WO2022043147A1 - Réactif de diagnostic pour la détection d'anticorps du sars-cov-2 - Google Patents

Réactif de diagnostic pour la détection d'anticorps du sars-cov-2 Download PDF

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Publication number
WO2022043147A1
WO2022043147A1 PCT/EP2021/072889 EP2021072889W WO2022043147A1 WO 2022043147 A1 WO2022043147 A1 WO 2022043147A1 EP 2021072889 W EP2021072889 W EP 2021072889W WO 2022043147 A1 WO2022043147 A1 WO 2022043147A1
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Prior art keywords
protein
cov
sars
diagnostic reagent
polymer particles
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PCT/EP2021/072889
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German (de)
English (en)
Inventor
Thomas MASETTO
Matthias Grimmler
Christian KOCHEM
Leoni WEY
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Diasys Diagnostic Systems Gmbh
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Publication of WO2022043147A1 publication Critical patent/WO2022043147A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to a diagnostic reagent for detecting SARS-CoV-2 antibodies in a sample and a method for producing this reagent.
  • 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.
  • 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.
  • S1 amino acids 13-685
  • RBD receptor-binding domain
  • S2 amino acids 686-1273
  • 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.
  • an infection can also be detected indirectly using the specific antibodies against SARS-CoV-2 produced in the body.
  • PCR polymerase chain reaction test
  • 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.
  • 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.
  • ELIA electrochemical immunoassays
  • Elecsys® immunoassay from Hoffmann La Roche AG, Basel
  • N nucleocapsid
  • 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.
  • 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.
  • a diagnostic reagent for detecting SARS-CoV-2 antibodies in a sample 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.
  • 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.
  • other heterogeneous assay methods such as CLIA require the use of a large number of different reagents and work-up steps.
  • 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.
  • Quantum 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.
  • diagnostic reagent according to the invention is particularly preferably used to detect the total SARS-CoV-2 antibody content of a sample.
  • 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.
  • the sample will be a sample of fresh whole blood, which may have been appropriately processed for purposes of performing the analysis.
  • other liquid samples that may contain SARS-CoV-2 antibodies, such as standard solutions and calibrators, are also encompassed by the present 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.
  • the SARS-CoV-2 immunogenic protein is selected from the proteins listed in the following tables.
  • 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.
  • virus-specific structural proteins in SARS-CoV-2 are the spike protein, the membrane protein, the envelope small membrane protein and the nucleocapsid protein.
  • the polymer particles are present in an aqueous suspension.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • RBD binding domain region
  • NTD N-terminal domain
  • 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.
  • the surface-modified polymer particle has 2, preferably 3, more preferably >4 and most preferably >5 covalently bound different proteins.
  • 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.
  • 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 .
  • 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.
  • the cut-off for a positive antibody test can be in the range of ⁇ 100 ⁇ l/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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the suspension has a concentration of sugar or sugar alcohol dissolved therein in the range from 25 to 250 g/l.
  • concentration of sugar and/or sugar alcohol is within the range from 50 to 200 g/l.
  • 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.
  • 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.
  • 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.
  • the diagnostic reagent has protease inhibitors, namely series protease inhibitors and/or cysteine protease inhibitors and/or metalloprotease inhibitors and/or aspartic protease inhibitors.
  • 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.
  • the diagnostic reagent contains kosmotropic salts such as Na 2 SO 4 .
  • Cosmotropic salts increase the reactivity of the diagnostic reagent.
  • 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.
  • 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.
  • 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.
  • the polymeric particles consist entirely of a polymeric material or mixtures thereof. In other embodiments, the particles consist of multiple layers of different polymers.
  • 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 .
  • 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.
  • 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
  • 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.
  • 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.
  • the at least one protein covalently bound to the polymer particles is a recombinant protein.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • R2 contains NaNa, sodium benzoate, gentamycin sulfate, neomycin, and/or other common preservatives for preservation.
  • 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.
  • WHO reference First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) NIBSC code: 20/136
  • the immunoreactivity of a solution of the antibodies used is measured with a lot of the reagent.
  • 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.
  • 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:
  • step 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.
  • the surface groups of the surface-modified particle may be activated so that they have sufficient reactivity for the reaction with the at least one protein that is to be covalently bound.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • MES buffer 2-(N-morpholino)ethanesulfonic acid buffer with a pH value 5-7
  • 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.
  • a suitable buffer e.g. glycine or 2-ethanesulfonic acid buffer
  • 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.
  • 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 .
  • a suitable buffer eg: glycine or 2-ethanesulfonic acid buffer
  • the diagnostic reagents produced under 1 were then used to detect SARS-CoV-2 antibodies.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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%.

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Abstract

L'invention concerne un réactif de diagnostic pour la détection d'anticorps du SARS-CoV-2 dans un échantillon, et une méthode de préparation dudit réactif.
PCT/EP2021/072889 2020-08-28 2021-08-18 Réactif de diagnostic pour la détection d'anticorps du sars-cov-2 WO2022043147A1 (fr)

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WO2018206737A1 (fr) * 2017-05-09 2018-11-15 Immundiagnostik Ag Procédé de détermination de membre de la famille s100 de protéines liant le calcium par immunoturbidimétrie

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Publication number Priority date Publication date Assignee Title
WO2018206737A1 (fr) * 2017-05-09 2018-11-15 Immundiagnostik Ag Procédé de détermination de membre de la famille s100 de protéines liant le calcium par immunoturbidimétrie

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Title
ALVES DIANA ET AL: "Rapid Gel Card Agglutination Assays for Serological Analysis Following SARS-CoV-2 Infection in Humans", ACS SENSORS, vol. 5, no. 8, 16 July 2020 (2020-07-16), pages 2596 - 2603, XP055856942, ISSN: 2379-3694, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acssensors.0c01050> DOI: 10.1021/acssensors.0c01050 *
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