Kits and methods for the enrichment and detection of RNA viruses of the Coronaviridae family

Abstract

Kits and methods for the detection and enrichment of RNA viruses of the family Coronaviridae. The detection method comprises the steps of (a) coupling a binding agent that specifically recognizes and binds to a virus component to a carrier material, (b) incubating the carrier material with the thereon coupled binding agent with a virus-containing sample, (c) staining the viruses immobilised on the carrier material with a staining agent, and (d) detecting stained virus particles via a physical, chemical or biological detection means. The methods may be suitable for the rapid and efficient detection of coronaviruses, such as SARS-CoV-2. With the methods and kits, it is possible to perform rapid high-throughput tests in a large population. At the same time, the enrichment procedure makes it possible to enrich viral samples, e.g. from a throat swab of a patient, for use in a subsequent PCR.

Claims

1. A method for the detection of RNA viruses of the family Coronaviridae, comprising coupling a binding agent that is capable of binding to a virus component of said RNA virus of the family Coronaviridae to a carrier material. incubating the carrier material with the thereon coupled binding agent with a virus-containing sample, staining the viruses immobilised on the carrier material with a staining agent, detecting stained virus particles via a physical, chemical or biological detection means.

2. The method according to claim 1, wherein the binding agent is selected from the group consisting of i. CR3022 antibody, CR3022-RB antibody, spike antibody, spike S1 antibody, spike S2 antibody, envelope antibody, anti-M antibody, anti-S-glycoprotein antibody, ii. single-chain binders raised in camelids, cartilaginous fishes or jawless vertebrates, nanobodies, IgNAR, lampribodies, iii. virus receptors and cell entry receptors for RNA viruses of the family Coronaviridae, ACE2, neuropilin receptors, aminopeptidase N, dipeptidyl peptidase 4, CEACAM1, CEACAM5, DC-SIGN, L-SIGN, GRP78, CD147, hemagglutinin esteerase, carbohydrate receptors, sialic acids, sialosides, N-glycolylneuraminic acid, N-acetylneuraminic acid and their derivatives, heparan sulfate, mucins, iv. angiotensin-converting enzyme 2 (ACE2), an ACE2 construct, an ACE2 fusion protein, or a modified or mutant ACE2 polypeptide or fusion protein, v. binders obtained by in vitro or in silico selection based on proteinaceous scaffolds for molecular recognition, scaffold-protein affinity reagents (SPARs), adhirons, alphabodies, affibodies, affifins, affilins, anticalins, adnectins, avimers, affimers, Armadillo repeat proteins, DARPins, fynomers, Kunitz domains, PDZ domain scaffolds, knottins, monomers, peptide aptamers, monobodies, lectins, minibinders, miniproteins like LCB1, LCB1v1.3 and LCB1-Fc, vi. specific binders obtained by in vitro or in silico selection based on nucleic acid scaffolds, aptamers, SOMAmers, or vii. small molecules.

3. The method according to claim 1, wherein the RNA virus of the Coronaviridae family is selected from the group consisting of the human coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, MBRS-CoV, SARS-CoV, SARS-CoV-2.

4. The method according to claim 1, wherein the RNA virus of the Coronaviridae family is selected from the group consisting of feline, canine, porcine, bovine, bat, pangolin, ferret, mink, bird or dromedary camel coronavirus.

5. The method according to claim 1, wherein the carrier material is selected from the group consisting of polymethyl methacrylate (PMMA) microparticles, polyethylene (PE) microparticles, polypropylene (PP) microparticles, polystyrene (PS) microparticles, carboxylated or aminated latex particles, polydimethylsiloxane (PDMS) microparticles, cellulose acetate microparticles, cyclic olefin copolymer (COC) microparticles, protein A/G particles, agarose microparticles, magnetic microparticles, a hydrogel, a sol-gel, a porous polymer monolith, a porous silicone, beads or a membrane.

6. The method according to claim 1, wherein the staining agent is selected from the group consisting of SYTOX™, SYBR™, acridine orange (3-N,3-N,6-N,6-N-tetramethylacridine-3,6-diamine), thiazole orange, DAPI (4′,6-diamidino-2-phenylindole), 7-AAD (7-aminoactinomycin D), ethidium bromide, propidium iodide, an enzymatic or fluorescence-coupled staining agent or a membrane staining agent.

7. The method according to claim 1, wherein the physical detection means is a FACS analyzer, a microfluidic platform, a microscope, a camera and/or the human eye.

8. The method according to claim 1, wherein the biological detection means is a conjugate- or marker-coupled further secondary binding agent.

9. The method according to claim 1, wherein the sample is taken from body secretions such as saliva, blood serum, whole blood, sputum, urine, tear fluid, faeces, a rinse or swab (in particular from the mouth, nose and/or throat) or a gargle sample.

10. The method according to claim 1, wherein the binding agent is recombinant biotinylated Fc-ACE2.

11. The method according to claim 10, wherein the carrier material comprises streptavidin-agarose beads or streptavidine PMMA.

12. The method according to claim 10, wherein the staining agent is SYBR™ Gold.

13. The method according to claim 10, wherein the physical detection means is a confocal laser scanning microscope.

14. A kit for the detection of RNA viruses of the family Coronaviridae, comprising: a. a binding agent that specifically recognizes and binds to a virus component of said RNA virus of the family Coronaviridae, b. a carrier material that is coupled to the binding agent, c. a staining agent for staining viruses immobilised on the carrier material.

15. The kit according to claim 14, wherein the binding agent is selected from the group consisting of i. CR3022 antibody, CR3022-RB antibody, spike antibody, spike S1 antibody, spike S2 antibody, envelope antibody, anti-M antibody, anti-S-glycoprotein antibody, ii. single-chain binders raised in camelids, cartilaginous fishes or jawless vertebrates, nanobodies, IgNAR, lampribodies, iii. virus receptors and cell entry receptors for RNA viruses of the family Coronaviridae, ACE2, neuropilin receptors, aminopeptidase N, dipeptidyl peptidase 4, CEACAM1, CEACAM5, DC-SIGN, L-SIGN, GRP78, CD147, hemagglutinin esteerase, carbohydrate receptors, sialic acids, sialosides, N-glycolytneuraminic acid, N-acetylneuraminic acid and their derivatives, heparan sulfate, mucins, iv. angiotensin-converting enzyme 2 (ACE2), an ACE2 construct, an ACE2 fusion protein, or a modified or mutant ACE2 polypeptide or fusion protein, v. binders obtained by in vitro or in silico selection based on proteinaceous scaffolds for molecular recognition, scaffold-protein affinity reagents (SPARs), adhirons, alphabodies, affibodies, affifins, affilins, anticalins, adnectins, avimers, affimers, Armadillo repeat proteins, DARPins, fynomers, Kunitz domains, PDZ domain scaffolds, knottins, monomers, peptide aptamers, monobodies, lectins, minibinders, miniproteins like LCB1, LCB1v1.3 and LCB1-Fc, vi. specific binders obtained by in vitro or in silico selection based on nucleic acid scaffolds, aptamers, SOMAmers, or vii. small molecules.

16. The kit according to claim 14, wherein the RNA virus of the Coronaviridae family is selected from the group consisting of the human coronaviruses HCoV-229E. HCoV-NL63, HCoV-OC43, HCoV-HKU1, MERS-CoV, SARS-CoV, SARS-CoV-2.

17. The kit according to claim 14, wherein the RNA virus of the Coronaviridae family is selected from the group consisting of feline, canine, porcine, bovine, bird or dromedary camel coronavirus.

18. The kit according to claim 14, wherein the carrier material is selected from the group consisting of polymethyl methacrylate (PMMA) microparticles, polyethylene (PE) microparticles, polypropylene (PP) microparticles, polystyrene (PS) microparticles, carboxylated or aminated latex particles, polydimethylsiloxane (PDMS) microparticles, cellulase acetate microparticles, cyclic olefin copolymer (COC) microparticles, protein A/G particles, agarose microparticles, magnetic microparticles, a hydrogel, a sol-gel, a porous polymer monolith, a porous silicone, beads or a membrane.

19. The kit according to claim 14, wherein the staining agent is selected from the group consisting of SYTOX™, SYBR™, acridine orange (3-N,3-N,6-N,6-N-tetramethylacridine-3,6-diamine), thiazole orange, DAPI (4′,6-diamidino-2-phenylindole), 7-AAD (7-aminoactinomycin D), ethidium bromide, propidium iodide, an enzymatic or fluorescence-coupled staining agent or a membrane staining agent.

20. The kit according to claim 14, wherein the binding agent is recombinant biotinylated Fc-ACE2.

21. The kit according to claim 20, wherein the carrier material comprises streptavidin-agarose beads or streptavidine PMMA.

22. The kit according to claim 20, wherein the staining agent is SYBR™ Gold.

23. The kit according to claim 14, wherein a magnifying device is provided for making the staining agent visible to the human eye.

24. The kit according to claim 14, wherein the carrier material is individually labelled with an oligonucleotide that is specific for said carrier material.

25. A method for the enrichment of RNA viruses of the family Coronaviridae, comprising coupling a binding agent that is capable of binding to a virus component of said RNA virus of the family Coronaviridae to a carrier material, incubating the carrier material with the thereon coupled binding agent with a virus-containing sample.

26. The method according to claim 25, wherein the binding agent is selected from the group consisting of i. CR3022 antibody, CR3022-RB antibody, spike antibody, spike S1 antibody, spike S2 antibody, envelope antibody, anti-M antibody, anti-S-glycoprotein antibody, ii. single-chain binders raised in camelids, cartilaginous fishes or jawless vertebrates, nanobodies, IgNAR, lampribodies, iii. virus receptors and cell entry receptors for RNA viruses of the family Coronaviridae, ACE2, neuropilin receptors, aminopeptidase N, dipeptidyl peptidase 4, CEACAM 1, CEACAM5, DC-SIGN, L-SIGN, GRP78, CD147, hemagglutinin esteerase, carbohydrate receptors, sialic acids, sialosides, N-glycolylneuraminic acid, N-acetylneuraminic acid and their derivatives, heparan sulfate, mucins, iv. angiotensin-converting enzyme 2 (ACE2), an ACE2 construct, an ACE2 fusion protein, or a modified or mutant ACE2 polypeptide or fusion protein, v. binders obtained by in vitro or in silico selection based on proteinaceous scaffolds for molecular recognition, scaffold-protein affinity reagents (SPARs), adhirons, alphabodies, affibodies, affifins, affilins, anticalins, adnectins, avimers, affimers, Armadillo repeat proteins, DARPins, fynomers, Kunitz domains, PDZ domain scaffolds, knottins, monomers, peptide aptamers, monobodies, lectins, minibinders, miniproteins like LCB1, LCB1v1.3 and LCB1-Fc, vi. specific binders obtained by in vitro or in silico selection based on nucleic acid scaffolds, aptamers, SOMAmers, or vii. small molecules.

27. The method according to claim 25, wherein the RNA virus of the Coronaviridae family is selected from the group consisting of the human coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, MERS-CoV, SARS-CoV, SARS-CoV-2.

28. The method according to claim 25, wherein the RNA virus of the Coronaviridae family is selected from the group consisting of feline, canine, porcine, bovine, bird or dromedary camel coronavirus.

29. The method according to claim 25, wherein the carrier material is selected from the group consisting of polymethyl methacrylate (PMMA) microparticles, polyethylene (PE) microparticles, polypropylene (PP) microparticles, polystyrene (PS) microparticles, carboxylated or aminated latex particles, polydimethylsiloxane (PDMS) microparticles, cellulose acetate microparticles, cyclic olefin copolymer (COC) microparticles, protein A/G particles, agarose microparticles, magnetic microparticles, a hydrogel, a sol-gel, a porous polymer monolith, a porous silicone, beads or a membrane.

30. The method according to claim 25, wherein the binding agent is recombinant biotinylated Fc-ACE2.

31. The method according to claim 30, wherein the carrier material comprises streptavidin-agarose beads or streptavidine PMMA.

32. The method according to claim 30, wherein the staining agent is SYBR™ Gold.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1 shows SARS-CoV-2 virus particles bound to biotinylated Fc-ACE2 coupled to streptavidin PMMA beads. The virus particles are visualised by SYBR™ Gold staining.

[0109] FIG. 2 shows SARS-CoV-2 bound beads that are stained with a combination of different binding agents anti-spike [1A9], anti-nucleoprotein, convalescent serum and the staining agent SYBR™ Gold.

[0110] FIG. 3 shows secondary methods for the detection of coronavirus in a sample using quantitative PCR, flow cytometry or colorimetry.

[0111] FIG. 4 shows SARS-CoV-2 detection from a patient swab using ACE2 beads.

MODES FOR CARRYING OUT THE INVENTION

[0112] The invention is explained in more detail in the following embodiments.

Binding of SARS-CoV-2 Particles to PMMA Microparticles

[0113] 40 .Math.l streptavidin-PMMA beads were incubated with 6 .Math.g biotinylated Fc-ACE2 in a total volume of 100 .Math.l PBS. To remove unbound proteins, beads were washed with PBS in Mobicol mini-columns with 10 .Math.m filters inserted. The beads were resuspended in 0.5 ml PBS with 0.1% BSA.

[0114] 0.5 ml cell culture supernatant containing 10.sup.6 SARS-CoV-2 particles was thawed and added to 12.5 .Math.l PMMA beads (containing approximately 2000 beads) with pre-bound Fc-ACE2. Another source of SARS-CoV-2 particles resulted from a patient swab that was immersed in aqueous solution. The suspension consisting of virus particles and Fc-ACE2-coated beads was incubated for 2 hours at room temperature with constant gentle agitation. Beads with bound virus particles were collected and washed with PBS containing 0.1% BSA in Mobicol mini-columns with 10 .Math.m filters inserted. Beads were resuspended in 250 .Math.l PBS containing 0.1% BSA and fixed to inactivate virus by adding 250 .Math.l 4% PFA (dissolved in PBS), followed by 30 min incubation at room temperature. The beads were washed with PBS containing 0.1% BSA and with PBS containing 50 mM Tris/HCI pH 7.5.

Binding of SARS-CoV-2 Particles to Magnetic Beads and Subsequent Analysis

[0115] To coat magnetic beads with Fc-ACE2, 2 ml magnetic Streptavidin-Dynabeads (4.5 .Math.m diameter) at a concentration of 10.sup.7 beads / ml in PBS were incubated with 100 .Math.g biotinylated Fc-ACE2. To remove unbound protein, magnetic beads were washed with PBS. Beads were resuspended in 0.5 ml PBS with 0.1% BSA and were distributed to 0.2 ml PCR tubes in either low or high amounts. The tubes containing low amount of magnetic beads contained only 1000 beads / tube and were used for experiments that aim at fluorescent detection of beads via flow cytometry. The tubes containing high amount of magnetic beads contained 750000 beads / tube and were used for experiments that aim at either qPCR analysis or detection via an enzyme-coupled colorimetric assay. In order to bind SARS-CoV-2 virus particles to magnetic beads 0.2 ml of either undiluted or the indicated ten-fold dilutions of the previously described cell culture supernatant was added to the Fc-ACE2-coated magnetic beads. The suspension was incubated for 2 hours at room temperature with constant gentle agitation. Beads with bound virus particles were collected, washed with PBS containing 0.1% BSA and PBS. Beads were either analysed by standard qPCR or fixed in 4% PFA (dissolved in PBS), followed by 30 min incubation at room temperature. The latter samples were washed with PBS containing 0.1% BSA and with PBS containing 50 mM Tris/HCI pH 7.5. Immunolabeling was performed as described below, followed either by standard FACS analysis or by an enzyme-based colorimetric assay as described below.

Visualisation of SARS-CoV-2 Particles Via Staining of the Viral Genome

[0116] Bound virus particles were stained with SYBR™ Gold at a final dilution of 1:50,000 in a final volume of 200 .Math.l PBS. 50 .Math.l of the bead suspension was transferred to a well of a .Math.-slide and images of the settled beads were taken with a confocal laser scanning microscope (Leica TCS SP5).

Visualisation of SARS-CoV-2 Particles via Immunolabeling

[0117] To block unspecific interaction beads with bound virus particles were incubated for 30′ in blocking buffer (PBS containing 0.1% (w/v) BSA and 0.1% (w/v) TX-100) at room temperature.

[0118] Labelling with mouse monoclonal antibodies was performed by a two-hour incubation with the primary antibody diluted in blocking buffer (mouse monoclonal SARS-CoV-2 spike antibody [1A9] applied at a final concentration of 2 .Math.g/ml or mouse anti-nucleocapsid antibody (MM05) applied at a final concentration of 4 .Math.g/ml) at room temperature. After two short washing steps (2× 250 .Math.l blocking buffer, 10′ each), the beads were incubated with secondary antibody (Alexa488-labelled anti-mouse IgG (H+L), obtained in goat, at a final concentration of 5 .Math.g/ml in blocking buffer). After two short washing steps (2× 250 .Math.l blocking buffer, 10′ each), the bead suspension was transferred to a well of a .Math.-slide and images of the settled beads were taken with a confocal laser scanning microscope (Leica TCS SP5).

[0119] Labelling with convalescent serum was performed by a two-hour incubation with convalescent serum applied at a 1:800 dilution in blocking buffer at room temperature. After two short washing steps (2× 250 .Math.l blocking buffer, 10′ each), the beads were incubated with a mouse monoclonal anti-Ig kappa chain antibody (clone L1C1) at a final concentration of 5 .Math.g/ml in blocking buffer. After two short washing steps (2× 250 .Math.l blocking buffer, 10′ each), the beads were incubated with secondary antibody (Alexa488-labelled anti-mouse IgG (H+L), obtained in goat, at a final concentration of 5 .Math.g/ml in blocking buffer). After two short washing steps (2× 250 .Math.l blocking buffer, 10′ each), the bead suspension was transferred to a well of a .Math.-slide and images of the settled beads were taken with a confocal laser scanning microscope (Leica TCS SP5).

Detection of SARS-CoV-2 Virus Particles Bound to Beads Via an Enzyme-Coupled Colorimetric Assay

[0120] To block unspecific interaction beads with bound virus particles were incubated for 30′ in blocking buffer (PBS containing 0.1% (w/v) BSA and 0.1% (w/v) TX-100) at room temperature. This was followed by a two-hour incubation with the primary mouse anti-nucleocapsid antibody (MM05) applied at a final concentration of 4 .Math.g/ml in blocking buffer at room temperature. After two short washing steps (2× 250 .Math.l blocking buffer, 10′ each), the beads were incubated with secondary goat anti-mouse IgG (H+L) conjugated with horse radish peroxidase (HRP), at a final concentration of 1:500 in blocking buffer at room temperature. After extensive washing steps (8x 200 .Math.l blocking buffer, 10′ each), the bead suspension was transferred into new tubes and washed with 2× 200 .Math.l PBS. Finally, beads were resuspended in 80 .Math.l 1-Step Ultra TMB-ELISA Substrate Solution and incubated at room temperature. As soon as the colorimetric reaction reached an endpoint, aliquots were transferred into new tubes. Equal volumes of 2 M sulfuric acid (H.sub.2SO.sub.4) were added which stopped the enzymatic reaction and resulted in a colour change to yellow.

Materials

[0121] Biotinylated human Fc-ACE2: ACE2 (Gin 18 - Ser 740, Q9BYF1-1) - Fc (Pro 100 - Lys 330, P01857) - Avi; ACROBiosystems (Cat. AC2-H8F9) [0122] Streptavidin PMMA beads, 21 .Math.m functionalised monodisperse PMMA microparticles (PolyAn Cat. 105 21 020) [0123] Magnetic Streptavidin-Dynabeads (4.5 .Math.m diameter) = Invitrogen Exosome-Streptavidin Isolation/Detection Reagent; ThermoFisherScientific (Cat. 10608D) [0124] Mobicol mini columns with inserted small 10 .Math.m filter and Luer-Lock cap; MoBiTec (Cat. M105010S and Cat. M3009) [0125] SYBR™ Gold nucleic acid stain, concentrate in DMSO; ThermoScientific (Cat. S11494) [0126] SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9]; Biozol (Cat. GTX-GTX632604); manufacturer: Genetex; host: mouse; clone 1A9, isotype IgG1 [0127] SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody, Mouse MAb; Hölzel Diagnostika Handels GmbH (Cat. 40143-MM05-100) [0128] Mouse monoclonal Anti-Ig kappa chain antibody clone L1C1, raised against B lymphoma cells of human origins, SantaCruz Biotechnologies (Cat. sc-59265) [0129] Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488; ThermoScientific (Cat. #A-11029) [0130] Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, HRP; ThermoScientific (Cat. #A16078) [0131] 1-Step Ultra TMB-ELISA Substrate Solution, ThermoScientific (Cat. #34028) [0132] .Math.-slide angiogenesis glass bottom; Ibidi cat. 81507

DETAILED DESCRIPTION OF THE FIGURES:

[0133] FIG. 1 shows the specific enrichment of SARS-CoV-2 virus particles on the surface of Fc-ACE2 coated beads and instantaneous staining of bound virus particles with a nucleic acid dye as an experimental method and diagnostic tool.

[0134] The specific interaction between human ACE2 and the spikes of SARS-CoV-2 mediates viral binding to human cells and their subsequent infection. To test whether this interaction can be used for the enrichment of intact viruses on carrier materials, streptavidin-PMMA-Beads were coated with biotinylated Fc-ACE2. The specificity of such Fc-ACE2-coated beads was evaluated by incubating them with cell culture supernatants containing the indicated virus particles (adenovirus, influenza virus or SARS-CoV-2). Streptavidin-PMMA-Beads served as additional control for specificity. After incubation beads were washed to remove unbound material and fixed with PFA. Bound virus particles were fluorescently stained with the nucleic acid dye SYBR™ Gold. Fluorescence microscopic analysis was performed by confocal laser scanning microscopy and single confocal sections recorded with identical microscopy settings are shown. The transmitted light images serve to locate the beads. The beads shown in FIG. 1 are highly representative for other beads processed and analyzed under identical experimental conditions.

[0135] Binding of SARS-CoV-2 was not detected on empty beads (streptavidin-PMMA-beads) that served as negative control. Thus, SARS-CoV-2 enrichment on Fc-ACE2-coated beads was dependent on the presence of its specific binder, Fc-ACE2. While SARS-CoV-2 binding was very efficient yielding characteristic and bright fluorescent dots neither influenza virus nor adenovirus particles were detected on beads coated with Fc-ACE2. In conclusion, beads coated with Fc-ACE2 allowed for the highly specific enrichment of SARS-CoV-2 and can therefore serve as a diagnostic tool to detect the presence of intact and therefore potentially infectious virus particles. As shown here, an easy and rapid way to detect bound virus particles is to perform subsequent staining of viral genome with a nucleic acid dye. Due to electrostatic interactions with the bead surface SYBR™ Gold displays a very dim and unspecific background staining. As shown in FIG. 1, such background staining can be easily discerned from the characteristic punctate and very bright pattern of stained virus particles. The surface of the beads used as well as the buffer conditions can be further optimized to suppress unspecific staining and thereby strongly increase the signal-to-noise ratio.

[0136] In summary, FIG. 1 shows that SARS-CoV-2 virus particles could be specifically enriched on carrier materials like beads coated with a binding agent (here ACE2) that specifically bind to viral surface proteins. Bound virus particles were readily detected by instantaneous staining of viral genomes using SYBR™ Gold. Although images shown here have been recorded with confocal laser scanning microscope, analysis can be performed using more simple and standard fluorescent microscopes or even mobile, handheld devices to allow rapid point-of-care diagnostic. Fc-ACE2-coated beads thus allow the selective enrichment of SARS-CoV-2 viruses and form the basis for sensitive diagnostics, e.g. via staining of the viral genome using SYBR™ Gold (FIG. 1), via immunolabeling of the viruses (FIGS. 2, 3 and 4) or via nucleic acid-based diagnostics such as established PCR-based detection methods (FIG. 3).

[0137] FIG. 2 shows methods of immunolabelling of SARS-CoV-2 particles which have been specifically enriched on beads coated with Fc-ACE2.

[0138] In addition to staining the viral genome, immunolabeling of bound viruses presents an alternative method for detecting virus particles bound to a carrier material. The use of antibodies is expected to increase both the specificity and sensitivity of the virus detection. The feasibility of such a method was tested on beads on which viruses had been enriched.

[0139] The experiment shown in the upper panel makes use of streptavidin-PMMA-Beads beads or streptavidin-PMMA-Beads coated with recombinant biotinylated Fc-ACE2. Both types of beads were incubated with a cell culture supernatant containing SARS-CoV-2. After washing away unbound material beads were fixed with PFA. The described procedure resulted in two types of beads, streptavidin-PMMA-Beads without SARS-CoV-2 particles (Empty Beads) and Fc-ACE2 coated beads with bound SARS-CoV-2 (Beads with SARS-CoV-2). Bound SARS-CoV-2 particles were immunolabelled with commercially available monoclonal antibodies recognizing either the spike protein or the nucleocapsid protein of SARS-CoV-2. A third source of antibodies (polyclonal human antibodies) for immunolabelling was serum obtained from a patient who recovered from a previous infection with SARS-CoV-2 (convalescent serum). In the course of immunofluorescence, the beads covered with SARS-Cov-2 were incubated with the indicated antibodies against SARS-CoV-2 proteins. Fluorescently labelled polyclonal antibodies were used as secondary antibodies. Stained virus particles were visualized by confocal laser scanning microscopy and single confocal sections are shown. Importantly, the beads that were covered with SARS-CoV-2 particles were recorded with the same microscopy settings as the corresponding empty beads, which served as the negative controls. As beads lacking SARS-CoV-2 virus particles did not result in any detectable fluorescent staining, the various ways to immunolabel SARS-CoV-2 particles were considered highly specific.

[0140] The experiment demonstrates that readily available proteinaceous binders (here antibodies) against viral proteins can be used to specifically detect bound virus particles. Although resulting in an even brighter and more specific staining, the procedure is inherently not as rapid as a nucleic acid stain shown in FIG. 1. However, experimental protocols can be optimized for fast staining that can be completed in the range of minutes. Similarly, sensitivity of such assay can be improved further, for example by introducing additional fluorescent staining agents or combining different staining methods.

[0141] The lower panel shows maximum projections of recorded stacks of confocal sections (different planes in z). This visualization of microscopy data unveils the characteristic dot-like staining pattern of bound virus particles. The various methods used to detect bound virus particles yielded very similar results, demonstrating the robustness of workflows based on the fluorescent detection of virus particles.

[0142] FIG. 3 shows alternative methods of detection of SARS-CoV-2 which has been enriched on beads coated with Fc-ACE2.

[0143] To test alternative methods of labeling and detection, streptavidin magnetic beads coated with recombinant biotinylated Fc-ACE2 were incubated with ten-fold serial dilutions of a cell culture supernatant containing SARS-CoV-2. After washing away unbound material beads were either directly analyzed by qPCR or fixed with PFA. The graph qPCR Titration illustrates that beads coated with Fc-ACE2 enrich SARS-CoV-2 virus particles that can subsequently be detected and quantified by nucleic acid based-diagnostics such as established PCR-based detection methods.

[0144] In order to perform flow cytometry analysis Fc-ACE2 coated magnetic streptavidin beads with bound SARS-CoV-2 were fixed and virus particles were specifically immunolabelled with the commercially available monoclonal antibodies recognizing the nucleocapsid protein of SARS-CoV-2. Fluorescently labelled polyclonal antibodies were used as secondary antibodies and beads with bound virus particles were analyzed by flow cytometry. The graph shown relates measured values of fluorescence to the amount of virus particles bound to beads. This experiment demonstrates that classical flow cytometry or related methods represent a highly suitable method for detecting and quantifying virus particles bound to beads.

[0145] The use of enzymatic activities such as that of horseradish peroxidase, luciferase or alkaline phosphatase allows a targeted amplification of the signal for the highly sensitive detection of viruses bound to the carrier material. To test the applicability of such method, bound virus particles were specifically immunolabelled with a monoclonal antibody recognizing the nucleocapsid protein of SARS-CoV-2. The secondary antibodies were polyclonal antibodies coupled to horse radish peroxidase. The actual method of detection is based on an enzymatic reaction mediated by horse-radish peroxidase. The colour change after addition of the substrate TMB is a highly sensitive diagnostic feature and its intensity depends on the amount of bound SARS-CoV-2 virus particles. FIG. 3 shows that such colorimetric assay can be used for quantification either before or after stopping the reaction by addition of H2SO4.

[0146] In summary, FIG. 3 demonstrates three additional methods to detect virus particles specifically bound to beads. Besides being sensitive and specific, the presented methods are highly suitable for quantifying virus particles bound to beads over a wide range of concentrations.

[0147] FIG. 4 shows the detection of SARS-CoV-2 virus in patient sample and demonstrates that a microscopic approach is able to reliably enrich and detect single virus particles even from patient samples.

[0148] A nasopharyngeal swab taken from a diagnosed COVID19 patient was immersed in buffer to dissolve virus particles in buffered aqueous solution. The supernatant containing solubilized virus particles was transferred either on streptavidin-PMMA-Beads (empty beads) or streptavidin-PMMA-Beads coated with biotinylated Fc-ACE2 (ACE2 beads). Unbound material was washed away, beads were fixed with PFA and processed like in FIG. 2 by incubation with the monoclonal anti-nucleocapsid antibody followed by an incubation with fluorescently labelled secondary antibodies. Fluorescence was visualized by confocal laser scanning microscopy. Maximum projections of recorded stacks of confocal sections (different planes in z) are shown. Like in the lower panel of FIG. 2 this type of visualization of microscopy data unveils the characteristic dot-like staining pattern of bound virus particles.

[0149] Since beads are used to enrich for viruses, sensitivity largely depends on the sample volume and the actual amount of beads used in the assay. As demonstrated here, specific enrichment combined with subsequent staining of bound virus particles can be used to unequivocally diagnose a real sample taken from a diagnosed patient.