BE1028465B1 - SEROLOGICAL RAPID DETECTION TEST FOR COVID-19 INFECTION - Google Patents

Abstract

The invention relates to a test or a kit for the rapid serological detection of antibodies specific for SARS-CoV-2 on a solid support fixing at least two different antigenic structures of SARS-CoV-2.

Description

RAPID SEROLOGICAL DETECTION TEST FOR COVID-19 INFECTION Field of the invention

[1] The present invention is in the field of the detection of viral infections and relates in particular to a new test or a kit for the rapid serological detection of infection by severe acute respiratory syndrome type 2 (SARS-CoV-2 or COVID-19 infection) as well as its method of obtaining. — Technological background underlying the invention

[2] The SARS-CoV-2 coronavirus which emerged at the end of 2019 and which has been dispersed globally, has led the pandemic called COVID-19 has already had a significant impact on our health system and on our lives.

[3] Currently, epidemiological surveillance of the infection is of paramount importance to prevent the appearance of new cases. Many tests or serological kits for this infection have been proposed and marketed, but these are focused on the detection of antibodies directed against a single antigen.

[4] The majority of these tests or kits make it possible to distinguish immunoglobulins of the IgM type from IgG, but the added value of the detection of these IgM immunoglobulins is questionable due to the loss of specificity of the tests including IgM (Van Elslande et al, Clin. Microbiol. Infect. https.//doi ora/10.1016/.cmi.2020.05.023).

[5] While most sensitive tests or kits aim to detect antibodies directed against the nucleocapsid protein (NP protein), it has been shown by some authors that the detection rate of IgM immunoglobulins directed against the Spike protein is the highest compared to the NP protein, in the days following the infection after the appearance of the symptoms of the pathology (Liu et al, J. Clin. MIcrobiol, doi: 10.1128/JCM.00461-20).

[6] This Spike protein of SARS-CoV-2 has two domains S1 and S2 and the entry of the virus into the target cells is obtained by the interaction of the RBD (Receptor Binding Domain) which belongs to the S1 domain of the Spike protein with the cell surface ACE2 domain (Wrapp D. et al, Science, 367, 1260-1263 (2020).

[7] Antibodies against RBD have been shown to be able to neutralize virus entry into cells (Huo. et al. hitps///doLorg/10.10186/.chom.2020.08.010).

[8] To obtain very high sensitivity, a test or kit must detect either IgM and IgG immunoglobulins directed against the Spike protein or either against the RBD domain of the Spike protein and IgG immunoglobulins IgG directed against the NP protein.

[9] However, qualifying the immune response against SARS-CoV-2 and identifying the presence of anti-RBD antibodies will not only qualify the immune response against the virus, but also predict the protection obtained against the virus (S Yin et al, J. Infect., doi: 10.1016/j.jinf.2020.06.076.;

[10] Salazar E et al, hitps://doi.org/10.1101/2020.08.08. 138990).

[11] Among the 205 rapid serological tests identified by FIND (https: //wvww. finddx.org/covid- 19/pipeline/Pavance=all&type=Kapid+diagnostic+tests&test target=Antibody&st atus=all&section=show-all&action=default ), no test or kit proposed is based on the detection of two different antigens and their immune identification.

Aims of the invention

[12] The present invention aims to provide a new type of test or serological kit for infection by SARS-COV-2 in a patient, as well as its method of production which do not have the disadvantages of the state of the art. .

[13] A particular object of the invention is to obtain such a test or such a kit having a high detection sensitivity.

[14] Another object of the invention is to provide a test or a kit and its synthesis process which improve the epidemiological analysis of patients, by detecting patients who may present an immune response, but also present, or not , adequate and sufficient immunity to be protected against further infection.

[15] A complementary object of the invention is to obtain a test or a kit and its method of obtaining which facilitate detection even from complex samples, incorporating cells or cellular debris, such as whole blood and which may interfere with the detection and reading of the results of the test carried out on these samples. Characteristic elements of the invention

[16] The present invention relates to a test or a kit for the serological detection of the COVID-19 infection generated by SARS-CoV-19 in an individual, by the detection of antibodies in this individual directed against SARS-CoV-2 .

[17] According to the invention, the test or kit of the invention comprises a solid support including at least two different and specific antigenic structures of SARS-CoV-2, preferably the nucleocapsid (NP) protein of a SARS virus (SARS-CoV-1 or SARS-CoV-2), an antigenic fraction of the S1 domain of the Spike protein, preferably the RBD of the Spike protein or one or more antigenic portion(s) of this domain or this nucleocapsid.

[18] These antigenic structures are described in the following documents F. Wu et al, (Nature, Vol 570, page 265-270 March 2020) and D. Wrapp et al (Science, 367 P.1260 -1263, March 2020) and on the sites of Genbank NC_045512 and Swiss prot PODTECS.

[19] The test or kit of the invention may be of any type, if it is based on serological detection between an antibody and its corresponding antigenic structure, in particular ELISA type tests or kits. , RIA or immuno-chromatographic and incorporating different types of enzymatic, colorimetric, metallic, radioactive markers, etc.

[20] Preferably, the test or the kit of the invention is an immunochromatographic test of the dipstick or lateral flow device type, this test or this kit comprising the elements of a device or instrument similar to a sheet with on a application zone, the support and suitable materials for contact with the sample to be analyzed, on a detection zone, the support and suitable materials such as nitrocellulose, incorporating the aforementioned antigenic structures and on a detection zone adsorption, support and suitable materials to facilitate the migration of the antibodies present in the sample towards the antigenic structures.

[21] Advantageously, the inventors have improved this test or this immuno-chromatographic kit by adding, between the application zone and the detection zone, to said solid support and to the appropriate materials, preferably on nitrocellulose, a sufficient quantity of a detergent.

[22] According to the invention, the detergent added to the solid support is a nonionic detergent, such as triton-X100, tween20 or tergitol, in order to lyse any cells present in a sample. This incorporation of the detergent facilitates reading and unexpectedly strengthens the detection signal.

[23] Preferably, the test or the kit of the invention comprises other elements, in particular a labeled conjugate, such as antibodies labeled for the recognition of the antibodies of the individual tested and attached to the structure or structures ( s) antigen(s) mentioned above.

[24] Advantageously, the antigenic structures, preferably the RDB, can also be present in the test in a free manner (that is to say not fixed on the solid support) and labeled in order to amplify the detection signal, when they bind to the second specific hypervariable part of the antibodies of the sample, the other hypervariable part of the antibodies of the sample binding the antigenic structure fixed on the solid support of the test or of the kit.

[25] According to the invention, the marker is preferably chosen from the group consisting of metallic particles, metallic colloids, in particular colloidal gold particles, latex particles, specific color microparticles or a fluorescent marker, such as Cyaninine-5, Aminocoumarin, Acridine….

[26] According to a variant of the invention, the test or the kit of the invention can also incorporate the elements of a second test or detection kit incorporating reagents for detecting SARS-CoV-2 antigens and as described in the patent application BE 2020/5229 incorporated herein by reference.

[27] Another aspect of the invention also relates to the process for obtaining the test or the kit of the invention as described in the claims.

Definitions

[28] The expression "COVID-19 serological test or kit" refers to any instrument, test, assay or diagnostic device used to obtain the serological detection of antibodies specific for infection by this SARS-CoV-2 virus and which can identify whether a tested individual (human patient) is infected with this virus and advantageously what type of antibody is produced by said individual.

[29] The test or kit of the invention includes other reagents and elements, such as vials or tubes, buffers and instructions for handling the test of the invention.

[30] The expression "sheet-like device or instrument" refers to rapid disposable tests, called "reactive strips or strips or dipsticks" and "lateral flow devices", possibly incorporated into an immunoassay kit. chromatography.

[31] The term “antigenic portion of a protein or of a domain of a protein” means a portion of several amino acids of this protein which is capable of generating a humoral immune reaction against this portion bound on a solid support. and which is capable of specifically binding antibodies of an individual infected with SARS-CoV-2. Description of the invention

[32] The inventors have discovered, in an advantageous and useful manner, that since the antibodies against the RBD domain are potentially protective and the patients tested have only developed antibodies against a single SARS-CoV-2 antigen, the association according to the invention in a single test or a serological kit of the two antigens makes it possible to increase the sensitivity of a detection.

[33] The identification of the responses against the two antigens constituted by the RBD domain and the NP protein, will make it possible to obtain the information that a person has not only been contaminated and produced an immune response, but in the event that the response is positive against the RBD domain of the Spike protein, that person will potentially be protected against further infection.

[34] If we consider the epidemiological future of this infection by SARS-CoV-2 where vaccines will be available, these being probably directed against the Spike protein, the test or the kit of the invention will make it possible to verify that the future vaccine has produced the desired antibodies (against the RBD domain) but will also make it possible to demonstrate an infection, if anti-nucleoprotein antibodies appear in the patient.

[35] According to the invention, a biological sample from the patient is any physiological fluid obtained from the patient and likely to contain the infectious virus or the antibodies directed against this virus, in particular whole blood or serum, feces, urine as well as bronchoalveolar or nasopharyngeal fluid.

[36] The Applicant has produced two immuno-chromatographic serological rapid tests or kits identified as COVID-19NP/RBD Sero-Strip and COVID-19 NP/RBD Sero-K7 tests (which is a lateral flow device type test), which make it possible to detect and differentiate in a sample, preferably whole blood or serum, specific antibodies directed against the RBD and the NP protein of SARS-CoV-2 in a single test.

[37] The nitrocellulose membrane of the rapid test according to the invention is fixed by antigenic reagents (including the recombinant SARS (SARS-CoV-1 or SARS-CoV-2) nucleocapsid (NP) protein and the SARS RBD (Receptor Binding Domain) of the Spike protein), capable of reacting with antibodies present in the sample and which will be revealed by the addition of a colloidal gold conjugate. The sample can be brought into direct contact with the sample present in a test tube. When four drops of buffer and the test strip are added together in the test tube, migration begins on the test.

[38] Then, the labeled and solubilized conjugate reacts with the antibodies present in the sample, migrates with the diluted sample by passive diffusion. The conjugate (preferably consisting of a rat antibody labeled with colloidal gold) and the sample come into contact with the antigenic reagents adsorbed on the nitrocellulose. If the sample contains anti-RBD antibodies, the conjugate-antibody complex will remain attached to the first RBD test line and a red line will form . If the sample contains anti-NP antibodies, the conjugate-antibody complex will remain attached to the second NP test line and a second red line will form.

[39] The solution continues to migrate to react with a third reagent which binds a migration control conjugate producing a control line confirming that the test is working correctly. The result can advantageously be visible in less than 15 minutes.

Claims (11)

SET OF DEMANDS BE2020/5514 1. Serological test or kit for detecting antibodies specific to SARS-CoV-2, comprising a solid support fixing by nitrocellulose, at least two different antigenic structures consisting of the nucleocapsid protein (NP) or an antigenic portion of the nucleocapsid (NP) of SARS and the Receptor Binding Domain (RBD) of the Spike protein of SARS-CoV-2 and comprising fixed on a portion of the nitrocellulose the RBD and on another portion of the nitrocellulose the nucleocapsid. 2. Test or kit according to claim 1, which is an Elisa test. 3. Test or kit according to claim 1, which is an immuno-chromatographic test, preferably of the dipstick or lateral flow device type. 4. Immuno-chromatographic test or kit according to claim 3, combined with a second immuno-chromatographic test comprising reagents for detecting SARS-CoV-2 antigens. 5. Immunochromatographic test or kit according to any one of the preceding claims 3 to 4, further comprising a labeled antibody conjugate. 6. Immunochromatographic test or kit according to any one of the preceding claims 3 to 5, comprising the labeled free RBD and/or the NP protein or an antigenic portion of the labeled NP protein. 7. Immuno-chromatographic test or kit according to any one of the preceding claims 3 to 6, in which the label is chosen from the group consisting of metallic colloids, gold particles, latex particles or microparticles having a specific color or a fluorescent marker. 8. Immuno-chromatographic test or kit, according to any one of the preceding claims 3 to 7, comprising three zones or portions, including an application zone, a detection zone and an adsorption zone, and comprising between the zone of application and the detection zone, a portion of nitrocellulose impregnated with a detergent. 9. Process for obtaining the test or the kit according to any one of the preceding claims, in which on a solid support is fixed, by nitrocellulose, at least two different antigenic structures constituted by the protein of the nucleocapsid (NP) or an antigenic portion of the SARS nucleocapsid (NP) and the Receptor Binding Domain (RBD) of the Spike protein of SARS-CoV-2. 10. Process for obtaining according to claim 9, in which the test or the kit is (#F2020/5514 test or an immuno-chromatographic kit. 11. Method according to claim 10, in which a sufficient quantity of a detergent is deposited on the solid support between the application zone and the detection zone of the immuno-chromatographic test.