WO2022043147A1

WO2022043147A1 - Diagnostic reagent for detection of sars-cov-2 antibodies

Info

Publication number: WO2022043147A1
Application number: PCT/EP2021/072889
Authority: WO
Inventors: Thomas MASETTO; Matthias Grimmler; Christian KOCHEM; Leoni WEY
Original assignee: Diasys Diagnostic Systems Gmbh
Priority date: 2020-08-28
Filing date: 2021-08-18
Publication date: 2022-03-03
Also published as: DE102020122593A1

Abstract

The invention relates to a diagnostic reagent for detecting SARS-CoV-2 antibodies in a sample, and to a method for preparing said reagent.

Description

Diagnostic reagent for the detection of SARS-CoV-2 antibodies

OBJECT OF THE INVENTION

The invention relates to a diagnostic reagent for detecting SARS-CoV-2 antibodies in a sample and a method for producing this reagent.

BACKGROUND OF THE INVENTION

SARS-CoV-2 is a virus discovered at the end of 2019 that can cause the infectious disease COVID-19 in humans. COVID-19 is characterized by non-specific symptoms, a relatively long incubation period and a high level of transmissibility from person to person even before symptoms of the disease appear. In many cases, life-threatening pneumonia develops during the course of the COVID-19 disease.

SARS-CoV-2 belongs to the Coronaviridae (coronavirus) virus family. These are RNA viruses with a viral envelope and a capsid.

Structures (spikes) that protrude beyond the surface of the virus envelope are characteristic of the appearance of the corona virus. The SARS-CoV-2 spike protein comprises a subunit (S1; amino acids 13-685) which contains the receptor-binding domain (RBD; amino acids 319-541) with which the virus can dock to a cell , and a subunit (S2; amino acids 686-1273) which, as a fusion protein, causes the viral envelope and cell membrane to fuse. Another membrane protein on the outside of the virus envelope of SARS-CoV-2 is the envelope protein (envelope small membrane protein).

The membrane protein, which is also anchored in the virus envelope, is directed inwards. Inside the virus envelope is a capsid containing a helical nucleoprotein complex. This consists of the nucleocapsid protein. The reliable identification of people infected with SARS-CoV-2 through diagnostic tests is a basic requirement for determining the acute course of infection and, associated with this, for the development of suitable measures to contain the SARS-CoV-2 pandemic. The tests are also the basis for the adequate diagnosis and treatment of those affected, reporting, monitoring and screening measures.

In addition to direct virus detection using a polymerase chain reaction test (PCR), an infection can also be detected indirectly using the specific antibodies against SARS-CoV-2 produced in the body. These immunological tests offer the advantage that infections that have been overcome can also be detected, i.e. a statement on the specific antibody status can be made. This can be used, for example, to check the vaccination efficiency or vaccination status of a person.

Methods based on an enzymatic color reaction are already known for the detection of SARS-CoV-2 antibodies. In these so-called "enzyme-linked immunosorbent assays" (ELISA), a microtiter plate is first provided with an antigen of the virus. After adding the sample, the antibodies against SARS-CoV-2 contained therein couple to the antigen.

This is followed by the addition of secondary antibodies that have a specificity for the bound antibodies and to which an enzyme is coupled. Finally, a substrate for the secondary antibody-coupled enzyme is added. The concentration of the dye formed during the conversion of the substrate allows conclusions to be drawn about the original concentration of the antibody sought in the solution being examined using photometric methods.

However, corresponding ELISAs require a high processing effort, i.e. many pipetting and washing steps, so that these are time-consuming. In addition, they require a large number of special instruments, so that the performance of ELISAs is not readily possible in every analysis laboratory. In addition, assay development is relatively laborious and there is a risk of limited antibody reactivity since binding of the enzyme to the antibody can limit the spatial availability of sites on the antibody intended to bind to the antigen.

So-called “electrochemiluminescent immunoassays” (ECLIA), such as the Elecsys® immunoassay from Hoffmann La Roche AG, Basel, are also known a recombinant antigen of the nucleocapsid (N) protein is used to detect SARS-CoV-2 antibodies.

The measuring principle of ECLIAs is based on chemical compounds that are easy to oxidize on an electrode and then reduce using cyclic voltammetry. At the appropriate voltage, the reduction is sufficiently exothermic to place the compound in an excited, photon-emitting state. This emission is monitored photometrically. The binding of antibodies prevents close contact with the electrode and thus changes the emission. In most cases, ruthenium complexes such as ruthenium-(II)-tris(bipyridyl) ²⁺ are bound as tracers to the immune complex to be determined.

The ECLIA reagents are usually complex and expensive to produce. Furthermore, the sensitive reagents used in ECLIA and/or ELISA immunoassays often have only limited stability.

Both the ELISA and the ECLIA method require method-specific equipment and high personnel costs. Both of these are either not available at all in many clinical chemistry laboratories, or only to an insufficient extent due to the high volume of samples during the pandemic.

TASK

Against this background, the object of the present invention was therefore to provide an antibody test for the detection of SARS-CoV-2 antibodies in a sample, which can be produced in large quantities in a cost-effective and reproducible manner, which requires no method-specific equipment and only a small amount of work , high reagent stability and high specificity and can be used in practically all clinical chemistry laboratories.

DESCRIPTION OF THE INVENTION

This object is achieved according to the invention by a diagnostic reagent for detecting SARS-CoV-2 antibodies in a sample, the reagent having an aqueous suspension of surface-modified polymer particles with at least one protein covalently bound to them, the at least one covalently bound protein having a one a) immunogenic protein from SARS-CoV-2 or b) a fragment of an immunogenic protein from SARS-CoV-2, the bi) a homologous sequence section of the binding domain region or the N-terminal domain of the spike protein with a sequence match > 80 % and/or bz) which has at least 50% of the number of amino acids of the corresponding immunogenic protein of SARS-CoV-2, has a homologous sequence section with a sequence identity >80%.

The diagnostic reagent claimed here is suitable for detecting SARS-CoV-2 antibodies with a photometric detection system. The diagnostic reagent is preferably suitable for detecting SARS-CoV-2 antibodies using the PETIA method.

The invention therefore also includes a photometric method, preferably a PETIA method, for detecting SARS-CoV-2 antibodies using the diagnostic reagent according to the invention.

The assay according to the invention is a homogeneous assay that is suitable for detecting SARS-CoV-2 using a photometric detection system. The assay according to the invention can therefore be used in the PETIA method.

In the “PETIA method” (Particle Enhanced Turbidimetric Immuno Assay), a turbidimetric evaluation is carried out via the decrease in the transmission of light through the reaction liquid. The more antibodies are contained in a sample, the greater the agglutination occurring between the particles occupied by the at least one protein, which leads to greater turbidity of the liquid and thus to a reduction in transmission. No additional enzyme or other means of detection are required, and the measurement can be carried out with a simple photometer system, which is available in practically every diagnostic laboratory. In contrast, other heterogeneous assay methods such as CLIA require the use of a large number of different reagents and work-up steps. For this reason, particles with a size of >1000 nm are usually used in these processes, so that the loss of material during the necessary multiple washing steps is low. The diagnostic reagent claimed here is used to detect SARS-CoV-2 antibodies. This includes both qualitative and quantitative detection, i.e. detection of whether a sample contains SARS-CoV-2 antibodies and, if necessary, detection of the total content of SARS-CoV-2 antibodies in the sample is.

"Qualitative detection" in this context means that the decrease in transmission when measuring the mixture of diagnostic reagent and sample indicates the presence of SARS-CoV-2 antibody molecules that are bound to the polymer particles and conclusions can be drawn from this can be drawn on the presence of SARS-CoV-2 specific antibodies in the sample.

In comparison, "quantitative detection" means that the decrease in transmission allows conclusions to be drawn about the amount of SARS-CoV-2 antibody molecules that are bound to the polymer particles and from this conclusions about the amount of SARS-CoV-2 Antibodies can be drawn in the sample. The diagnostic reagent according to the invention is particularly preferably used to detect the total SARS-CoV-2 antibody content of a sample.

In the context of the present invention, a “sample” is understood to mean any material prepared for analysis purposes that contains or could contain a proportion of SARS-CoV-2 antibodies to be analyzed.

In most cases, the sample will be a sample of fresh whole blood, which may have been appropriately processed for purposes of performing the analysis. However, other liquid samples that may contain SARS-CoV-2 antibodies, such as standard solutions and calibrators, are also encompassed by the present invention.

In the context of the invention, “immunogenic proteins from SARS-CoV-2” are understood to mean all immunogenic viral proteins from SARS-CoV-2 that are able to trigger an immune response in the human body.

In certain embodiments of the invention, the SARS-CoV-2 immunogenic protein is selected from the proteins listed in the following tables.

SUBSTITUTE SHEET (RULE 26) In certain embodiments of the invention, the immunogenic protein is a virus-specific structural protein, virus-specific “structural proteins” being understood here to mean virus-specific proteins with a scaffolding function and/or with a docking function. According to the invention, the virus-specific structural proteins in SARS-CoV-2 are the spike protein, the membrane protein, the envelope small membrane protein and the nucleocapsid protein.

In the reagent according to the invention, the polymer particles are present in an aqueous suspension. In this context, the term “aqueous suspension” denotes a suspension of the polymer particles loaded with the protein in water or an aqueous solution in which preferably sugar, sugar alcohol and/or buffer substances are dissolved. The term “suspension” is to be interpreted narrowly in connection with the present invention and means that almost all particles are floating in the aqueous phase and are not sedimented. A suspension within the meaning of the present invention is therefore present when at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% of the polymer particles are free and monomerically floating.

The diagnostic reagents according to the invention can be used very easily on all standard chemical analyzer platforms since the determination of the reactivity only requires the measurement of the absorbance. They can therefore be used in almost all analytical laboratories, even those that are less well equipped, without additional instrumental effort.

The reagents according to the invention are distinguished by a reagent blank value which is almost constant over the lifetime of the reagent. Both are due to the high colloidal stability of the particles. The reagents according to the invention can therefore be stored for a long period of time without any problems. Due to the comparatively simple production process, the reagents according to the invention can also be produced in large numbers very quickly and inexpensively.

Furthermore, the diagnostic reagent according to the invention is characterized by a high storage stability.

The high storage stability is expressed, among other things, in the fact that the turbidity of the suspension is stable compared to the initial value even after several months. In particular, the suspension of the present invention is characterized in that the absorption of the suspension at 660 nm deviates by less than 5% from the initial value on day zero within 20, 30, 60 or even 90 days from the time the suspension was prepared. In certain embodiments, the deviation is even less than 3% or even less than 2%. In certain embodiments of the invention, the slight deviation in the degree of turbidity within the aforementioned periods of time can also be measured at other wavelengths in the range from 340 to 800 nm.

In a preferred embodiment of the invention, the immunogenic protein of SARS-CoV-2 or its fragment, to which the at least one covalently bound protein has a homologous sequence section with a sequence identity >80%, is the nucleocapsid protein, the membrane protein, or the spike protein, preferably the spike protein. Since the spike protein is particularly characteristic of SARS-CoV-2, ie there is a high deviation in the sequence order compared to other corona viruses, diagnostic reagents according to the invention that are based on interactions with this protein or with a fragment of this protein are special specific.

The sequence identity of the at least one covalently bound protein to the immunogenic protein of SARS-CoV-2 or its fragment is preferably ≧80%, preferably ≦85%, more preferably 90%, even more preferably ≦95 and most preferably >98%.

In a preferred embodiment, the at least one covalently bound protein has a sequence section homologous to an immunogenic protein of SARS-CoV-2 with a sequence identity > 80%, preferably ≧85%, more preferably 90%, even more preferably >95 and most preferably > 98% up. Particularly preferably, the at least one covalently bound protein has a sequence section homologous to the spike protein with a sequence identity >80%, preferably >85%, more preferably >90%, even more preferably >95 and most preferably >98%. In a further preferred embodiment, the at least one covalently bound protein has a sequence section homologous to the nucleocapsid protein with a sequence identity >80%, preferably >85%, more preferably >90%, even more preferably >95 and most preferably >98%.

In a particularly preferred embodiment, the sequence length of the at least one covalently bound protein is essentially the same as that of the immunogenic protein of SARS-CoV-2 or the fragment of the immunogenic protein of SARS-CoV-2, to which it has a homologous sequence segment, where essentially match means that the difference in the number of amino acids based on the number of amino acids of the protein with the higher number of amino acids is <20%, preferably <15%, more preferably <10%, even more preferably <5%, and most preferably <2%. In this case, the sequence identity is particularly preferably 80%, preferably 85%, even more preferably 90%, even more preferably 95% and most preferably 98%. A slight deviation in length and homology improves the detection of the antibodies and thereby increases the sensitivity of the diagnostic reagent according to the invention.

Particularly preferably, the at least one covalently bound protein is essentially the same as the spike protein in terms of sequence length and has a sequence section homologous to this protein with a sequence identity > 80%, preferably 85%, more preferably > 90%, even more preferably > 95 % and most preferably > 98%. The at least one covalently bound protein is particularly preferably the spike protein.

Particularly preferably, the at least one covalently bound protein essentially has the same sequence length as the nucleocapsid protein and more preferably has a sequence segment homologous to this protein with a sequence identity >80%, preferably 85%, more preferably 90%, even more preferably ≦95% and most preferably 98%. The at least one covalently bound protein is particularly preferably the nucleocapsid protein.

In a further preferred embodiment, the at least one covalently bound protein has a sequence section homologous to a fragment of an immunogenic protein of SARS-CoV-2 with a sequence identity > 80%, preferably > 85%, more preferably > 90%, even more preferably > 95 % and most preferably > 98%.

In a preferred embodiment, the fragment has a sequence section homologous to the binding domain region (RBD; amino acids 319-541) or the N-terminal domain (NTD; amino acids 1-290) of the spike protein with a sequence identity > 80%, preferably > 85 %, more preferably >90% and most preferably >95%. These two sections are particularly characteristic of SARS-CoV-2 and diagnostic reagents according to the invention, which are based on interactions with these sequence sections, are therefore particularly specific. In another preferred embodiment, the fragment has a minimum length, namely at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the number of amino acids of the corresponding fragment of the immunogenic protein of SARS-CoV-2.

The at least one covalently bound protein particularly preferably has or consists of one of the immunogenic proteins of SARS-CoV-2, preferably one of its structural proteins, the spike protein being particularly preferred here.

In a preferred embodiment of the invention, the surface-modified polymer particle has 2, preferably 3, more preferably >4 and most preferably >5 covalently bound different proteins.

Basically, the higher the homology of the at least one covalently bound protein to an immunogenic protein of SARS-CoV-2 or its fragment, the higher the specificity. This means that high homology reduces the number of false positive tests due to the presence of antibodies to similar coronaviruses.

In a preferred embodiment of the invention, the polymer particles have an average particle size in the range of 50 to 600 nm, preferably 100 to 500 nm, more preferably 150 to 500 nm, even more preferably 150 to 450 nm, and most preferably 200 to 400 nm .

According to the invention, the average particle size of the polymer particles is in the range from 50 to 600 nm. In certain embodiments within the claimed range, the average particle size is preferably >190 nm, >240 nm or even >300 nm Particle size preferably < 410 nm, < 360 nm or even < 300 nm.

In certain embodiments, there is a monomodal particle size distribution with a maximum. In other embodiments, there is a bimodal particle size distribution with two maxima. The inventors have found that, on the one hand, the cut-off for a positive antibody test can be in the range of <100 µl/ml, and on the other hand, particularly in people who have been vaccinated against SARS-CoV-2 , greatly increased antibody titers in the range of 5,000 to 10,000 U/ml or even more can be achieved.

In order to achieve the greatest possible measurement accuracy both in the lower range of 0 to 100 U/ml and in the middle of 100 to 1,000 U/ml and in the upper range of 1,000 to 10,000 U/ml, a bimodal particle size distribution is preferred for specific embodiments of the invention. It has been found that particles of different sizes can complement each other optimally with their respective measurement accuracy in different titer ranges.

In the embodiments with a bimodal particle size distribution, the distance between the two maxima is preferably in the range from 100 to 300 nm, more preferably in the range from 125 to 275 nm, particularly preferably in the range from 150 to 250 nm.

In the embodiments with a bimodal particle size distribution, the ratio of the height of the maximum with the smaller particle size to the height of the maximum with the larger particle size is in the range from 1:2 to 1:5, preferably in the range from 1:3 to 1:4.

In the case of the present invention, the particle size of the polymer particles is determined by means of dynamic light scattering at 25° C., for example with the Malvern Zetasizer Pro. The average particle size refers to the number average. It is also particularly preferred to use a mixture of particles with different average particle sizes, e.g. a mixture of particles with a smaller diameter in the range from 150 to 200 nm and particles with a larger diameter of 400 to 500 nm.

Surprisingly, with the present invention it was possible to achieve better storage stability than would have been expected, despite the high particle size for a homogeneous assay. Larger particles usually have a greater tendency towards sedimentation and are therefore more difficult to resuspend after a longer storage period. Surprisingly, however, an extremely storage-stable suspension with constant reactivity can be obtained even with relatively large particles, in particular by adding sugar or sugar alcohol and setting a basic pH.

In a preferred embodiment of the invention, the suspension has a concentration of sugar or sugar alcohol dissolved therein in the range from 25 to 250 g/l. In certain embodiments, the concentration of sugar and/or sugar alcohol is within the range from 50 to 200 g/l. In some embodiments the concentration is 50 to 150 g/l, in other embodiments 150 to 250 g/l.

The sugar or sugar alcohol is preferably selected from sucrose, mannitol, sorbitol, xylitol, maltitol, raffinose, rhamnose, threalose, and combinations thereof. If a combination of sugars/sugar alcohols is used, the amounts stated above relate to the sum of the proportions of the sugars/sugar alcohols in this combination. The particles are stabilized by the sugar molecules.

The storage stability of the diagnostic reagent can be increased by further additives. In a preferred embodiment of the invention, the diagnostic reagent contains detergents for stabilization, with non-ionic or zwitteronic detergents being particularly preferred. Particularly preferred are tert-octylphenylpolyoxyethylene (Triton-X-100) or 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS).

So-called “blockers” can also be added to the diagnostic reagent, which prevent non-specific interactions with the surface-modified polymer particle and the at least one protein covalently bound to it. These can be, for example, sweeteners such as acesulfame, advantame, aspartame. The addition of a serum that contains coronaviruses other than SARS-CoV-2 can also serve to increase sensitivity, since this blocks the corresponding interaction sites and the reagent blank value is adjusted accordingly. If a coronavirus-positive serum is again added to such a reagent, no change in reactivity can be detected.

In a preferred embodiment, the diagnostic reagent has protease inhibitors, namely series protease inhibitors and/or cysteine protease inhibitors and/or metalloprotease inhibitors and/or aspartic protease inhibitors.

The addition of chaotropic compounds, ie chemical substances that disturb the order of the hydrogen bonds in the water, can also be advantageous. KSCN, KCl, urea, guanidinium HCl, (NH4) ₂ SO4 or CaCl ₂ are particularly preferred here.

In another preferred embodiment, however, the diagnostic reagent contains kosmotropic salts such as Na ₂ SO ₄ . Cosmotropic salts increase the reactivity of the diagnostic reagent. In a preferred embodiment of the invention, the composition has a thiol component, which is preferably selected from DTT (di-thio-treitol), beta-mercaptoethanol, thiosuccinic acid, amino acids such as proline, arginine or leucine, or mixtures of the aforementioned.

In a preferred embodiment of the invention, the diagnostic reagent has a pH in the range from 8 to 10. In certain embodiments, the pH of the diagnostic reagent is 9.0 ± 0.5. The pH of the diagnostic reagent is preferably adjusted by adding a buffer to the particles, such as a buffer selected from EPPS, HEPPS, tricine, Tris, glycylglycine, bicine, TAPS, boric acid, ethanolamine, CHES, glycine and CAPS.

In a preferred embodiment of the invention, the polymer of the polymer particle is selected from (meth)acrylate polymer, dextran-epichlorohydrin copolymer, polystyrene, melamine resin and silica. Polystyrene is particularly preferred, since this has low intrinsic absorption and a large number of different surface functionalizations are available on the market for this. In certain embodiments, the polymeric particles consist entirely of a polymeric material or mixtures thereof. In other embodiments, the particles consist of multiple layers of different polymers.

In specific embodiments, the particles may have a core and one or more layers coated onto that core. The core and one or more layers coated thereon may be composed of a polymeric material, various polymeric materials, or a non-polymeric material, provided that the polymeric particles are composed predominantly of polymeric material.

A polymer particle in the sense of the present invention is present when the particles consist of at least 80% by weight, at least 90% by weight, at least 95% by weight or 100% by weight of a polymer material or of a combination of different polymer materials exist.

The overall density of the polymer particle is preferably in the range from 0.9 to 1.1 g/cm ³ and particularly preferably in the range from 1.0±0.5 g/cm ³ .

In a preferred embodiment of the invention, the protein is covalently bonded by directly linking the protein to the functional surface groups of the upper surface-functionalized polymer particles formed, wherein the functional groups are preferably selected from a carboxyl group (-COOH), primary amine group (-RNH ₂ ), aromatic amine group (-ArNH ₂ ), chloromethyl group (-CH ₂ CI), an aromatic chloromethyl group (-ArCH ₂ CI), amide group (-CONH ₂ ), hydrazide group (-CONHNH ₂ ), aldehyde group (-CHO), hydroxyl group (-OH), thiol group (-SH), epoxy group and biotin-avidin.

Direct linking in the context of the present invention means that the at least one covalently bound protein is bound directly to the surface group of the polymer particle, i.e. there is no linker between the surface group of the particle and the at least one covalently bound protein.

By dispensing with such a linker, the production of diagnostic reagents can be significantly simplified and particle-enhanced assays are obtained in a quick and cost-effective manner.

In another preferred embodiment of the invention, at least one linker is arranged between the at least one covalently bound protein and the surface group of the polymer particle. The linker is preferably selected from the group consisting of lysine, silane and glycol.

Greater dynamics and/or mobility of the bound protein can be achieved by using a linker. In addition, branching and thus multiplication of the bond can also be achieved by the linker.

In a preferred embodiment of the invention, the at least one protein covalently bound to the polymer particles is a recombinant protein.

In a preferred embodiment of the invention, the at least one protein covalently bound to the polymer particles has a protein tag, preferably a His tag, a GST tag or an mFc tag. Tags can serve to support detection methods that can be used in addition to or as an alternative to absorption spectrometry (e.g. fluorescence labeling with GFP or flash tags), but they can also fulfill other functions, such as introducing a specific reactivity into the protein (e.g. GST - Day).

The diagnostic reagent according to the invention can also be part of a set of chemicals, which in addition to the surface-modified polymer particles with at least one SARS-CoV-2 protein covalently bound to this can have at least one reaction buffer, at least one control, at least one calibrator and/or at least one sample dilution matrix as a further component.

The reaction buffer can contain substances which, for example, influence the reactivity but should not be stored together with the suspension of the particles since they influence the storability of the particles.

The sample diluent matrix is preferably an aqueous matrix with physiological pH, salts for dilution-safe processing of samples so that they can be “diluted” into the linear, analytical range of the assay.

A kit of chemicals according to the invention preferably contains the diagnostic reagent divided into two reagent components, the individual components being as follows:

Reagent component 1 (R1 ):

R1 contains a buffer system with preferably one or more of the following components glycine, HEPES, MES, TRIS, MOPS, PBS or other common systems used for biological components.

The ionicity of R1 is preferably in the range of 20-1000 mM, particularly preferably 75-250 mM. Salts such as NaCl, KCl, MgCk, Na2SO4, NaHSÜ4 or other common salts can be used to adjust the ionicity. The ionic strength and choice of salt effects the elimination of interferences/equalization serum-plasma.

The pH at R1 is preferably in the range from 4 to 10, particularly preferably in the range from 5 to 8.

In order to eliminate non-specific interference interactions, R1 can be polyacrylate (preferably with a concentration of 0.05-5% by weight, more preferably 0.5-2% by weight, particularly preferably 1-1.5% by weight) in certain embodiments. -%) contain. In order to eliminate non-specific particle aggregations, R1 can, in certain embodiments, contain sugar (selected from mannitol, sucrose, sorbitol and PEG (polyethylene glycol) or combinations thereof (preferably at a concentration of 0.2-10% by weight, more preferably 1-5 % by weight, particularly preferably 2-3% by weight).

In certain embodiments, R1 can contain detergents selected from Tween 20, SDS, Tritonen, Thesit or other common detergents, in certain embodiments optionally in combination with guanidinium chloride (preferably with a concentration of 0.1 - 3% by weight, more preferably 0.2-1% by weight) and/or with BSA, gelatin, or other known blockers.

In certain embodiments, R1 contains NaNa, sodium benzoate, gentamycin sulfate, neomycin, and/or other common preservatives for preservation.

R2 contains the antigen component bound to latex particles or fragments of the antigen. Depending on the purity and immunogenicity, the concentration of the antigen component bound to the latex particles or of the bound fragments of the antigen is in the range from 0.01 to 0.75% by weight, preferably in the range from 0.02 to 0.5% by weight. %.

R2 contains a buffer system with preferably one or more of the following components: glycine, HEPES, MES, TRIS, MOPS, PBS, or other common buffer systems used for biological components.

The ionicity of R2 is preferably in the range of 20-1000 mM, particularly preferably 75-250 mM. Salts such as NaCl, KCL, MgCk, Na2SO4, NaHSO4 or other common salts can be used to adjust the ionicity.

The pH at R2 is preferably in the range from 4 to 11, preferably in the range from 7 to 10.

In order to eliminate non-specific particle aggregations, R2 can, in certain embodiments, contain sugar (selected from mannitol, sucrose, sorbitol and PEG (polyethylene glycol)) or combinations thereof (preferably at a concentration of 0.2-10% by weight, more preferably 1-5 % by weight, particularly preferably 2-3% by weight). For surface wetting, in certain embodiments, R2 can contain detergents selected from Tween 20, SDS, Tritonen, Thesit and other common detergents, in special embodiments optionally in combination with unreactive proteins and/or with BSA, gelatin, or other known blockers.

In certain embodiments, R2 contains NaNa, sodium benzoate, gentamycin sulfate, neomycin, and/or other common preservatives for preservation.

In certain embodiments of the invention, the at least one calibrator is a sales calibrator generated using the WHO reference (First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) NIBSC code: 20/136) with a correlation coefficient r > 0.99.

To produce the sales calibrator, the immunoreactivity of a solution of the antibodies used is measured with a lot of the reagent. At the same time, the WHO reference material is measured with the same reagent and its immunoreactivity is registered. The antibody solution and reference material are then serially diluted to generate a dose-response curve. The highest concentration of antibody solution that is not in Prozone (Antigen Excess) is taken as the highest point of the new standardized calibration curve. Its immunoreactivity is compared to the reference material. The concentration of the antibody solution is now given in BAU/mL (unit of the reference material) and no longer in mg/mL or AU (artificial units). The new calibrator is now WHO standardized.

In preferred embodiments, the at least one calibrator and/or the at least one control contains recombinant monoclonal antibodies that bind Spike-1 RBD. The advantages of calibrators and controls with recombinant monoclonal antibodies are simple production, higher purity and better batch homogeneity.

The invention also relates to a method for producing a diagnostic reagent, comprising the following steps:

A) Provision of a) surface-functionalized polymer particles, preferably in the form of an aqueous suspension, and b) a solution or suspension of at least one protein having a sequence homologous to an immunogenic protein of SARS-CoV-2 or a fragment of an immunogenic protein of SARS-CoV-2 with a sequence identity > 80%, preferably > 90%, has, wherein the fragment has a sequence section homologous to the binding domain region or the N-terminal domain of the spike protein with a sequence identity > 80% and/or has at least 50% of the number of amino acids of the corresponding immunogenic protein of SARS-CoV-2 ,

B) mixing components a) and b) to obtain a suspension,

C) Reacting the suspension from step B) at a pH in the range 3 to 8, preferably in the range 5 to 7, to covalently bind the protein to the polymer particles.

In certain embodiments, it may be necessary for the surface groups of the surface-modified particle to first have to be activated so that they have sufficient reactivity for the reaction with the at least one protein that is to be covalently bound. For example, since carboxylic acid and amines only have a low conversion rate due to the competing acid/base proton exchange, an acid group is converted into an activated, i.e. electrophilic form, such as the acyl chloride, anhydride or an active ester. Carbodiimides or hydroxybenzotriazole aminium/uronium or phosphonium salts can be used to form an active ester.

In a preferred embodiment of the invention, the reaction of the surface-functionalized polymer particles with the at least one protein in step C) takes place at a temperature in the range from 20 to 29° C., in the range from 30 to 34° C. or in the range from 35 to 45 °C, preferred is a reaction in the range between 30 to 45 °C.

In a preferred embodiment of the invention, the surface-functionalized polymer particles react with the at least one protein in a defined pH range and at a defined temperature over a period of 20 to 100 hours. In certain embodiments, the reaction occurs over a period of >30, >40, >50, or even >60 hours. The covalent binding of the at least one protein via the functional groups on the surface of the polymer particles preferably takes place at a pH of 3 to 6. In certain embodiments, the covalent binding takes place at a pH of 3 to 5 or 5 to 7 In certain embodiments, the pH of the reaction leading to the covalent attachment of the antibodies via the functional groups on the surface of the polymer particles is 4.0 ± 0.5.

The adjustment of the pH during the reaction, which leads to the covalent binding of the at least one protein via the functional groups on the surface of the polymer particles, is preferably carried out using inorganic or organic buffer substances such as borate, citrate, phosphate, malate, maleate , succinate, acetic acid/acetate. Hydrochloric acid/caustic soda is preferably used to set/adjust the pH value.

After the periods of time specified above have elapsed, the pH of the suspension of the surface-functionalized polymer particles now loaded with the at least one protein is increased to the range from 8 to 10. One or more of the alkalizing agents or buffer substances specified above can be added for this purpose.

The pH in the suspension is preferably adjusted using buffer substances with amine groups, such as EPPS, HEPPS, tricine, tris, glycylglycine, bicine, TAPS, boric acid, ethanolamine, CHES, glycine and CAPS. Hydrochloric acid/caustic soda is preferably used to set/adjust the pH value.

The invention also relates to a diagnostic reagent obtainable by the method according to the invention.

EXAMPLES

The invention will now be explained further on the basis of specific embodiments of diagnostic reagents according to the invention and on the basis of the attached figures.

exemplary embodiments

1. Preparation of diagnostic reagents according to the invention

Various experiments are presented below in which particles for diagnostic reagents according to the invention were produced.

For this purpose, at least one protein according to claim 1 was covalently bonded to at least one polymer particle made of polystyrene under the conditions of the method according to the invention. Table 1 shows the proteins successfully coupled to the particles. The corresponding proteins were obtained, for example, from the manufacturers Biozol, Sino Biological, Shanxi Kangjianen Biotechnology, Eurofins Genomic, Sekbio, Hytest, Invivo or Icosagen.

1.1. Production of diagnostic reagents according to the invention from carboxyl-modified polystyrene particles

For coupling, carboxyl-modified polystyrene particles in aqueous suspension (concentration 2-20 mg/ml) with an average particle size of -250 nm were first centrifuged and resuspended in 0.05 M 2-(N-morpholino)ethanesulfonic acid buffer with a pH value 5-7 (hereafter "MES buffer") washed several times. After the last centrifugation step, the centrifuge was resuspended in 0.05 M MES buffer (concentration 2-20 mg/ml) and carbodiimide (target concentration 24 mg/ml) was added. After an incubation time of 1 h at room temperature, the particles were washed again with 0.05 M MES buffer and finally resuspended in 0.05 M MES buffer. The proteins/protein fragments listed in Table 1 were added to this suspension at 25-40° C. (target concentration 20-120 mg/ml). The progress of the reaction was monitored by centrifugation and analysis of the supernatant using a Western blot, SDS-Page. If the supernatant still contained protein/protein fragments, the reaction was continued. After the reaction had ended, the particles with proteins coupled thereto were centrifuged and resuspended in 0.025 M glycine buffer (pH 5-7) with 0.5-1% by weight surfactant (Tween-20, Thermo Fisher) and finally incubated again for 1 h. The finished particles were then centrifuged and suspended in a suitable buffer (e.g. glycine or 2-ethanesulfonic acid buffer) (target pH 6- 10) to achieve a target concentration of the particle in the diagnostic reagent of 0.5-20 mg/mL. The particles were stored at 2-8 °C.

Table 1: Overview of the proteins coupled to polystyrene particles

¹ Recombinant nucleocapsid protein

² Recombinant spike protein (RBD)

³ nucleocapsid protein

⁴ S1 -Receptor Binding Domain (319-541)

⁵ Ni sepharose purification

⁶ Ion exchange column

1.2 Production of diagnostic reagents according to the invention from chloromethyl-modified polystyrene particles

For coupling, chloromethyl-modified polystyrene particles (concentration 2-20 mg/ml) with an average particle size of -250 nm were first centrifuged and resuspended in 0.05 M 2-(N-morpholino)ethanesulfonic acid buffer with pH 6 -8 (hereafter "MES buffer") washed several times. After the last centrifugation step

SUBSTITUTE SHEET (RULE 26) resuspended the centrifuge in 0.05 M MES buffer (concentration 2-20 mg/ml). The proteins/protein fragments listed in Table 1 were added to this suspension at 23° C. (target concentration 20-120 mg/ml). The progress of the reaction was monitored by centrifugation and analysis of the supernatant using a Western blot, SDS-Page. If the supernatant still contained protein/protein fragments, the reaction was continued. After the reaction had ended, the particles with proteins coupled thereto were centrifuged and resuspended in 0.025 M glycine buffer (pH 5-7) with 0.5-1% by weight surfactant (Tween-20, Thermo Fisher) and finally incubated again for 1 h . The finished particles were then centrifuged and suspended (target pH 6-10) in a suitable buffer (eg: glycine or 2-ethanesulfonic acid buffer) to achieve a target concentration of the particle in the diagnostic reagent of 0.5-20 mg/ml . The particles were stored at 2-8 °C.

1 . Determination of reactivity - detection of SARS-CoV-2 antibodies

The diagnostic reagents produced under 1 were then used to detect SARS-CoV-2 antibodies. For this purpose, a serum from a patient infected with SARS-CoV-2 was used as a sample and the serum from a patient not infected with SARS-CoV-2 was used as a control experiment. An example of a calibration curve can be found in FIG.

To determine the reactivity, 90 μl of a reaction buffer were mixed with 30 μl of one of the reagents listed in Table 1 and 10 μl of the sample, and the absorption at 658 nm was determined using a BioMajesty® 6010 (Jeol®, Japan). The absorbance value given in Table 2 (Abs 0 h or Abs 96 h) indicates the difference in absorbance between the sample of the SARS-CoV-2 infected patient and the control experiment. As can be seen from the table, the reactivity of the diagnostic reagents according to the invention is virtually unchanged even after 96 hours.

Table 2: Overview of the reactivities of diagnostic reagents according to the invention

SUBSTITUTE SHEET (RULE 26) The change in the reagent blank value, ie the inherent color of the reagent mixture of the control experiments, was also analyzed over time. If the particles of a diagnostic reagent agglutinate over time, the absorption of the reagents increases and the particles can no longer be used for reliable detection. As can be seen from the table below, the reagents according to the invention have a reagent blank value which is almost constant over time, ie the diagnostic reagents according to the invention are colloidally stable and can be used over a long period of time without problems.

Table 3: Overview of the reagent blank values of diagnostic reagents according to the invention

3. Use of particles with different particle diameters

Reagents of the invention were prepared according to the protocol outlined above, coupling the protein S-RBD, HP811-60, to carboxyl-modified polystyrene latices of mean diameter 95 nm, 258 nm and 351 nm. The reactivity was determined as in the example above. However, the absorption was measured at different wavelengths. The results can be found in the table below. It becomes clear that different wavelengths can easily be used to determine the reactivity.

Table 4: Samples with different mean particle diameters

SUBSTITUTE SHEET (RULE 26) 4. Determination of long-term stability

To determine the long-term stability, the reactivity and the reagent blank value of the diagnostic reagent were determined according to Table 1, entry #7 over a period of >25 days. As can be seen from FIGS. 2 and 3, both the reactivity and the reagent blank value are almost constant.

5. Comparison with electrochemiluminescence immunoassay

To compare the diagnostic reagents according to the invention with known electrochemiluminescent immunoassays (“ECLIA”), 30 samples tested positive with the ECLIA assay Elecsys developed by Hoffmann La-Roche AG, Basel were mixed with diagnostic reagents according to the invention and the reactivity was determined. The Elecsys assay uses a recombinant antigen of the nucleocapsid protein to detect SARS-CoV-2 antibodies. The table below shows the comparison of the diagnostic results.

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

The result for the true positive rate (TPR, sensitivity) is:

TP / TP+FN x 100 = 42 / 43 x 100 = 100.0%

The result for the false positive rate (FPR) and the specificity is:

FP / FP+TN x 100 = 0 / 5 = 0% (specificity = 100 - FPR = 100%)

6. Dilution of a sample according to the invention

A DS018 sample was diluted in the ratios 1:2, 1:4, 1:8 and 1:16 and the value obtained for the concentration of SARS-CoV-2 antibodies by determining the absorption and comparing it with the calibration curve was compared with the target value . The correlation shown in FIG. 4 shows the linear behavior with dilution.

7. Determination of measurement accuracy

For this purpose, the concentration of the SARS-CoV-2 antibodies for the sample DS011 was determined 20 times in a row. The results can be found in the table below. The coefficient of variation [CV%] was 3.79%.

SUBSTITUTE SHEET (RULE 26)

8. Investigation of bimodal particle size distributions

The following results of investigations into embodiments with a bimodal particle size distribution show that particles of different sizes complement each other optimally with their respective measuring accuracy in different titer ranges.

The results are shown again graphically in FIGS. 5 to 7.

SUBSTITUTE SHEET (RULE 26) 9. Comparison of WHO reference material vs. monoclonal antibodies as calibrator

The results of investigations into embodiments with a bimodal particle size distribution, shown graphically in FIG. 8, show that particles of different sizes complement each other optimally with their respective measuring accuracy in different titer ranges.

SUBSTITUTE SHEET (RULE 26)

Claims

29

Patent claims Diagnostic reagent for the detection of SARS-CoV-2 antibodies in a sample, the reagent having an aqueous suspension of surface-modified polymer particles with at least one covalently bound protein, characterized in that the at least one covalently bound protein has a an a) immunogenic protein from SARS-CoV-2 or b) a fragment of an immunogenic protein from SARS-CoV-2, which bi) has a sequence section homologous to the binding domain region or the N-terminal domain of the spike protein with a sequence match of 80 % and/or bg) which has at least 50% of the number of amino acids of the corresponding immunogenic protein of SARS-CoV-2, has a homologous sequence section with a sequence identity >80%. Diagnostic reagent according to claim 1, wherein the immunogenic protein of SARS-CoV-2 is a structural protein, preferably the nucleocapsid protein or the spike protein, preferably the spike protein. The diagnostic reagent of claim 1, wherein the fragment of an immunogenic protein of SARS-CoV-2 is the Si subunit of the spike protein or the region binding domain of the Si subunit or the N-terminal domain of the spike protein. Diagnostic reagent according to any one of the preceding claims, wherein the polymer particles have an average particle size in the range of 100 to 500 nm, preferably 150 to 400 nm. A diagnostic reagent as claimed in any one of the preceding claims wherein the suspension has a pH in the range 8-10. 30

6. Diagnostic reagent according to any one of the preceding claims, wherein the polymer of the polymer particles is selected from (meth)acrylate polymer, dextran-epichlorohydrin copolymer, polystyrene, melamine resin and silica.

7. Diagnostic reagent according to any one of the preceding claims, wherein the covalent bond of the protein is formed by directly linking the protein to the functional surface groups of the surface-functionalized polymer particles and wherein the functional groups are selected from a carboxyl group (-COOH), primary amine group (-RNH2 ), aromatic amine group (-ArNHa), chloromethyl group (-CH2CI), aromatic chloromethyl group (-ArCH ₂ CI), amide group (-CONH2), hydrazide group (-CONHNH2), aldehyde group (-CHO), hydroxyl group (-OH), thiol group (-SH), epoxy group and biotin-avidin.

8. Diagnostic reagent according to any one of the preceding claims, wherein the at least one protein covalently bound to the polymer particles is a recombinant protein and/or the at least one protein covalently bound to the polymer particles has a protein tag.

9. A kit of chemicals comprising a diagnostic reagent according to any one of the preceding claims.

10. Set of chemicals according to claim 9, comprising, in addition to the surface-modified polymer particles with at least one SARS-CoV-2 protein covalently bound to them, a reaction buffer, a control, a calibrator and/or a sample dilution matrix as further components.

11 . A kit of chemicals according to any one of claims 9 and 10, wherein the diagnostic reagent is divided into at least the following two reagent components:

Reagent component 1 with a buffer system and with a pH in the range of 4 to 10 and

Reagent component 2 with the surface-modified polymer particles with at least one SARS-CoV-2 protein covalently bound to them, with a buffer system and with a pH in the range from 4 to 11.

12. Set of chemicals according to any one of claims 9 to 11, which contains at least one calibrator containing recombinant monoclonal antibodies which bind Spike-1 RBD.

13. A method for producing a diagnostic reagent according to any one of the preceding claims, comprising the following steps:

A) Provision of a) surface-functionalized polymer particles, preferably in the form of an aqueous suspension, and b) a solution or suspension of at least one protein which is a to an immunogenic protein of SARS-CoV-2 or a fragment of an immunogenic protein of SARS- CoV-2 homologous sequence with a sequence identity of 80%, preferably s 90%, wherein the fragment has a sequence section homologous to the binding domain region or the N-terminal domain of the spike protein with a sequence identity of 80% and/or at least 50% has the number of amino acids of the corresponding immunogenic protein of SARS-CoV-2,

B) mixing components a) and b) to obtain a suspension,

C) Reacting the suspension from step B) at a pH in the range 3-6 to covalently bind the protein to the polymer particles.

14. The method according to claim 13, wherein the reaction of the surface-functionalized polymer particles with the at least one protein takes place at a temperature in the range from 20 to 29°C, in the range from 30 to 34°C or in the range from 35 to 45°C and/ or over a period of 20 to 100 hours.

15. Diagnostic reagent for detecting SARS-CoV-2 antibodies in a sample, wherein the reagent can be obtained by a method according to any one of claims 13 or 14.