label can be used to detect antibodies against SEQ ID NO1 of the immunoglobulin classes IgA and / or IgG and / or IgM, preferably IgA, more preferably against SEQ ID NO1 and against another coronavirus antigen. In a preferred embodiment, the label is selected from the group comprising a fluorescent, a radioactive or an enzymatically active label, preferably one that has a colorimetric reaction catalyzed. FITC or another fluorescein derivative can be used as a fluorescent label. A protein with peroxidase activity can be used as an enzymatically active label. Preferably the secondary antibody recognizes mammalian, more preferably human, antibodies. Preferably the secondary antibody is a monoclonal antibody. If more than one antibody from more than one immunoglobulin class is detected, two secondary antibodies can be used, preferably one that binds an antibody of the immunoglobulin class IgA and one that binds an antibody of the immunoglobulin class IgG. The two secondary antibodies can be present in a mixture or separately, preferably separately in order to detect the separate detection of antibodies against different immunoglobulin classes, such as IgA, IgM and IgG antibodies, preferably IgG and IgA, in order to detect e.g. . B. Obtain information on the course of the disease based on the fact that antibodies of the immunoglobulin class IgA appear earlier than antibodies of the immunoglobulin class IgG in many patients. Alternatively, a secondary antibody that binds to antibodies from more than one immunoglobulin class, e.g. B. from the group comprising IgG, IgA and IgM, preferably a secondary antibody that binds against antibodies of the immunoglobulin classes IgG and IgA. A secondary antibody can be a polyclonal or monoclonal antibody. In a preferred embodiment, the secondary antibody is derived from a mammal that is not a human, but binds to human antibodies of a specific immunoglobulin class, preferably IgA, IgG and / or IgM. A variety of secondary antibodies, optionally with labels such as a fluorescent label, are commercially available.

According to the present invention, a kit is provided which comprises a polypeptide comprising SEQ ID NO1 or a variant thereof, preferably coated on a diagnostically useful carrier, more preferably a microtiter plate, and further comprises one or more than one reagent, preferably all reagents from the group comprising a calibrator, a positive control, a negative control, a washing buffer, a means for detecting the presence of an antibody against SEQ ID NO1, preferably an antibody of the immunoglobulin classes IgA, IgM and / or IgG, preferably a secondary antibody, which specifically binds to antibodies, preferably antibodies of the immunoglobulin classes IgA, IgM and / or IgG, wherein the secondary antibody can comprise a detectable label, a sample buffer, a detection solution, preferably a chromogen / substrate solution, a stop solution and a protective film.

In a preferred embodiment, a calibrator is a reagent which binds to a polypeptide comprising SEQ ID NO1 or a variant thereof and is preferably recognized by secondary antibodies which recognize antibodies of the immunogobulin classes IgA, IgM and / or IgG. The calibrator can be an antibody of the immunoglobulin class IgA against SEQ ID NO1.

In a preferred embodiment, a positive control is a solution which comprises a compound such as an antibody against SEQ ID NO1, preferably from the group comprising antibodies of the immunoglobulin classes IgA, IgG and IgM, more preferably IgA, from the sample of a patient who suffers from SARS-CoV-2 in an amount that gives a positive result using the method according to the present invention. In a further preferred embodiment, a positive control is a reagent that captures all antibodies from the immunogobulin class to be detected, e.g. B. a secondary antibody, preferably an antibody against human antibodies of the immunoglobulin classes IgA, IgG or IgM. In a preferred embodiment, a negative control is a reagent in which such a compound is absent, and it could comprise serum from a healthy person, or a reagent that does not bind to the immunoglobulin class of the antibody to be detected, but more preferably to antibodies binds to another immunoglobulin class that cannot be detected, such as IgG, IgA or IgM.

A wash buffer can be used to wash the microtiter plate after incubation to remove nonspecific antibodies and could be PBS. The means for detecting the presence of an antibody could be a secondary antibody which binds to antibodies of the immunoglobulin class to be detected, preferably human antibodies of the immunoglobulin classes IgA, IgM and / or IgG, and is labeled, preferably with an enzyme, more preferably with an enzyme that has peroxidase activity. The sample buffer can be used to dilute patient samples and it can be PBS.

The detection solution can generate a signal in the presence of the labeled secondary antibody and is preferably a color-developing solution and more preferably 3,3 ', 5,5'-tetramethylbenzidine / H ₂ O ₂ . The stop solution can be added to a reaction to halt the reaction of the detection solution and can comprise a strong acid, e.g. B. 0.5 M sulfuric acid. The protective film can be placed on the microtiter plate or on an incubation tray to prevent evaporation.

Any reagent used in accordance with the present invention may contain a preservative, e.g. B. azide.

The present invention encompasses a number of novel nucleic acid and polypeptide sequences, more specifically

• 1 zeigt einen Zeitverlauf der Konzentrationen von Antikörpern gegen SEQ ID NO1 (IgA, IgG) und gegen N-Protein (IgG, IgM), überwacht in zwei Patienten (1), wie in Beispiel 3 beschrieben.
• 2 zeigt die Ergebnisse eines ELISA, bei dem die Anwesenheit oder Abwesenheit von Antikörpern der Immunglobulin-Klasse IgA gegen S1 (Quadrate), der Immunglobulin-Klasse IgG gegen SEQ ID NO1 (Dreiecke) und der Immunglobulin-Klasse IgG gegen N-Protein (Kreise) bestimmt wurde, wie in Beispiel 3 beschrieben ist.
• 3 zeigt eine IFA unter Verwendung von Aceton-fixierten S1-exprimierenden oder mit Kontrollplasmid transfizierten HEK293-Zellen (A) inkubiert direkt mit anti-His-Tag-Antikörpern (1:200) oder Patientenserum 3 (PS3, 1:100) im ersten Schritt und anti-Maus-lgG-FITC oder anti-Human-lgG-FITC im zweiten Schritt, (B) inkubiert 30 Minuten lang mit PBS vor der zweischrittigen Inkubation mit PS3, wie beschrieben in A, oder (C) inkubiert 30 Minuten lang mit PBS, gefolgt von Inkubation mit PS3 (1:100) im ersten Schritt, anti-Human-lgG-Biotin (1:200) im zweiten Schritt und ExtrAvidin-FITC (1:2000) im dritten Schritt. S1-exprimierende Zellen waren mit einem pTriEx-Vektor unter Verwendung von Standardverfahren transfiziert worden, der SEQ ID NO2 exprimiert.

The present invention is further illustrated by the following examples, sequences and figures, from which further features, embodiments, aspects and advantages of the present invention can be taken. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials as described herein. All publications, patent applications, patents, and other sources mentioned herein are incorporated herein by reference in their entirety.

• 1 shows a time course of the concentrations of antibodies against SEQ ID NO1 (IgA, IgG) and against N-protein (IgG, IgM), monitored in two patients ( 1 ), as described in Example 3.
• 2 shows the results of an ELISA in which the presence or absence of antibodies of the immunoglobulin class IgA against S1 (squares), the immunoglobulin class IgG against SEQ ID NO1 (triangles) and the immunoglobulin class IgG against N-protein (circles) was determined as described in Example 3.
• 3 shows an IFA using acetone-fixed S1-expressing or HEK293 cells (A) transfected with control plasmid, incubated directly with anti-His-tag antibodies (1: 200) or patient serum 3 (PS3, 1: 100) in the first step and anti-mouse IgG-FITC or anti-human IgG-FITC in the second step, (B) incubated for 30 minutes with PBS before the two-step incubation with PS3 as described in A, or (C) incubated for 30 minutes with PBS followed by incubation with PS3 (1: 100) in the first step, anti-human IgG-biotin (1: 200) in the second step and ExtrAvidin-FITC (1: 2000) in the third step. S1 expressing cells were transfected with a pTriEx vector expressing SEQ ID NO2 using standard methods.

Example 1: Detection of antibodies against SEQ ID NO1 using an ELISA immunassay

rehearse

Eight specimens from patients prepared using PCR as described by Corman et al. ( Corman et al. (2020) Diagnostic detection of 2019-nCoV by real-time RT-PCR, https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2 ) tested positive for SARS-CoV-2 and obtained 6 to 14 days after infection, and 14 samples from patients obtained earlier after infection were available.

In addition, a number of samples relating to various coronaviruses were available, including 18 samples from patients infected with MERS, three samples from patients infected with SARS-CoV-1, four patients with NL63, three patients with 229E, six patients with OC43, and three Patients with HKU1.

Production of microtiter plates coated with antigen

SEQ ID NO2 was expressed in HEK293T cells by standard cloning of SEQ ID NO4 into a pTriEx-1 plasmid with an artificial signal sequence and a C-terminal His-tag, which leads to the expression of SEQ ID NO2, and after removal of the Signal peptides, for SEQ ID NO3. The transfected cells were cultured at 37 ° C. and 8.5% CO ₂ in Dulbeccos-modified Eagle medium with 10% fetal calf serum, 100 U / ml penicillin and 0.1 mg / ml streptomycin for three to 5 days. The cells were harvested, resuspended in 20 mM Tris-HCl pH 7.4, 10% (w / v) sucrose, 5 mM EDTA, 1 mM PMSF and stored at -80 ° C. until further use.

In order to produce SEQ ID NO3, the supernatant of the cell culture was adjusted to 5 mmol / l tris chloride pH 8.0, 164 mmol / l sodium chloride, 50 mmol / l magnesium chloride, 20 mmol / l imidazole, 0.1% Triton X-100, Clarified by centrifugation for 30 minutes at 17,600xg, 4 ° C. and applied to a Nickel Rapid Run column (Agarose Bead Technologies, Miami, FL, USA) which was treated with 5 mmol / l Tris-chloride pH 8.0, 300 mmol / L sodium chloride, 20 mmol / l imidazole had been adjusted and eluted by increasing the imidazole concentration up to 150 mmol / l. All fractions contained in SEQ ID NO3 were added together and concentrated by means of ultrafiltration (VivaSpin, Sartorius, Göttingen, Germany). The final preparation was stored at -80 ° C until further use.

The final protein preparation of SEQ ID NO3 was treated with or without 16 mmol / l dithiotreitol and incubated for 10 minutes at 70 ° C. or at room temperature, followed by SDS gel electrophoresis and Coomassie staining. The protein identity was verified by mass spectrometry.

For use in a microtiter plate ELISA, the purified protein was diluted in PBS to a final concentration of approximately 1.5 µg / ml and used to coat ELISA microtiter plates (Nunc, Roskilde, Denmark) overnight.

Experimental approach

The samples were diluted 1: 101 in IgG sample buffer, applied to microtiter plates and incubated as described for commercially available EUROIMMUN ELISA test kits, using commercially available reagents (e.g. EI 2260-9601 G / A, which is a buffer that has essentially physiological conditions with regard to salt concentration and pH). Instructions EI 2260-9601 G / A were followed. In short: 60 minutes at 37 ° C; three washing steps using washing buffer; Addition of 100 µl peroxidase-labeled anti-human IgG conjugate (rabbit) or anti-human IgA conjugate (rabbit) per well; Incubation for 30 minutes at 37 ° C; three washing steps using EUROIMMUN washing buffer; Addition of 100 µl chromogen / substrate solution (TMB / H ₂ O ₂ ) per well; Incubation for 30 minutes at room temperature; Addition of 100 μl stop solution (0.5 M sulfuric acid); Measurement of the optical density at 450 nm against 630 nm as a reference.

The calibration was carried out using commercially available calibrators (product number EI 2606-9601 A, EUROIMMUN Medizinische Labordiagnostika AG). A ratio was calculated by dividing the absorbance of the control or patient serum by the absorbance of the calibrator. Results below 0.8 were considered negative results, results between 0.8 and 1.1 were considered borderline, and results greater than 1.1 were considered positive.

Results

The primary data from 79 patients are shown in Table 1: Raw data Cut-off OD Raw data Cut-off OD 0.100 0.200 IgG (OD) Dilution 1: IgG (ratio) IgA (OD) Dilution 1: IgA (ratio) pos: ≥ 1.1 pos: ≥ 1.1 borderline: 0.8-1.0 - - borderline: 0.8-1.0 1 Calibrator 1.911 300 19.1 3.055 600 15.3 2 Calibrator 1.593 600 15.9 2.204 1200 11.0 3 Calibrator 1.077 1200 10.8 1.068 2400 5.3 4th Calibrator 0.697 2400 7.0 0.529 4800 2.6 5 Calibrator 0.441 4800 4.4 0.314 9600 1.6 6th Calibrator 0.248 9600 2.5 0.155 19200 0.8 7th Calibrator 0.132 19200 1.3 0.078 38400 0.4 8th Calibrator 0.066 38400 0.7 0.051 76800 0.3 9 SARS-CoV-2, late phase 0.157 1.6 1.402 7.0 10 SARS-CoV-2, late phase 0.075 0.8 0.377 1.9 11 SARS-COV-2, late phase 0.276 2.8 9,999 50.0 12 SARS-COV-2, late phase 0.027 0.3 0.027 0.1 13 SARS-COV-2, late phase 0.023 0.2 0.064 0.3 14th SARS-COV-2, late phase 0.119 1.2 1.142 5.7 15th SARS-COV-2, late phase 0.031 0.3 0.203 1.0 16 SARS-COV-2, late phase 0.079 0.8 0.385 1.9 17th SARS-COV-2, <day 6 0.021 0.2 0.055 0.3 18th SARS-COV-2, <day 6 0.019 0.2 0.084 0.4 19th SARS-COV-2, <day 6 0.021 0.2 0.225 1.1 20th SARS-COV-2, <day 6 0.018 0.2 0.192 1.0 21st SARS-COV-2, <day 6 0.033 0.3 0.599 3.0 22nd SARS-COV-2, <day 6 0.029 0.3 0.331 1.7 23 SARS-COV-2, <day 6 0.013 0.1 0.030 0.2 24 SARS-COV-2, <day 6 0.017 0.2 0.097 0.5 25th SARS-COV-2, <day 6 0.027 0.3 0.214 1.1 26th SARS-COV-2, <day 6 0.016 0.2 0.021 0.1 27 SARS-COV-2, <day 6 0.014 0.1 0.073 0.4 28 SARS-COV-2, <day 6 0.014 0.1 0.042 0.2 29 SARS-COV-2, <day 6 0.015 0.2 0.030 0.2 30th SARS-COV-2, <day 6 0.015 0.2 0.079 0.4 31 MERS 1 0.013 0.1 0.042 0.2 32 MERS 2 0.008 0.1 0.017 0.1 33 MERS 3 0.011 0.1 0.038 0.2 34 MERS 4 0.008 0.1 0.011 0.1 35 MERS 5 0.008 0.1 0.027 0.1 36 MERS 6 0.008 0.1 0.035 0.2 37 MERS 7 0.009 0.1 0.013 0.1 38 MERS 8 0.017 0.2 0.036 0.2 39 MERS 9 0.010 0.1 0.024 0.1 40 MERS 10 0.007 0.1 0.012 0.1 41 MERS 11 0.020 0.2 0.026 0.1 42 MERS 12 0.008 0.1 0.026 0.1 43 MERS 13 0.015 0.2 0.021 0.1 44 MERS 14 0.012 0.1 0.036 0.2 45 MERS 15 0.024 0.2 0.066 0.3 46 MERS 16 0.021 0.2 0.080 0.4 47 MERS 17 0.008 0.1 0.039 0.2 48 MERS 18 0.029 0.3 0.104 0.5 49 SARS-1 0.214 2.1 0.285 1.4 50 SARS-1 0.596 6.0 0.227 1.1 51 SARS-1 0.128 1.3 0.260 1.3 52 OC43 0.035 0.4 0.126 0.6 53 OC43 0.029 0.3 0.098 0.5 54 OC43 0.016 0.2 0.041 0.2 55 OC43 0.011 0.1 0.048 0.2 68 NL63 0.013 0.1 0.023 0.1 69 NL63 0.027 0.3 0.039 0.2 70 NL63 0.036 0.4 0.057 0.3 71 NL63 0.019 0.2 0.030 0.2 72 229E 0.041 0.4 0.045 0.2 73 229E 0.022 0.2 0.024 0.1 74 229E 0.030 0.3 0.034 0.2 75 OC43 0.050 0.5 0.100 0.5 76 OC43 0.079 0.8 0,201 1.0 77 HKU1 0.023 0.2 0.054 0.3 78 HKU1 0.019 0.2 0.055 0.3 79 HKU1 0.010 0.1 0.029 0.1

Conclusions

The results show that antibodies against SEQ ID NO1 can be used to diagnose SARS-CoV-2 infection in samples from human patients.

A comparison of the data obtained with secondary antibodies that recognize antibodies of the immunoglobulin classes IgG or IgA shows that the detection of antibodies of the immunoglobulin class Class IgA is more sensitive, at least in an early phase of the disease: 4/14 patient samples that were taken in the early phase of the infection, before the end of six days after the onset of the disease, could be correctly identified as positive if antibodies of the immunoglobulin Class IgA were detected, whereas the detection of antibodies of the immunoglobulin class IgG in the same samples gave negative results.

Both tests showed cross-reactivity with samples from SARS-CoV-1 patients, but with virtually none of the samples from patients infected with MERS, NL63, 229E, OC43 and HKU1. A distinction between SARS-CoV-1 and SARS-CoV-2 is possible based on the different time-resolved immunoglobulin class signature, in particular the later appearance of antibodies of the immunoglobulin class IgA in SARS-CoV-1 ( Hsue, PR, Huang, LM, Chen, PJ, Kao, CL, and Yang PC (2004) Chronological evolution of IgM, IgA, IgG and neutralization antibodies after infection with SARS-associated coronavirus, Clinical Microbiology and Infection, 10 (12) , 1062-1066 ).

Several post-published publications by independent researchers have confirmed the inventors' findings. Jääskelainen et al. drew the conclusion that of six commercially available serological tests, the EUROIMMUN assay based on the detection of antibodies of the immunoglobulin class IgA against S1 showed the highest sensitivity (Jääskelainen, AJ, Kuivanen, S., Kekäläinen, E., Ahava , MJ, Loginov, R., Kallio-Kokko, H., Vapalahti, O., Jarva, H., Kurkela, and Lappalainen, M. (2020), J. Clin. Virology 129, 104512).

Beavis et al. confirmed that the sensitivity of the IgA-based test according to the present invention is superior in an early phase of the disease, whereas the IgG-based assay is superior in later phases, more precisely at least four days after diagnosis by PCR (Beavis, KG, Mathushek, SM, Abeleda, APF, Bethel, C., Hunt, C., Gillen, S., Moran, A., and Tesic, V. (2020) Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies, J. Clin. Virology 129, 104468).

Overall, the test according to the present invention can be used for the early detection of diagnostically relevant antibodies against SEQ ID NO1. In some patients, truly positive results can be achieved within six days of the onset of symptoms. Therefore, the test helps to close, or at least narrow, the diagnostic gap between the time that PCR-based tests can be used and the time that immunoassays can be used.

Example 2: An extended ELISA study with the aim of further characterizing the diagnostic reliability and relevance of the tests for the detection of an antibody against SEQ ID NO1

To determine the sensitivity, the presence or absence of antibodies of the immunoglobulin class IgA was detected in 166 samples from 152 European patients using EUROIMMUN product no. EI 2606-9601 A. The presence of a SARS-CoV-2 infection was previously according to using the RT-PCR Corman VM, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25 (3): pii = 2000045 (2020-01-23) based on a sample from an early stage of the infection.

Briefly, this test is based on an ELISA using a microtiter plate coated with an antigen comprising purified SEQ ID NO1. After incubation with samples and extensive washing steps using physiological buffers, a secondary antibody against antibodies of the immunoglobulin class IgA, which is provided with an enzymatically active label, is used to specifically target antibodies of the immunoglobulin class IgA against SEQ ID NO1 with a Labeling followed by incubation with a chromogenic substrate. The details can be found in the instructions that come with the product.

The serological characterizations were carried out with samples taken during the course of the infection.

With samples taken up to day 10 after the onset of symptoms or direct detection, a sensitivity of 60.2% for antibodies of the immunoglobulin class IgA against SEQ ID NO1 could be determined. The sensitivity was 98.6% when using samples obtained after day 10.

The results and immune responses, however, are highly individual. For example, a minority of patients may not show an IgA, IgG or IgM response or none of the aforementioned immune responses at all.

Example 3: Persistence of diagnostically relevant antibodies over time in SARS-CoV-2 patients, shown by means of ELISA

The time course of concentrations of antibodies against SEQ ID NO1 (IgA, IgG) and N-protein (IgG, IgM) was monitored in two patients ( 1 ). Both presented with mild disease, but the infection had been confirmed by RT-PCR. Typical specific symptoms such as temporary loss of smell were observed. The EUROIMMUN products EI 2606-A and -G and EI 2606-2-G and -M were used according to the manufacturer's instructions. The principle and the essential components of the tests are as shown in Examples 1 and 2; however, different antigens (polypeptide comprising SEQ ID NO1 and N-protein) as well as different secondary antibodies (binding to IgA, IgG or IgM) were used, depending on the target and the immunoglobulin class to be detected.

No samples were available from the very early phase of the disease. However, the course of the disease was monitored for more than four months. Initially, IgA and IgG against SEQ ID NO1 and IgG, but no IgM against N-protein, were detectable. In both patients after four months at least IgA or IgG against SEQ ID NO1 was detectable, whereas IgG against N-protein was absent or only weakly detectable two months after the first positive PCR.

These results show that antibodies against SEQ ID NO1 are present for longer than antibodies against N-protein, at least in some patients. The concentration of IgA antibodies decreases faster than the concentration of IgG antibodies, but IgA antibodies can still be dominant after they disappear due to their initially higher signal.

Monitoring another time course with a third patient is in 2 shown. Essentially, antibodies of the immunoglobulin class IgA against S1 (squares), antibodies of the immunoglobulin class IgG against SEQ ID NO1 (triangles) and antibodies of the immunoglobulin class IgG against N-protein (circles) were tested using the EUROIMMUN products EI 2606 -A and -G or EI 2606-2.

Example 4: ELISA-based monitoring of various antibodies over time

The time course of the concentrations of IgA and IgG antibodies against SED ID NO1 and IgG and IgM antibodies against N-protein was determined using ELISA kits EI 2606 A / G and EI 2606-2 G / M (EUROIMMUN Medical Laboratory Diagnostics AG). Four samples each from two patients suffering from a SARS-CoV-2 infection were examined. The results are shown in Table 2: sample Day after the first positive PCR EUROIMMUN GER EUROIMMUN ELISA SARS-CoV-2 SARS-CoV-2 NCP EI 2606 A EI 2606 G EI 2606-2 G EI 2606-2 M C200412DA C200428DB various various IgA IgG IgG IgM relationship relationship relationship relationship pos: ≥ 1.1 pos: ≥ 1.1 pos: ≥ 1.1 pos: ≥ 1.1 borderline: 0.8- <1.1 borderline: 0.8- <1.1 borderline: 0.8- <1.1 borderline: 0.8- <1.1 Patient 1 16 5.3 1.9 1.5 0.1 Patient 1 34 4.1 3.2 1.3 0.1 Patient 1 63 1.6 3.3 1.0 0.1 Patient 1 146 0.6 1.8 0.4 0.1 Patient 2 16 11.0 5.7 1.3 0.1 Patient 2 34 10.6 7.0 1.4 0.1 Patient 2 63 6.3 6.0 1.3 0.2 Patient 2 146 4.0 2.3 0.6 0.2

The IgA signal is strongest in both of the monitored patients and can be detected within a time window of at least 47 days. IgG against SEQ ID NO1 can be detected over the entire period of monitoring. In contrast to this, IgG against N-protein are only detectable at the first two (patient 1) or three (patient 2) points in time, only slightly above the cut-off value. IgM against N-protein cannot be detected at any time.

It can be concluded that antibodies against SEQ ID NO1 persist significantly longer than antibodies against N-protein, with IgG against SEQ ID NO1 being detectable over the entire period of 130 days. This confirms the higher sensitivity of the test according to the present invention.

Example 5: Detection of antibodies against SEQ ID NO1 using a chemiluminescence immunoassay (ChLIA)

A EUROIMMUN Random Access RA 10 Analyzer (YG 0710-0101) was used according to the manufacturer's instructions and with the basic settings.

Tosyla activated magnetic beads (M-280, Invitrogen) were coated using the manufacturer's instructions. Briefly, the beads were washed in coating buffer (0.1 M sodium phosphate pH 7.4) for 10 minutes at 37 ° C on an IKA roller 10 (supplied by VWR), followed by magnetic concentration of the beads and removal of supernatant . Recombinant polypeptide, purified according to Example 1, was added in the same buffer, followed by the addition of 1 M ammonium sulfate and incubation under the same conditions for 19 hours, followed by two washing steps using washing buffer (PBS pH 7.4 0.5% BSA 0.2% Tween-20), blocking in the same buffer for 4 hours at 37 ° C and two further washing steps. The beads were stored in the same buffer for at least 16 hours at 4 ° C (PBS pH 7.4 0.1% BSA 0.2% Tween-20).

The test was carried out by mixing magnetic beads with sample buffer (BSA / Tween-20 in Tris-HCI EDTA). 20 μl of bead suspension (1 mg / ml) was contacted with 5 μl of sample in a total volume of 200 μl in sample buffer. After the ten-minute incubation, the beads were washed three times in sample buffer, followed by the addition of 160 µl IgG / IgA / IgM conjugate (EUROIMMUN Medizinische Labordiagnostik AG, LK 0711-10010, essentially a secondary antibody marked with acridinium ester) and incubation For 10 minutes at 37 ° C. After three washes, alkaline hydrogen peroxide was quickly added and mixed to trigger emission of light, followed by instant luminescence detection for 10 seconds. The results are shown in Table 3: IgA ELISA IgG ELISA IgG ChLIA IgM ChLIA IgA ChLIA relationship relationship Ratio (not valid) Ratio (not valid) Ratio (not valid) pos: ≥ 1.1 pos: ≥ 1.1 pos: ≥ 1.1 pos: ≥ 1.1 pos: ≥ 1.1 borderline: 0.8- <1.1 borderline-0.8- <1.1 borderline: 0.8- <1.1 borderline: 0.8- <1.1 borderline: 0.8- <1.1 Patient 1 8.2 / 7.8 / 8.5 2.4 / 2.2 / 2.6 0.9 0.2 1.9 2 14.1 / 12.9 / 11.2 6.9 / 6.0 / 5.7 3.2 0.5 3.7 3 13.1 5.4 5.6 0.4 1.6 4th 0.3 0.3 0.1 0.1 0.2 5 0.3 0.1 0.1 0 0.2 6th 0.3 0.0 0.1 0 0.3 7th 0.4 0.1 0.4 0.1 0.2 8th 0.5 0.5 / 0.76 0.3 0.1 0.5

Patients 1 to 3 suffered from SARS-CoV-2 infection, whereas patients 4 to 8 were negative controls.

Overall, highly consistent results were achieved with ELISA and ChLIA.

Example 6: Indirect immunofluorescence test (IFA) with HEK-S1 cells which expressed SEQ IQ NO2

An IFA was performed using glass slides with a biochip array with recombinant HEK293 cells expressing SARS-CoV-2 S1 protein or with HEK293 control cells to show that this method could be used to detect antibodies against SEQ ID NO1 can be used.

Each biochip mosaic was incubated with 35 µl serum samples diluted 1: 100 PBS at room temperature for 30 minutes, washed with PBS-Tween and immersed in PBS-Tween for 5 minutes. In the second step, fluorescein isothiocyanate (FITC) -labeled goat anti-human IgG (EUROIMMUN Medical Labordiagnostika AG, Lübeck) was added and incubated for 30 minutes at room temperature. The slides were then washed with a PBS-Tween rinse and then immersed in PBS-Tween for 5 minutes. The glass slides were embedded in PBS-buffered glycerol containing DABCO (approximately 10 μl per field) and examined by means of fluorescence microscopy. Alternatively, the slides were incubated with PBS for 30 minutes prior to serum incubation, and bound human IgGs were detected by incubation with anti-human IgG-biotin (1: 200, 109-065-098, Dianova) for 30 minutes, followed by a washing step as described above and incubation for 30 minutes with ExtrAvidin-FITC (1: 2000, E2761, Sigma-Aldrich), followed by a further washing step. The samples were classified as positive or negative based on the fluorescence intensity of the transfected cells in direct comparison with cells transfected to the control and control samples. The results were evaluated by two independent observers using a EUROStar II microscope (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany). The reagents were obtained from Merck, Darmstadt, Germany or Sigma-Aldrich, Heidelberg, Germany, unless otherwise stated.

An S1 IgG ELISA positive patient serum (PS) showed a positive reaction with S1 (SEQ ID NO2), but not with an HEK cell transfected as a control ( 2 ), whereas none of the 49 S1 IgG ELISA negative control sera reacted. The signal intensity of the patient serum was improved by incubating the HEK-S1 cells with PBS before the serum incubation and detection of human IgG antibodies and by anti-human IgG biotin / ExtrAvidin-FITC. A total of 15/24 S1-IgG-ELISA-positive patient sera showed a positive reaction in HEK-S1-IFA with anti-human-IgG-biotin / ExtrAvidin-FITC.

Example 7: Vaccination Studies

On day 1, a healthy subject received an injection of 12.86 μg of recombinant S1 protein in physiological PBS (SEQ ID NO3) into the quadriceps muscle. Alum adjuvant (Twinrix for adults, EMRA-MED Arzneimittel GmbH) was used according to the manufacturer's instructions. On days 9, 21 and 28 the subject received an injection of a further 12.86 μg S1 protein in physiological PBS buffer and 10 μl Imject alum adjuvant in 500 μl sodium chloride solution.

Blood samples were taken on days 10, 23 and 29.

The presence of IgG and IgA antibodies against SARS-CoV-2 S1 and against N protein was determined in serum samples from the healthy subject using serological kits (EUROIMMUN Medical Labordiagnostika AG, EI 2606-9601 A, EI 2606-9601 G , EI 2606-9601-2 G) according to the manufacturer's instructions.

Samples from healthy blood donors and from patients with SARS-CoV-2 infections were used as controls. Table 1: Antibodies to SARS-CoV-2 antigens in a subject vaccinated using S1 protein Day IgA (S1) (Cut off: <0.8) IgG (S1) (Cut off: <0.8) IgG (NCP) (Cut off: <0.8) IgM (NCP) (Cut off: <0.8) 10 0.5 0.5 - - 23 1.0 0.2 - - 29 3.5 6.2 0.6 0.7

Samples were obtained from two patients suffering from SARS-CoV-2 infection (positive controls) 17 and 19 days after the onset of symptoms (usually 5-6 days after infection). The antibody concentrations are shown in Table 2. Table 2: Antibodies against SARS-CoV-2 antigens in two patients suffering from SARS-CoV-2 infection IgA (S1) (Cut off: <0.8) IgG (S1) (Cut off: <0.8) IgG (NCP) (Cut off: <0.8) IgM (NCP) (Cut off: <0.8) Patient 1 2.6 1.8 1.7 1.3 Patient 2 1.3 6.3 3.5 11.4

Samples were obtained in four healthy subjects who did not receive the vaccine (negative controls). The antibody concentrations are shown in Table 3. Table 3: Antibodies against SARS-CoV-2 antigens in healthy subjects IgA (S1) (Cut off: <0.8) IgG (S1) (Cut off: <0.8) IgG (NCP) (Cut off: <0.8) IgM (NCP) (Cut off: <0.8) Patient 1 0.0 0.1 0.0 0.5 Patient 2 0.2 0.3 0.1 0.4 Patient 3 0.1 0.1 0.1 0.2 Patient 4 0.1 0.1 0.1 0.1

These results show that vaccinated and infected subjects or subjects treated with a vaccine that does not contain SEQ ID NO1 or a variant thereof can be distinguished using an assay based on the detection of an antibody to SEQ ID NO1. Antibodies to the S1 antigen in the vaccine can be detected in both, but antibodies to the N protein can only be detected in infected patients and not in vaccinated subjects.

QUOTES INCLUDED IN THE DESCRIPTION

This list of the documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (15)

Use of a polypeptide comprising SEQ ID NO1 or a variant thereof which can bind to an antibody against SED ID NO1 from a sample from a patient for the production of a diagnostic kit. Use of a polypeptide comprising SEQ ID NO1 or a variant thereof, which can bind to an antibody against SED ID NO1 from a sample from a patient, for screening donor blood for contamination with a coronavirus, preferably SARS-CoV-2. Use after one of the Claims 1 to 2 , the variant having a sequence identity of at least 88, preferably 90, 92, 94, 96, 97, 98 or 99% to SEQ ID NO1 or a fragment thereof. Use after one of the Claims 1 to 3 , the variant having at least 10, 15, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500 or 600 consecutive amino acids of the sequence of SEQ ID NO1. Use after one of the Claims 1 to 4th wherein the polypeptide is immobilized on the solid phase of a carrier. Use after Claim 4 , wherein the carrier is selected from the group comprising a glass carrier, preferably for microscopy, a biochip, a microtiter plate, a lateral flow device, a test strip, a membrane; preferably line blot; a chromatography column, a microarray and a bead, preferably a magnetic bead. Use after one of the Claims 1 to 6th wherein the polypeptide is recombinant, isolated and / or purified. Use of an antibody against SEQ ID NO1 for diagnosing a SARS-CoV-2 infection or for the differential diagnosis of a coronavirus infection. Use of an antibody against SEQ ID NO1 to screen donor blood, preferably for contamination with a coronavirus SARS-CoV-2. Use after one of the Claims 8 to 9 , wherein the antibody is an antibody of an immunoglobulin class from the group comprising IgA, IgG and IgM. Use after one of the Claims 8 to 10 , an antibody of the immunoglobulin class IgA against SEQ ID NO1 and an antibody of the immunoglobulin class IgG against SEQ ID NO1 being used for diagnosis or differential diagnosis. Use after one of the Claims 1 to 11 the antibody (s) being human. Use after one of the Claims 8 to 12 , wherein the differential diagnosis comprises differentiating between a SARS-CoV infection, preferably SARS-CoV-2 infection, and an infection with MERS, NL63, 229E, OC43 and HKU1. Use of an antibody, optionally a recombinant antibody which binds to SEQ ID NO1, which is preferably recognized by a secondary antibody which binds to antibodies of the immunoglobulin class IgA, IgG or IgM, preferably IgA, as a calibrator for the early diagnosis of SARS-CoV -2 infection. Use of a recombinant antibody that binds to SEQ ID NO1, which is preferably recognized by a secondary antibody that binds to antibodies of the immunoglobulin class IgA, as a calibrator for screening donor blood for contamination with a coronavirus, preferably SARS-CoV-2.

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