In vitro diagnostic method for detecting the presence of a target by using stabilized membrane proteins

Abstract

The present teachings relate to in vitro diagnostic methods, kits, membrane proteins, and test devices for detecting the presence and / or absence of a target in a biological sample, wherein the target binds to at least one epitope of an isolated membrane protein or to a fragment of the isolated membrane protein that includes at least one epitope of the isolated membrane protein that binds to the target.

Description

[Technical Field]

[0001] The present invention relates to novel methods and test devices for the use of isolated stabilized membrane proteins for the detection of biological targets, such as host cellular viral receptors, as detector molecules for the detection of viral infections. [Background technology]

[0002] The analysis of biological samples to determine their characteristics and obtain information about various biological targets is required in both biology and medicine.In particular, various methods can be used to analyze biological samples to detect the presence, absence, concentration, and / or spatial distribution of biological targets.For example, the detection of proteins in tissue sections or cytological preparations can be carried out using histochemistry, immunohistochemistry (IHC), or immunofluorescence.However, many of the existing techniques for detecting targets in biological samples have limitations in terms of sensitivity, accuracy, and / or multiplexing properties.

[0003] For example, clinical detection of viruses is usually achieved using any one of a variety of methods. For example, viral particles or nucleic acids can be isolated from biological samples (e.g., nasopharyngeal aspirates, throat swabs, blood fluids, fecal material, etc.). Retrospective diagnosis can be made by serology. While hemagglutination inhibition (HAI) and enzyme immunoassay (EIA) can also be used to provide type-specific diagnoses, complement fixation (CFT) is the most widely used method. For more rapid diagnosis, either antigen detection or RNA detection can be performed. Antigen detection can be performed by IFT or EIA, but to achieve the highest level of sensitivity and specificity, RNA detection by reverse transcriptase polymerase chain reaction (RT-PCR) is used. However, the latter is expensive and technically advanced.

[0004] For example, approximately two-thirds of HBV infections are asymptomatic, without clear clinical symptoms of hepatitis. Therefore, laboratory diagnosis is essential for reliable detection of HBV infection. In early HBV infection, HBV DNA (detectable by PCR) is the first positive marker of infection (although usually not until several weeks after exposure). Several weeks later, viral antigens appear as the first serological markers (HBsAg, HBcAg, HBeAg, detected by ELISA). Once symptoms of acute hepatitis B appear, virus-specific antibodies become detectable in immunocompetent individuals (anti-HB, anti-HBc, anti-HBe ELISA). Although antibodies can often only be detected some time after the onset of disease, PCR provides a high degree of diagnostic certainty.

[0005] The drawbacks of state-of-the-art testing methods are: (i) different components of the virus (viral proteins, viral genome) are detected; (ii) PCR methods are also time- and material-intensive; (iii) most detections are performed in laboratories and require at least one business day; and (iv) rapid tests often lack sensitivity and accuracy.

[0006] Therefore, it is an object of the present disclosure to provide an in vitro diagnostic method for detecting pathogenic viruses that exhibits high specificity and sensitivity and can be performed in a short time without using a laboratory. Summary of the Invention

[0007] The present disclosure relates to a novel method and test device for the use of isolated, stabilized membrane proteins for the detection of biological targets, such as viral receptors on host cells, as molecular detectors for the detection of viral infections. For example, viral binding to the receptor allows the virus to infect and thus represents a highly conserved and invariant process. Rapid tests based on this method are characterized by high specificity, sensitivity, and rapid performance without the need for a laboratory.

[0008] Methods for detecting a target in a biological sample according to the present disclosure generally include: (a) contacting the biological sample with an isolated membrane protein and a polymer and / or copolymer capable of solubilizing and stabilizing the membrane protein; and (b) detecting the formation of a target-protein complex in the biological sample comprising the target and the membrane protein or a fragment thereof.

[0009] In particular, the method of the present disclosure uses a viral receptor on the host cell as a detection agent. Virus binding to the receptor allows the virus to infect, and thus represents a highly conserved and invariant process. Rapid tests based on this method are characterized by high specificity, sensitivity, and rapid performance without the need for a laboratory.

[0010] Host cell receptors are often water-insoluble membrane proteins. To separate them from biological membranes and bring them into solution, modern solubilizing detergents (e.g., SDS, nonionic glucosides or maltosides, Triton X-100, CHAPS) are used. These detergents disrupt the lipid bilayer surrounding membrane proteins. However, without the surrounding lipid bilayer of biological membranes, protein unfolding often occurs, especially in human membrane proteins, leading to irreversible disruption of function. Specificity for viral binding is also lost in this process. For example, in combination with phospholipids, non-aromatic, detergent-free polymers (DIBMA = diisobutylene / maleic acid copolymer) maintain the native lipid bilayer into which membrane proteins are usually incorporated. Using DIBMA, the prior art has already shown that membrane proteins can be functionally and detergent-free solubilized and stabilized by encasing them in the polymer in so-called nanodiscs. Thus, DIBMA or a DIBMA variant can be used to stabilize host cell viral receptors in Nanodiscs so that viral binding specificity is maintained and used as receptors.

[0011] Thus, in a first aspect, the present disclosure relates to an in vitro diagnostic method for detecting the presence and / or absence of a target in a biological sample, said target binding to at least one epitope of an isolated membrane protein or at least a fragment of said isolated membrane protein comprising at least one epitope of said isolated membrane protein that binds to said target, The above method is (a) contacting the biological sample with the isolated membrane protein or fragment thereof, wherein the isolated membrane protein or fragment thereof is solubilized and stabilized by a polymer and / or copolymer, particularly in polymer and / or copolymer Nanodiscs; (b) detecting in the biological sample the formation of a target-protein complex comprising the target and the membrane protein or fragment thereof. Includes:

[0012] In a second aspect, the present disclosure relates to an in vitro diagnostic kit for detecting the presence and / or absence of a target in a biological sample, comprising: (a) at least one isolated membrane protein or a fragment thereof comprising at least one epitope that binds to a target, wherein said isolated membrane protein or fragment thereof is in the presence of a polymer and / or copolymer that can solubilize and stabilize said membrane protein, in particular wherein said membrane protein is contained in nanodiscs of the polymer and / or copolymer; (b) a reagent for detecting the formation of a target-protein complex between the target and the membrane protein or fragment thereof in the biological sample, wherein the isolated membrane protein or fragment thereof and the reagent are present in an amount sufficient to detect the formation of the target-protein complex.

[0013] In a third aspect, the present disclosure relates to a solubilized and stabilized isolated membrane protein, or fragment thereof, for use in the treatment of disease, particularly selected from the group consisting of viral-based diseases, malignant diseases, or chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic and extrinsic asthma; skin diseases including atopic dermatitis, polymorphous light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic intestinal disease, wherein said isolated membrane protein or fragment thereof is solubilized and stabilized by polymers and / or copolymers, particularly in polymer and copolymer Nanodiscs.

[0014] In a fourth aspect, the present disclosure relates to a test device for early and rapid detection of a target in a biological sample, said device comprising a test strip, said test strip comprising: at least one sample application site, in particular a sample pad; At least one test zone and one control zone Including, The test zone comprises an immobilized isolated membrane protein or fragment thereof, which is solubilized and stabilized by polymers and / or copolymers, particularly in polymer and copolymer nanodiscs.

[0015] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read in conjunction with the accompanying drawings, in which like features represent like parts throughout the drawings. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1 is a scheme showing that many pathogenic viruses use their integral membrane protein WNTCP in the membrane of hepatocytes with its natural function as a bile acid transporter (left), and is used by hepatitis B virus for binding and internalization via endocytosis as an import port (right). [Figure 2] FIG. 2 is a scheme of the test procedure in which the Hepatitis B virus (HBV) specifically binds to the membrane protein NTCP, which is functionally stabilized via a polymer and attached via a linker to, for example, a rapid test membrane. [Figure 3] FIG. 3 shows two designs of embodiments of a test device for rapid detection of targets according to the present disclosure. [Figure 4] Figure 4 shows a scheme of a typical surface plasmon resonance (SPR) assay, an optical-based, label-free detection technique for real-time monitoring of binding interactions between two or more molecules. [Figure 5] FIG. 5 is a scheme showing the kinetic profile of an analyte-target binding reaction. [Figure 6] FIG. 6 is a graph showing the kinetic data and equations for the SPR assay. [Figure 7] Figure 7 is a graph showing Covid measurements of LMNG and ACE2. [Figure 8] Figure 8 is a graph showing Covid measurements of DIBMA and ACE2. [Figure 9] Figure 9 is a graph showing Covid measurements of SMA and ACE2. [Figure 10] FIG. 10 shows a test strip for a standard lateral flow. [Figure 11] FIG. 11 shows A) a test device according to the present disclosure in which, in contrast to the standard device of FIG. 10, the primary antibody is changed to a stabilized membrane protein, and B) a test device according to the present invention in which, in contrast to the standard device of FIG. 10, the nanoparticle-labeled primary antibody is changed to a stabilized membrane protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure relates to an in vitro diagnostic method for detecting the presence and / or absence of a target in a biological sample, wherein said target binds to at least one epitope of an isolated membrane protein or at least a fragment of said isolated membrane protein comprising at least one epitope of said isolated membrane protein that binds to said target; The above method is (a) contacting the biological sample with the isolated membrane protein or fragment thereof, wherein the isolated membrane protein or fragment thereof is solubilized and stabilized by a polymer and / or copolymer, particularly in polymer and / or copolymer Nanodiscs; (b) detecting in the biological sample the formation of a target-protein complex comprising the target and the membrane protein or fragment thereof. Includes:

[0018] For example, virus-receptor interactions play a key regulatory role in viral host range, tissue tropism, and viral pathogenesis. Viruses utilize elegant strategies to bind to one or more receptors, overcome membrane protein barriers, gain entry, and gain access to necessary host cellular machinery. Viral attachment proteins can be viewed as "keys" that unlock host cells by interacting with "locks"—receptors—on the cell surface, and these lock-and-key interactions are crucial for viruses to successfully enter host cells. Many common themes have emerged in virus-receptor utilization within and across virus families, indicating that viruses often target specific classes of molecules to mediate these events. Common viral receptors include sialylated glycans, cell adhesion molecules such as members of the immunoglobulin superfamily and integrins, and phosphatidylserine receptors. Redundancy in receptor utilization suggests that viruses target specific receptors or "common locks" to exploit their cellular functions and also suggests evolutionary conservation.

[0019] Viral receptors are mostly integral membrane proteins, a specific class of proteins that insert in vivo into biological membranes and traverse their lipid bilayers. The surface of these proteins that naturally contacts the membrane (the transmembrane region) is particularly hydrophobic.

[0020] The manipulation of membrane proteins in aqueous solution is usually essential, as it is essential for their purification and for the study of their structure and function. This requires avoiding spontaneous aggregation of hydrophobic domains and maintaining a relatively non-polar environment around the transmembrane regions. The standard preparation of such proteins in aqueous solution involves micellar concentrations of detergents. The success of this process is based on the high affinity of transmembrane proteins for these amphiphilic and dispersing compounds. Nevertheless, this is a more complicated operation than for soluble proteins, especially due to the presence of stabilizing polymers.

[0021] These stabilizing polymers must be added to all solutions containing test proteins at concentrations above their critical micelle concentration (cmc). In addition to any cost issues imposed by detergent consumption, experiments are often complicated by the fact that membrane proteins are usually fragile and sensitive to their environment. For example, in the presence of excessive amounts of micelles, they can denature, while detergent failure generally leads to their precipitation.

[0022] Several patents, notably those cited in International Patent Publication Nos. 9,400,557; 115,505; EP-A-363 106; DE-A-3 527 139; JP-A-6,107,6500; U.S. Pat. Nos. 5,223,411; JP-A-0,227,0856, and JP-A-0,116,8653, describe the extraction, purification, and manipulation of membrane proteins in aqueous media, either dispersed in micellar systems or inserted into lipid bilayers.

[0023] Schafmeister et al., Science, 262, pp. 734-738, 1993, also describe the formation of complexes between membrane proteins and amphiphilic peptide polymers. The amphiphilic polymers involved are small polypeptides known as peptitergents, with a rigid structure (α-helix), one side of which is hydrophobic and the other is hydrophilic. Peptitergents maintain the solubility of bacteriorhodopsin. However, they are unsuccessful with porins, presumably because their rigidity limits their adaptability when confronted with various hydrophobic surfaces. The authors envision the use of peptitergents to promote the crystallization of membrane proteins.

[0024] Also, in the field of combinations between amphiphilic synthetic polymers and globular (water-soluble) proteins, reference is made to the work of F. Petit et al., Sci., 273, pp. 777-781, 1995, on modified amphiphilic polyacrylates with molecular weights between 150,000 and 200,0900. The aim of these studies was to examine the protein / polymer combinations (in particular gel formation, complexation kinetics and energetics) rather than to maintain the membrane proteins in dispersed solution.

[0025] As mentioned above, viral host cell receptors are water-insoluble membrane proteins. To isolate them from biological membranes and bring them into solution, modern solubilizing detergents (e.g., SDS, nonionic glucosides or maltosides, Triton X-100, CHAPS) are used. These detergents disrupt the lipid bilayer surrounding the membrane proteins. However, without the lipid bilayer surrounding the biological membrane, protein unfolding often occurs, especially in human membrane proteins, resulting in irreversible loss of function. The specificity of viral binding is also lost.

[0026] Surprisingly, it has been found that the use of polymers and / or copolymers capable of solubilizing and stabilizing membrane proteins and / or GPCRs, in particular in combination with isolated membrane proteins without any detergents, can be used in in vitro diagnostic methods for detecting the presence of targets in biological samples.

[0027] As mentioned above, the present disclosure also relates to an in vitro diagnostic kit for detecting the presence and / or absence of a target in a biological sample, comprising: (a) at least one isolated membrane protein or fragment thereof comprising at least one epitope that binds to a target, wherein the isolated membrane protein or fragment thereof is in the presence of a polymer and / or copolymer that is capable of solubilizing and stabilizing the membrane protein; (b) a reagent for detecting the formation of a target-protein complex between the target and the membrane protein or fragment thereof in the biological sample, wherein the isolated membrane protein or fragment thereof and the reagent are present in amounts sufficient to detect the formation of the target-protein complex.

[0028] The terms "polypeptide," "peptide," or "protein" are used interchangeably herein to designate a linear series of amino acid residues joined together by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The amino acid residues are preferably in the naturally occurring "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue so long as the desired functional property is retained by the polypeptide. Furthermore, amino acids include the 20 "standard" amino acids as well as modified and unusual amino acids.

[0029] In advantageous embodiments, membrane proteins according to the present disclosure, particularly water-insoluble membrane proteins, are isolated. When used in reference to a nucleic acid or protein, the term "isolated" refers to a nucleic acid sequence or protein that has been identified and separated from at least one contaminant (nucleic acid or protein, respectively) with which it is normally associated in its natural source.

[0030] The polymer used in this process can be a polymer, a homopolymer, or a copolymer, as defined in more detail below.

[0031] For example, the use of a non-aromatic, surfactant-free copolymer (DIBMA = diisobutylene / maleic acid copolymer) in combination with phospholipids was found to maintain the native lipid bilayer into which membrane proteins are normally incorporated.

[0032] Thus, membrane proteins can be functionally and detergent-free solubilized and stabilized by enveloping them with polymers in so-called Nanodiscs. Thus, it should be possible to use DIBMA or DIBMA variants to stabilize host cell viral receptors in Nanodiscs in a way that maintains virus-binding specificity and allows their use as captures in rapid assays.

[0033] The term "nanodisc" is well known in the art and is distinct from the nanodisc clathrates described herein. Nanodiscs are disc-shaped lipid bilayers encompassed by a protein scaffold. A specific exemplary protein scaffold is derived from the carboxy-terminal tail of apolipoprotein AI, an amphipathic α-helical protein separated by prolines (Bayburt, et al., 2004). Mixing lipid-free scaffold proteins with lipids results in self-assembled nanoparticles containing approximately 10 nm diameter lipid bilayers in which two copies of the scaffold protein are wrapped around the disk in an antiparallel fashion. The hydrophobic surfaces of the scaffold proteins function to isolate the hydrocarbon tails of the phospholipids from the solvent (Borhani, et al., 1997). The resulting particles are water-soluble and stable. Nanodiscs according to the present disclosure may be polymer-based lipid nanodiscs (see, e.g., U.S. Patent Publication No. 20190154698).

[0034] Detergents (e.g., SDS, n-octyl-β-d-glucopyranoside (OG), n-dodecyl-β-d-maltoside (DDM)) are widely used for solubilizing membrane proteins, although it is well known that different detergents have different weaknesses.

[0035] Short-chain non-ionic detergents, for example, can affect the functional properties of membrane proteins. It seems clear that removing the native lipid bilayer from a membrane protein can inhibit protein function. One method for mimicking natural lipid membranes is the use of MSP-nanodiscs and detergent-free polymer systems (styrene-maleic acid copolymer (SMA) (2) and diisobutylene-maleic acid (DIBMA)) (Oluwole, Abraham Olusegun, et al. "Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene / Maleic Acid Copolymer." Angewandte Chemie International Edition 56.7 (2017): 1919-1924.; Oluwole, Abraham Olusegun, et al. "Formation of lipid-bilayer nanodiscs by diisobutylene / maleic acid (DIBMA) copolymer." 33.50 (2017): 14378-14388). The latter allows for direct extraction of membrane proteins from cells without the intermediate step of detergent solubilization. The synthetic polymer must itself carry styrene or maleic groups in order to solubilize the protein.

[0036] As mentioned above, interaction with viral receptors is usually mediated by specific viral attachment proteins expressed on the surface of the virion (Marsh M., Helenius A. Virus entry: open sesame. Cell. 2006;124:729-740). The inherent differences in the shape (icosahedral or helical) and composition (enveloped or non-enveloped) of the viral coat affect the overall structure of the viral attachment protein. The attachment proteins of enveloped viruses are generally spike-like and extend from the surface of the virion, allowing the attachment protein to serve as the first point of contact with receptors on the cell membrane. Non-enveloped viruses can be naturally spherical without extensions, such as polyomaviruses, or decorated with viral proteins that extend from the virion surface, such as reoviruses. It seems rather clear that spike-like proteins are the first point of contact between the virus and the host cell, compared to viral capsid proteins, which are embedded in the surface of spherical viral capsids. However, although reoviruses possess spike-like proteins that bind to cellular receptors, additional receptor interactions exist that are mediated by components of the capsid. Furthermore, although the overall shape of the particle can influence the structure of the attachment protein and thereby the mechanism by which the virion binds to the cellular receptor, virus-receptor interactions have also been successfully modeled by pseudocoating virus particles with glycoproteins from unrelated viruses. Pseudotyping of virus particles has proven to be a powerful tool for analyzing virus-receptor interactions, tissue tropism, and immune function, particularly for highly pathogenic viruses such as human immunodeficiency virus (HIV) and Ebola virus (EBOV). Effective pseudotyping of virions that recapitulate the pattern of infectious native virions suggests that attachment protein structure and attachment protein-receptor stoichiometry are not essential for viruses to find and activate the appropriate receptor for infection.

[0037] The present disclosure further relates to an in vitro diagnostic method for detecting the presence of a target virus or a fragment thereof in a biological sample, wherein said virus or viral fragment comprises a viral attachment protein that binds to at least one epitope of a water-insoluble host cell membrane protein or at least a fragment of said membrane protein that comprises at least one epitope of said membrane protein that binds to the viral attachment protein; The above method is (a) contacting the biological sample with the isolated membrane protein or fragment thereof, wherein the isolated membrane protein or fragment thereof is contained in a DIBMA / lipid particle, particularly a styrene / maleic acid copolymer; (b) detecting in the biological sample the formation of a virus-protein complex comprising the target virus or a fragment thereof and the membrane protein. Includes.

[0038] Stabilization of membrane proteins is understood as the transition from the membrane environment to an aqueous solution. Preferably, the lipid environment of the membrane protein is not affected. The structure, binding properties, and function are essentially maintained. An example can be found in Anais Marconnet, Baptiste Michon, Christel Le Bon, Fabrice Giusti, Christophe Tribet, et al., "Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers." Biomacromolecules, American Chemical Society, 2020, 21, pp. 3459-3467. ff10.1021 / acs.biomac.0c00929ff. ffhal-03018338.

[0039] The phrase "stabilized membrane protein" refers to a membrane protein that has been treated, for example, to improve the heat resistance of the protein, or to cause the protein to retain activity (e.g., of a particular receptor) or maintain native conformation upon extraction from the membrane. Stabilizing a membrane protein with an amphiphile described herein can improve its T value by about 5°C, about 10°C, about 15°C, about 20°C, or about 25°C, for example, compared to a standard detergent such as DDM. Increasing the stability of an isolated protein is important to allow researchers sufficient time to test and characterize the protein.

[0040] Below is exemplified a general protocol for the purification of membrane proteins stabilized in copolymers (e.g., AASTY (a copolymer derived from styrene and acrylic acid), Ultrasolute Amphipol (polyacrylic acid partially bonded to amide functional groups by cycloalkylamines or cycloalkylalkylamines):

[0041] As explained above, solubilization, stabilization, and purification of membrane proteins from surrounding native membranes depend on many parameters. Most parameters can be optimized during the purification process for higher efficiency. These parameters include buffer conditions (e.g., salt, pH), choice of polymer, protein to solubilizer ratio, temperature, and time. First, cell lysis and centrifugation are performed, for example, by using the following parameters: add protease inhibitors (PIs) to the buffer, readjust the pH value, and then disrupt the cells (e.g., sonication, French press). Centrifuge at 9000 rcf for 30 minutes at 4°C, discard the pellet (cell debris), collect the supernatant, and centrifuge at 100 000 rcf for 1 hour at 4°C. After centrifugation, the supernatant is discarded and the pellet is homogenized. The membrane protein is then solubilized: the polymer forms synthetic nanodiscs around the protein, thereby maintaining the natural phospholipid environment and preserving the natural, and therefore functional, properties of the protein in a convenient one-step process (solubilization and stabilization). The surfactant, on the other hand, forms micelles around the hydrophobic belt, thus removing lipids from the surroundings. For the natural state, the unique lipid environment must be preserved.

[0042] In one embodiment, the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, SLC transporters, etc. As starting material for the method of the present invention, a solution of free polymer is used, which results from the solubilization, stabilization, and purification of the above membrane proteins from their natural surroundings by using the polymer.

[0043] In some advantageous embodiments, the membrane protein is a full-length membrane protein, particularly a water-insoluble membrane protein.

[0044] In some embodiments, the target detected by the methods / kits of the present disclosure comprises a portion or whole affinity substance that is an affinity moiety derived from an affinity substance selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypic antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions and derivatives, variants or combinations thereof.

[0045] In some advantageous embodiments, the methods / kits of the present disclosure are used for the diagnosis of diseases selected from the group consisting of viral-based diseases such as coronavirus disease 2019, malignant diseases, chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic asthma and extrinsic asthma; skin diseases including atopic dermatitis, polymorphous light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; and intestinal diseases including Crohn's disease and chronic intestinal disease.

[0046] Thus, the target to be detected may be a target virus or a fragment thereof in a biological sample, said virus or said viral fragment comprising a viral attachment protein that binds to at least one epitope of a membrane protein or at least a fragment of said membrane protein. In particular, the virus to be detected is a SARS virus, in particular SARS-CoV-2 or a variant thereof.

[0047] In some advantageous embodiments, the methods / kits of the present disclosure are used for the diagnosis of coronavirus diseases, such as coronavirus disease 2019 (COVID-19). In some further advantageous embodiments, the biological sample is derived from a human or animal, such as blood, urine, tissue, organ, saliva, hair, nail clippings, or any other cell- or bodily fluid-containing sample.

[0048] As noted above, the polymer can be a homopolymer or a copolymer. In one embodiment, the polymer contains hydrophilic groups, such as COOH, maleimide, OH, amines, ammonium salts, zwitterions such as phosphocholine, and hydrophobic groups, such as polymerized styrene groups, polymerized diisobutylene groups, or linear C-C 16 Aliphatic groups (such as methyl and ethyl) and branched C1-C 16 Aliphatic groups (such as isopropyl or t-butyl), and cyclic C5-C 12 It may have an aliphatic or aromatic group.

[0049] The molecular weight of the polymer used in the method of the present invention may be 1900 to 20000, for example, 2000 to 18000, or 2000 to 15000, or 4000 to 16000, or 4000 to 13000, or 5000 to 14000. The molecular weight may be measured by gel permeation chromatography or mass spectrometry.

[0050] Examples of polymers include, but are not limited to, styrene / maleic acid copolymers sold under the trade name "SMA," derivatives of styrene / maleic acid copolymers such as SMA 200 and 300, and styrene / maleimide copolymers such as SMA 502. These materials can also be functionalized on the COOH group with amines such as ethanolamine or ethylenediamine to form amides, or with alcohols such as glycerol to form esters. Polymers can also be functionalized with polyethylene glycol to form esters, or with aminated polyethylene glycol to form amides.

[0051] The polymer can be a diisobutylidene / maleic acid copolymer, such as DIBMA 10 and DIBMA 12 from Cube Biotech, or a derivative of a diisobutylidene / maleic acid copolymer, such as DIBMA Gly (a diisobutylidene / maleic acid copolymer partially modified with 1-aminoglycerol), DIBMA Glu (a diisobutylidene / maleic acid copolymer partially modified with glucosamine), Glyco DIBMA (a diisobutylidene / maleic acid copolymer partially modified with N-methyl-D-glucamine), or a diisobutylidene / maleimide copolymer. DIBMA copolymers can be functionalized with the same molecules as SMA.

[0052] Furthermore, the polymer may be a copolymer derived from styrene and acrylic acid, in particular having a molecular weight between 5,500 and 11,000 and an acrylic acid / styrene ratio of 45% / 55% to 55% / 45%, such as sold under the name "AASTY".

[0053] Modified polymers derived from polyacrylic acid can be used, in which 10-90% of the carboxylic acid groups can be modified to amides with cyclooctylamine, 2-cyclohexyl-ethylamine, etc. These substances are sold under the name "Amphipol Ultrasolve".

[0054] In addition to the polymer disclosure above, further polymer descriptions are provided below.

[0055] Polymers with hydrophilic and hydrophobic functional groups Examples of hydrophilic groups can be, but are not limited to, polymers of acrylic and methacrylic acid, maleic acid, carboxylic acid groups, amides with α,ω-alkylenediamines in general, ω-hydroxyalkylamines and ω-aminoalkylthiols, trimethylammonio-alkylamines, amides of carboxylic acid groups with aminoglycerol, TRIS, or Bis-Tris, amides with maltosamine, glucosamine, mannosamine, and other amino-functionalized carbohydrates, or taurine.

[0056] Mention may also be made of esters of carboxylic acid groups with polyethylene glycols, diols, triols, polyols, and carbohydrates.

[0057] Another example may be a maleimide, which has the nitrogen atom functionalized with an alkyl chain bearing an alcohol, thiol, amine, ammonium salt, or the like.

[0058] Alternatively, a zwitterionic molecule consisting of an ammonium group and a phosphate group can be linked to a carboxylic acid group, as described in U.S. Patent Publication Nos. 2020281855 or 2021171673.

[0059] Examples of hydrophobic groups include, but are not limited to, polymerized styrene and derivatives such as methylstyrene, diisobutylene, and linear and branched alkenes such as 2-propyl, hexyl, octyl, decyl, linked to carboxylic acid groups via ester or amide functionalities. Also suitable are maleimide groups bearing alkyl or aryl groups on the amino functional group.

[0060] An example of the synthesis of styrene-maleic acid copolymers can be found in Shintaro Sugai, Nobumichi Ohno, Conformational transitions of the hydrophobic polyacids, Bio-physical Chemistry, Volume 11, Issues 3-4, June 1980, Pages 387-395.

[0061] The use of SMA to construct complexes with lipids is described in International Patent Publication No. WO 2006 / 129127 and references therein. SMA can be purchased commercially from Orbiscope or Cube Biotech as SMALP 140, SMALP 200, or SMALP 300.

[0062] The synthesis of copolymers from diisobutylene and maleic anhydride is described by BASF in U.S. Patent No. 4,250,289. The hydrolysis of anhydride copolymers to diisobutylene-co-maleic acid is described by Lee, Nature Protocols Vol. 11, No. 7, 2016, pp. 1149-1162, which is described for SMA but can be applied to DIBMA without issue.

[0063] The synthesis of a DIBMA polymer with glucosamine functionalization of 50% of all carboxyl groups can be found in Bartholomaus Danielczak, Marie Rasche, Julia Lenz, Eugenio Perez Patallo, Sophie Weyrauch, Flori-an Mahler, Michael Tope Agbadaola, Annette Meister, Jonathan Oyebamiji Babalola, Carolyn Vargas, Cenek Kolar and Sandro Keller, A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs, DOI: 10.1039 / D1NR03811G (Paper) Nanoscale, 2022, 14, 1855-1867. DIBMA is available commercially from Cube Biotech as DIBMA 10 and DIBMA 12.

[0064] The preparation of poly(acrylic acid-co-styrene) copolymers is described in WO 2020 257637 and Simon Harrisson*, Francesca Ercole and Benjamin W. Muir, “Living spontaneous gradient copolymers of acrylic acid and styrene: one-pot synthesis of pH-responsive amphiphiles,” Polym. Chem., 2010, 1, 326-332.

[0065] In some cases, the copolymer is a copolymer derived from styrene and acrylic acid, or a copolymer derived from styrene and an acrylic acid derivative.All copolymer derivatives can be used in the copolymer of interest.Examples of derivatives include acrylate, methacrylate, acrylic acid ester, acrylamide, and N-substituted acrylamide.In certain cases, the acrylic acid ester or acrylamide is substituted with a zwitterionic species, as described in U.S. Patent Application No. 20190062469 (the disclosure of which is incorporated herein by reference).

[0066] In certain embodiments, the copolymer comprises an acrylic acid or acrylic acid derivative content of 30% to 70%, 35% to 65%, or 40% to 60%.

[0067] The synthesis of Amphipol Ultrasolute, a polyacrylic acid polymer partially modified with cycloalkylamines or cycloalkylalkylamines, is described in WO 115083 and Marconnet, A., Michon, B., Le Bon, C., Giusti, F., Tribet, C., & Zoonens, M. (2020). Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules. doi:10.1021 / acs.biomac.0c00929.

[0068] Additional polyacrylates modified with alkyl groups such as pentyl, hexyl, and tert-butyl, and their use in conjugation with membrane proteins, are described in U.S. Patent Publication No. 2020 / 0383918.

[0069] A polymethacrylate containing about 0.52 butyl methacrylate (BMA) in the copolymer and about 0.48 methylacryloxycholine (MAC) in the copolymer, with a degree of polymerization (DP) of about 39.00, is distributed by Avanti Polar Lipids under the brand name Polymethacrylate Copolymer (N-C4-52-6.9). Other polymethacrylates are described in Yasuhara K, Arakida J, Ravula T, Ramadugu SK, Sahoo B, Kikuchi JI, Ramamoorthy A. 2017. Spontaneous Lipid Nanodisc Formation by Amphiphilic Polymethacrylate Copolymers. J Am Chem Soc. 139(51):18657-18663.

[0070] Polyacrylate polymers modified with alkanes such as n-butyl, t-butyl, pentyl, neopentyl, and hexyl are described in Nathaniel Z. Hardin, Thirupathi Ravula, Giacomo Di Mauro, Ayyalusamy Ramamoorthy, Hydrophobic Functionalization of Polyacrylic Acid as a Versatile Platform for the Development of Polymer Lipid Nanodiscs, Small. 2019 March; 15(9): e1804813. doi:10.1002 / smll.201804813, and U.S. Patent Publication No. 2020383918.

[0071] Alternatively, linear hydrocarbons functionalized with hydrophobic groups and having a degree of polymerization of less than 100 are mentioned in U.S. Patent Publication No. 2022 093587. An example of a linear carbohydrate is inulin, and examples of the hydrophobic group are alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalkyl having 1 to 3 heteroatoms. The hydrophobic group is attached to the carbohydrate via an ether group, an ester group, or an amide group.

[0072] The present disclosure further relates to a solubilized and stabilized isolated membrane protein or fragment thereof for use in the treatment of disease, particularly a disease selected from the group consisting of viral-based diseases, malignant diseases, or chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic and extrinsic asthma; skin diseases including atopic dermatitis, polymorphous light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic intestinal disease, wherein said isolated membrane protein or fragment thereof is solubilized and stabilized by a polymer and / or copolymer, particularly in polymer and / or copolymer Nanodiscs.

[0073] The interaction of the copolymer-stabilized membrane protein and its interaction partner can be detected through, but not limited to, different analytical methods.

[0074] Examples of optical detection include SPR (surface plasmon resonance), RM (resonant mirror), GCI (grating coupled interferometry), ELISA (enzyme-linked immunosorbent assay) as direct ELISA, sandwich ELISA, competitive ELISA, or reverse ELISA, and LFA (lateral flow assay).

[0075] Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive dielectric constant materials in a particle stimulated by incident light. SPR is the basis of many standard tools for measuring the adsorption of substances on planar metal (usually gold or silver) surfaces or on the surfaces of metal nanoparticles. It is the fundamental principle behind many color-based biosensor applications and lab-on-a-chip sensors.

[0076] An excellent overview of this technique, as well as RM, dual polarization interferometry, and other methods, is provided in "Hikmat N. Daghestani and Billy W. Day; Theory and Applications of Surface Plasmon Resonance, Resonant Mirror, Resonant Waveguide Grating, and Dual Polarization Interferometry Biosensors; Sensors 2010, 10, 9630-9646; doi:10.3390 / s101109630."

[0077] An ELISA assay uses at least one antibody specific for a particular antigen. A sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either nonspecifically (via adsorption to a surface) or specifically (via capture by another antibody specific for the same antigen, in a "sandwich ELISA"). After the antigen is immobilized, a detection antibody is added, forming a complex with the antigen. The detection antibody may be covalently linked to an enzyme or may itself be detected by a secondary antibody linked to an enzyme via bioconjugation. Between each step, the plate is usually washed with a mild detergent solution to remove any nonspecifically bound proteins or antibodies. After the final wash step, the plate is developed by adding an enzymatic substrate to generate a visible signal representing the quality of the antigen in the sample (Wikipedia).

[0078] The principle of lateral flow assays is described below, where binding is indicated by a visual signal that can be generated by almost any dye, but mainly gold nanoparticles or fluorescently or magnetically labeled particles.

[0079] Alternatively, binding can be detected by calorimetry, such as ITC (isothermal titration calorimetry). Isothermal titration calorimetry (ITC) is an analytical technique that is a titration method for analyzing intermolecular interactions by calorimetry. The titration is performed at constant pressure and temperature, meaning that a single ITC experiment provides data on the enthalpy of binding, the equilibrium binding constant, and the stoichiometry, from which the entropy and Gibbs energy of binding can be calculated. Thus, a single ITC experiment provides direct access to the important thermodynamic potentials associated with the interaction process—Gibbs energy, enthalpy, and entropy.

[0080] Test Equipment As mentioned above, the present disclosure relates to a test device for the early and rapid detection of a target in a biological sample, said device including a test strip, said test strip comprising: at least one sample application site, in particular a sample pad; at least one test zone and one control zone, said test zone comprising: At least one test zone and one control zone comprising an immobilized isolated membrane protein, or fragment thereof, said isolated membrane protein, or fragment thereof, solubilized and stabilized by polymers and / or copolymers, in particular in nanodiscs of polymers and copolymers. Includes.

[0081] Thus, the present disclosure improves and simplifies diagnostic assays known in the art. A simple and rapid diagnostic testing system is provided. A test device according to the present disclosure may include a housing containing a test strip, the test strip including at least one sample application site (e.g., a sample pad), at least one test zone (test line), and one control zone (control line). The housing may include an opening and / or a transparent material. The opening and / or transparent material of the housing preferably facilitates receipt of the test sample and reading of results from the test zone and control zone.

[0082] The preferred transparent material of the housing allows for reading of the results from the test zone and control of the test strip. The transparent material of the housing may also have an enclosure that can be opened at the time of reading. The housing may also be made entirely of a transparent material. Preferred materials for the test strip are known in the art and include nitrocellulose membranes, absorbent cellulose pads, blood filters, or wicks. The test strip may further include a backing layer, such as a polyvinyl backing layer. The test strip materials, such as the nitrocellulose membrane and / or absorbent pad, may be assembled onto and connected to the backing layer by an adhesive, preferably a pressure-sensitive adhesive. In a preferred embodiment, the sample application site is a sample pad or a sample wick.

[0083] The test strip of the device according to the present disclosure may include test zones containing target antibodies (primary antibodies), which are preferably immobilized in each zone.

[0084] There is a preferred device according to the present disclosure, in which the test strip further comprises a zone containing a protein conjugate between the sample application site and the test zone. The protein conjugate may be a monoclonal or polyclonal antibody that binds to a target. The protein conjugate in the zone is preferably released when a liquid, such as a biological sample, flows through the zone. In a preferred embodiment, the protein conjugate in the protein conjugate-containing zone comprises a gold-protein conjugate. A device containing a gold-protein conjugate is preferred.

[0085] Furthermore, it is preferred that the test strip comprises a zone comprising said protein-conjugates, wherein the protein-conjugates comprise respective gold-protein conjugates, i.e., gold conjugates of anti-target antibodies that bind to the target and / or immobilized isolated membrane protein-gold conjugates, wherein the isolated membrane protein or fragments thereof are solubilized and stabilized by polymers and / or copolymers, in particular in polymer and / or copolymer Nanodiscs.

[0086] In a preferred embodiment, the protein-conjugate containing zone is a fiberglass gold release pad, which releases the gold protein-conjugate as a liquid, eg, a sample, flows through it.

[0087] It is further preferred that the control zone of each of the two test strips contains a non-specific capture antibody, preferably immobilized. A preferred non-specific capture antibody is an antibody that non-specifically captures the conjugated protein, and is an anti-mouse antibody when a mouse clone conjugate is used, or an anti-rabbit antibody when a rabbit clone is used. Another preferred capture antibody is an anti-(anti-human immunoglobulin) control antibody. Furthermore, when a mouse anti-human immunoglobulin colloidal gold conjugate is used, the control antibody is an anti-mouse immunoglobulin. Preferred embodiments of the test strip of the device of the present invention are shown in Figures 3 and 11.

[0088] A preferred test strip is A backing layer; a sample pad as a sample application site; a gold conjugate pad containing a gold-protein conjugate; a capture line (test line) as a test zone, said test zone comprising an immobilized isolated membrane protein or a fragment thereof that binds to said target, said isolated membrane protein or fragment thereof being solubilized and stabilized by polymers and / or copolymers, in particular in nanodiscs of polymers and copolymers; Control lines as control zones (The material of the test strip in the area of ​​the test zone and control zone is nitrocellulose membrane, The remaining materials of the test strip are an absorbent pad and a blood filter. Pressure-sensitive adhesive for assembling / attaching the sample pad, nitrocellulose membrane, blood filter, and absorbent pad to the backing layer Includes.

[0089] Another preferred first test strip comprises: A backing layer; a sample pad as a sample application site; The protein-conjugate-containing zone (conjugate pad) contains a primary antibody and / or an immobilized isolated membrane protein or fragment thereof that binds to said target, said isolated membrane protein or fragment thereof being solubilized and stabilized by polymers and / or copolymers, in particular in polymer and copolymer nanodiscs. a test zone (test line) comprising an immobilized isolated membrane protein or fragment thereof (said isolated membrane protein or fragment thereof is solubilized by a polymer and / or copolymer, in particular in nanodiscs of polymers and copolymers) or a primary antibody; a control line with immobilized nonspecific secondary antibody as a control zone; (The material of the test strip in the area of ​​the test zone and control zone is nitrocellulose membrane, The remaining materials of the test strip are an absorbent pad and a blood filter. Pressure-sensitive adhesive for assembling / attaching the sample pad, nitrocellulose membrane, and absorbent pad to the backing layer Includes:

[0090] In a preferred embodiment, the gold conjugates in the zone (conjugate pad) are modified with a compound selected from the group comprising chitosan, oligochitosan, glucosamine, polylysine, or other polymers or mixtures thereof.

[0091] In an advantageous embodiment, the test device comprises: a) a conjugate pad comprising a primary antibody against a target labeled with nanoparticles, and a test zone comprising an immobilized isolated membrane protein or fragment thereof that binds to the target, the isolated membrane protein or fragment thereof being solubilized and stabilized by polymers and / or copolymers, in particular in nanodiscs of the polymers and copolymers; or b) the conjugate pad comprises an immobilized isolated membrane protein or a fragment thereof that binds to a target, the isolated membrane protein or fragment thereof being solubilized and stabilized by a polymer and / or copolymer, particularly in nanodiscs of polymers and copolymers, and the test zone comprises a primary antibody against the target; It is defined as follows.

[0092] The effect of any of these compounds or mixtures thereof on the color intensity of colloidal gold is related to their addition during colloidal gold preparation but before conjugation with proteins, i.e., antibodies or antigens. Colloidal gold is conjugated with specific antibodies and / or antigens after modification with chitosan (and / or other modifiers). Chitosan and other modifiers affect the color intensity of colloidal gold, enhancing the color-discriminating properties of the human eye and thereby enabling detection at very low concentrations. Signal amplification ranges up to 10-fold. Colloidal gold is preferably prepared by reduction of a 1% aqueous solution of tetrachloroauric acid (HAuCl4) using aqueous trisodium citrate to produce spherical gold particles. After colloidal gold preparation, an aqueous solution of chitosan (or any other modifier or mixture) is added at the appropriate volume and concentration to convert the color from purple to violet, depending on the volume and concentration of the added modifier solution.

[0093] Therefore, the present disclosure also relates to a novel rapid test method and test device that can directly detect viruses from the first day of infection within 15 minutes. Many pathogenic viruses use integral membrane proteins of their host cells as receptors for attachment to cells and subsequent uptake and replication within the cells. Some of the membrane proteins on the surface of the host cell in question are thus "exploited" in their function as viral receptors, thus determining the characteristics of the virus to infect and replicate in specific types of cells. Although viruses themselves are highly diverse, binding to host cell receptors is a highly conserved and invariant process.

[0094] Thus, stabilized and solubilized host cell membrane proteins are incorporated into the rapid test, thereby acting as highly specific scavengers against pathogenic viruses, thereby constructing an innovative lateral flow rapid test. HBV virus, for example, attaches itself to human hepatocytes via the membrane protein NTCP (= sodium taurocholate cotransporting polypeptide or bile acid transporter) anchored in the host cell membrane, where it multiplies and thus induces hepatitis disease (Figure 1).

[0095] The present invention will now be further described by reference to the following examples and figures, which should not be construed as limiting the invention thereto.

[0096] Methods and Examples It should be understood that the following examples are for illustrative purposes only and are not to be construed as limiting the present disclosure in any manner. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0097] Example 1: Purification of membrane proteins stabilized in copolymers (e.g., poly(acrylic acid-co-styrene), i.e., AASTY, Ultrasolute Amphipol) The solubilization, stabilization, and purification of membrane proteins from the surrounding native membrane depend on many parameters, most of which can be optimized during the purification process for higher efficiency. These parameters include: buffer conditions (salt, pH, etc.), the selected polymer, the ratio of protein to solubilizing agent, temperature, and time.

[0098] Cell lysis and centrifugation Add protease inhibitors (PI) to the buffer, readjust the pH, then disrupt the cells (e.g., sonicate, French press). Centrifuge at 9,000 rcf for 30 minutes at 4°C, discard the pellet (cell debris), and collect the supernatant. Centrifuge the supernatant at 100,000 rcf for 1 hour at 4°C, discard the supernatant, and homogenize the pellet.

[0099] Membrane protein solubilization The polymer forms synthetic nanodiscs around the protein, thereby maintaining the natural phospholipid environment and preserving the natural and therefore functional properties of the protein in a convenient one-step process (solubilization and stabilization). The surfactant, on the other hand, forms micelles around the hydrophobic belt, thus removing lipids from the surroundings. For the natural state, the intrinsic lipid environment must be preserved.

[0100] If the solubilization efficiency is low, it is recommended to screen parameter variations to improve the yield of total solubilized protein. A standard protocol is described below: A solubilizing agent is added to the protein solution. The ideal concentration may vary, a good starting point is: 0.5%-5% SMA / DIBMA 0.1-2.5% UltraSolute Amphipol AASTY 0.1-2.5%. Solubilize at 4°C for 3 to 24 hours with stirring. Higher temperatures can be screened for optimization. Centrifuge at 100,000 rcf for 1 hour at 4°C. The pellet is discarded and the supernatant is collected. The solubilized membrane proteins in the polymer nanodiscs (supernatant) are used for affinity chromatography.

[0101] Example 2: Affinity Chromatography Polymers can interfere with protein binding to the binding matrix, so reducing the polymer concentration is advantageous. A polymer concentration of 0.25% is essential for sufficient binding. Further reduction of the polymer to 0.025%-0.050% can be advantageous for improving binding efficiency. Purify the protein of choice via a preferred protocol depending on the affinity tag.

[0102] Example 3: Diagnostics with copolymer-supported membrane proteins Protocol for a standard ELISA assay using copolymer-solubilized proteins The copolymer-stabilized membrane protein of choice must be in a purified state (several days at 4°C). The day before the ELISA assay, a sterile 96-well plate should be coated with protein (a concentration of 500 ng / well to 1 μg / well is a good starting point for optimization). Dispense 100 μl of protein solution into each well, excluding possible controls such as a blank or a coating consisting of 1% BSA in TBS to visualize any possible nonspecific interactions.

[0103] Furthermore, it is recommended that duplicate measurements be made for each condition tested.

[0104] The plate is covered with parafilm or a plastic lid to prevent evaporation or drying and left at 4° C. overnight.

[0105] The next day, a washing buffer is prepared by dissolving 0.05% Tween 20 in TBS buffer [50 mM Tris, 100 mM NaCl; pH 7.0]. The coated plate is washed three times (300 μl per well): twice with TBS containing Treen 20 and once with TBS without additional detergent. Between buffer changes, the plate is allowed to stand for approximately 5 minutes each time.

[0106] After washing, 300 μl of 3% milk powder in TBS or 3% BSA in TBS is added to each well and incubated at room temperature for 1 hour to block the plate. The choice between milk powder and BSA depends on the coating protein, ligand, and antibody utilized in the assay.

[0107] After this blocking step, the washing steps described above are repeated in the same manner.

[0108] Dilute the selected ligand, which can be a protein, antibody, etc., to an optimal concentration. Serial dilutions using the same buffer in which the protein was stored are recommended.

[0109] 100 μl of each dilution of ligand is dispensed into the corresponding wells and incubated for 1 hour at room temperature.

[0110] The washing step is repeated once more after this incubation.

[0111] After these washing steps, 100 μl of the first antibody targeted to the ligand is added to each well and incubated for 1 hour at room temperature.

[0112] The dilution should be chosen based on the manufacturer's suggestions. If this information is not available, a dilution of 1:1000 using 3% milk powder or BSA is recommended.

[0113] The incubation of the first antibody can be omitted if a conjugate of two antibodies is used in the assay.

[0114] Once again, the same washing step is carried out, followed by incubation again with 100 μl of the second antibody for 1 hour at room temperature, where the antibody is always diluted 1:1000 using 3% milk powder in TBS.

[0115] After the second antibody incubation, the plates are washed again, this time twice with 0.05% Tween 20 in TBS buffer, twice with plain TBS, and a final washing step with MiliQ water (300 μl per well for each washing step).

[0116] The visualization / development buffer should be as fresh as possible, so it is recommended to mix the components for the solution immediately after the last water wash step. For the visualization / development buffer, mix 9 mL of MilliQ water, 1 mL of 1 M sodium acetate (pH 6.0, adjusted with 1 mM citric acid), 62.5 μL of 3,3',5,5'-tetramethylbenzidine (TMB, 10 mg / mL in DMSO), and 3.5% HO and use immediately by dispensing 100 μL of this solution into each well.

[0117] The incubation time for the visualization / development buffer is flexible but should be at least 10 minutes and less than 90 minutes, however, the shorter the incubation time, the more visible the differences between different samples.

[0118] The enzymatic reaction is stopped by adding 100 μL of 10% sulfuric acid. Mixing of the solution in the wells can be increased by gently tapping the plate frame. After 5 minutes, results can be obtained by measuring the plate at 450 nm using a standard photometer.

[0119] Example 4: Protocol for SPR assay using copolymer-solubilized membrane protein (COV spike protein) Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative and positive dielectric constant materials in a particle stimulated by incident light (Figure 4). SPR is the basis of many standard tools for measuring the adsorption of substances on planar metal (usually gold or silver) surfaces or on the surfaces of metal nanoparticles. It is the fundamental principle behind many color-based biosensor applications and lab-on-a-chip sensors. It should be emphasized that SPR is not a planar resonance; it is a polariton or surface wave-like phenomenon.

[0120] SPR mechanism of action Optical methods for measuring changes in the mass of biomolecules. Biomolecules of interest are bound to a metal film within the flow chamber. The analyte is directed through a flow chamber. Upon binding of the analyte, the refractive index of the metal film changes. Change in the angle of reflection of light "Surface plasmon resonance phenomenon" The more coupled, the larger the angular offset. More signals are measured

[0121] How is SPR used? The protein is bound to one channel of the gold plate. For example, EDC / NHS coupling The ligand / protein partner is prepared in increasing concentrations. The ligand is then directed into both channels to allow binding. Binding is detected by an increased signal. After a defined time, ligand-free buffer is also introduced and dissociation is measured. Dissociation is detected by a decreasing signal (Figure 5).

[0122] How are kd and ka values ​​determined? The signal depends mainly on the following factors: Coupling constant (ka) Dissociation constant (kd) Amount of bound ligand Analyte concentration. Using known ligand concentrations and concentrations of several different analytes, k a and k d can be calculated (FIG. 6). Example of a suitable equation*:

number

[0123] Example 5: Measurements in COVID and ACE The ACE receptor ectodomain is fused to the chip. Sars-CoV-2 spike protein stabilized with either LMNG, DIBMA, or SMA is washed on the ACE-fused chip. Binding and dissociation capacities are measured at different concentrations: SPR Biosensor SR7500DC(Reichert) Chip: Indigo Chip (Cube Biotech GmbH https: / / cube-biotech.com / ), prepared by chemical modification of a dextran chip (SCR 200R-DCM5, Xantec GmbH, Dusseldorf, Germany) according to the method from Example 7 of WO 2020 / 109162. Wash the chip with ACE2 buffer until the baseline is stable. Immobilize the ACE2 ectodomain in the left channel of the chip (approximately 600 RU). 60μg / mL, flow rate 0.5ml / min Wash the chip with ACE2 buffer until the baseline is stable. Exchange the buffer for COVID buffer and wash both channels until the baseline is stable. Analyte: COVID spike protein in DIBMA Prepare COVID sample concentrations: 0 nM; 0,488 nM; 0,96 nM; 1,9 nM; 3,9 nM; 7,8 nM; 15,6 nM; 31,25 nM; 72,5 nM; 125 nM; 250 nM; 500 nM Start measurement 25μL / min Bonding time: 6 minutes Dissociation time: 11 minutes The experiment is referenced twice: an empty channel without ACE2 and an injection of running buffer (buffer blank)

[0124] The results of the measurements are shown in Figures 7, 8 and 9.

[0125] Overview and Comparison The results in Table 1 are shown below: COVID in LMNG showed weaker dissociation (lowest kd value) than samples solubilized in polymer. COVID in LMNG and COVID in SMA show similar degrees of binding, and therefore COVID in SMA is active. COVI in DIMBA showed the weakest binding (lowest ka value). COVID in SMA showed the strongest dissociation (highest kd value)

[0126] [Table 1]

[0127] This experiment shows that COVID in SMA successfully stabilizes the spike protein in a conformation that can bind the ACE receptor on the chip.

[0128] Advantages of membrane proteins in polymers / copolymers / nanodiscs:

[0129] When SPR is performed with two full-length membrane proteins, both proteins cannot be stabilized in detergent because washing with detergent buffers can result in permanent dissociation of the coated Protein A from the chip. The copolymer stabilizes the membrane proteins in their native lipid environment without the need to add the copolymer to every buffer.

Claims

1. An in vitro diagnostic method for detecting the presence and / or absence of a target in a biological sample, wherein said target binds to at least one epitope of an isolated membrane protein or to a fragment of said isolated membrane protein comprising at least one epitope of said isolated membrane protein that binds to said target, (a) contacting the biological sample with the isolated membrane protein, or fragment thereof, wherein the isolated membrane protein, or fragment thereof, is solubilized and stabilized by a polymer and / or copolymer, particularly in polymer and / or copolymer Nanodiscs; (b) detecting in said biological sample the formation of a target-protein complex comprising said target and said membrane protein, or a fragment thereof; A method comprising:

2. 2. The method of claim 1, wherein the membrane protein is selected from the group consisting of membrane receptor proteins such as G protein-coupled receptors (GPCRs), membrane enzymes, cell adhesion proteins, and transporter proteins such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, and SLC transporters.

3. The method according to any one of claims 1 to 2, wherein the membrane protein is a full-length membrane protein.

4. The method according to any one of claims 1 to 3, wherein the membrane protein is a water-insoluble membrane protein.

5. 5. The method of any one of claims 1 to 4, wherein the polymer / copolymer that solubilizes and stabilizes the membrane protein has hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions like phosphocholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, t-butyl) and cyclic (C5-C12) aliphatic or aromatic groups.

6. The method of any one of claims 1 to 5, wherein the molecular weight of the polymer / copolymer is from 1,900 to 20,000.

7. 7. The method according to any one of claims 1 to 6, wherein the polymer / copolymer is selected from the group consisting of diisobutylene / maleic acid copolymers, styrene-maleic acid copolymers, (acrylic acid-co-styrene) copolymers and polyacrylic acids, in particular styrene / maleic acid copolymers, partially bonded to cyclic alkylamines or cycloalkylalkylamines.

8. The method of any one of claims 1 to 7, wherein the membrane protein or fragment thereof is comprised in a polymer / lipid particle, such as a DIBMA / lipid particle.

9. The method of claim 8, wherein the DIBMA in the DIBMA / lipid particles is DIBMA modified with glucosamine and / or aminoglycerol.

10. 10. The method of any one of claims 1 to 9, wherein the target comprises an affinity substance in its entirety or in part which is an affinity moiety derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptidic allergen, a recombinant allergen, an allergen-idiotype antibody, an autoimmune inducing structure, a tissue rejection inducing structure, an immunoglobulin constant region, and derivatives, variants or combinations thereof.

11. 11. The method of any one of claims 1 to 10, used for the diagnosis of a disease selected from the group consisting of viral-based diseases such as coronavirus disease 2019, malignant diseases, chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic asthma and extrinsic asthma; skin diseases including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; and intestinal diseases including Crohn's disease and chronic intestinal disease.

12. 12. The method of any one of claims 1 to 11, wherein the target is a target virus or a fragment thereof in a biological sample, and the virus or viral fragment comprises a viral attachment protein that binds to at least one epitope of the membrane protein or to at least a fragment of the membrane protein.

13. 13. The method of claim 12, wherein the virus is a SARS virus, in particular SARS-CoV-2 or a variant thereof.

14. 14. The method of any one of claims 1 to 13, used for the diagnosis of a coronavirus disease, such as coronavirus disease 2019 (COVID-19).

15. 15. The method of any one of claims 1 to 14, wherein the biological sample is derived from a human or animal, such as blood, urine, tissue, organ, saliva, hair, nail clippings, or any other cell or body fluid containing sample.

16. In vitro diagnostic kit for detecting the presence and / or absence of a target in a biological sample: (a) at least one isolated membrane protein, or a fragment thereof, comprising at least one epitope that binds to a target, in the presence of a polymer and / or copolymer that can solubilize and stabilize said membrane protein, in particular wherein said membrane protein is comprised in nanodiscs of the polymer and / or copolymer; (b) a reagent for detecting the formation of a target-protein complex between the target and the membrane protein, or a fragment thereof, in the biological sample, wherein the isolated membrane protein, or a fragment thereof, and the reagent are present in an amount sufficient to detect the formation of the target-protein complex.

17. 17. The kit of claim 16, wherein the membrane protein is selected from the group consisting of membrane receptor proteins such as G protein-coupled receptors (GPCRs), membrane enzymes, cell adhesion proteins, and transporter proteins such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, and SLC transporters.

18. The kit according to any one of claims 16 to 17, wherein the membrane protein is a full-length membrane protein.

19. The kit according to any one of claims 16 to 18, wherein the membrane protein is a water-insoluble membrane protein.

20. The kit according to any one of claims 16 to 19, wherein the polymer / copolymer capable of solubilizing and stabilizing the membrane protein and / or GPCR has hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions like phosphocholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, t-butyl) and cyclic (C5-C12) aliphatic or aromatic groups.

21. The kit according to any one of claims 16 to 20, wherein the molecular weight of the polymer / copolymer is from 1,900 to 20,000.

22. 22. The kit according to any one of claims 16 to 21, wherein the polymer / copolymer is selected from the group consisting of diisobutylene / maleic acid copolymer, styrene / maleic acid copolymer, (acrylic acid-co-styrene) copolymer and amphipol, in particular styrene / maleic acid copolymer.

23. The kit of any one of claims 16 to 22, wherein the membrane protein or fragment thereof is comprised in a polymer / lipid particle, such as a DIBMA / lipid particle.

24. 24. The kit of claim 23, wherein the DIBMA in the DIBMA / lipid particles is DIBMA modified with glucosamine and / or aminoglycerol.

25. 25. The kit of any one of claims 16 to 24, wherein the target comprises an affinity substance in its entirety or in part which is an affinity moiety derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptidic allergen, a recombinant allergen, an allergen-idiotype antibody, an autoimmune inducing structure, a tissue rejection inducing structure, an immunoglobulin constant region, and derivatives, variants or combinations thereof.

26. 26. The kit of any one of claims 16 to 25, wherein the method is used for the diagnosis of a disease selected from the group consisting of viral-based diseases such as coronavirus disease 2019, malignant diseases, chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic asthma and extrinsic asthma; skin diseases including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; and intestinal diseases including Crohn's disease and chronic intestinal disease.

27. 27. The kit of any one of claims 16 to 26, wherein the target is a target virus or a fragment thereof in a biological sample, and the virus or viral fragment comprises a viral attachment protein that binds to at least one epitope of the membrane protein or to at least a fragment of the membrane protein.

28. 28. The kit of claim 27, wherein the virus is a SARS virus, in particular SARS-CoV-2 or a variant thereof.

29. 29. The kit of any one of claims 16 to 28, wherein the kit is used for the diagnosis of a coronavirus disease, such as coronavirus disease 2019 (COVID-19), and in particular for membranes.

30. 30. The kit of any one of claims 16 to 29, wherein the biological sample is derived from a human or animal, such as blood, urine, tissue, organ, saliva, hair, nail clippings, or any other cell- or body fluid-containing sample.

31. 1. A solubilized and stabilized isolated membrane protein, or fragment thereof, for use in the treatment of disease, in particular selected from the group consisting of viral-based diseases, malignant diseases, or chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic and extrinsic asthma; skin diseases including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic bowel disease, wherein the solubilized and stabilized isolated membrane protein, or fragment thereof, is solubilized and stabilized by polymers and / or copolymers, in particular in polymer and / or copolymer Nanodiscs.

32. 32. The membrane protein for use according to claim 31 , wherein the membrane protein is selected from the group consisting of membrane receptor proteins such as G protein-coupled receptors (GPCRs), membrane enzymes, cell adhesion proteins, and transporter proteins such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases and SLC transporters.

33. The membrane protein for use according to any one of claims 31 to 32, wherein the membrane protein is a full-length membrane protein.

34. The membrane protein for use according to any one of claims 31 to 33, wherein the membrane protein is a water-insoluble membrane protein.

35. The membrane protein for use according to any one of claims 31 to 31, wherein the membrane protein is a full-length membrane protein.

36. The membrane protein for use according to any one of claims 31 to 35, wherein the membrane protein is a water-insoluble membrane protein.

37. 37. The membrane protein for use according to any one of claims 31 to 36, wherein the polymer / copolymer capable of solubilizing and stabilizing the membrane protein and / or GPCR has hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions like phosphocholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, t-butyl) and cyclic (C5-C12) aliphatic or aromatic groups.

38. A membrane protein for use according to any one of claims 31 to 37, wherein the molecular weight of the polymer / copolymer is between 1,900 and 20,000.

39. 39. The membrane protein for use according to any one of claims 31 to 38, wherein the polymer / copolymer is selected from the group consisting of diisobutylene / maleic acid copolymer, styrene-maleic acid copolymer, (acrylic acid-co-styrene) copolymer and amphipol, in particular styrene / maleic acid copolymer.

40. 40. The membrane protein for use according to any one of claims 31 to 39, wherein the membrane protein or fragment thereof is comprised in a polymer / lipid particle, such as a DIBMA / lipid particle.

41. 41. The membrane protein for use according to claim 40, wherein the DIBMA in the DIBMA / lipid particle is glucosamine and / or glycerol DIBMA.

42. 42. A membrane protein for use according to any one of claims 31 to 41, wherein the target comprises an affinity substance in its entirety or in part which is an affinity moiety derived from an affinity substance selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotype antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions and derivatives, variants or combinations thereof.

43. 43. The membrane protein for use according to any one of claims 31 to 42, wherein the complex is used for the treatment of a disease selected from the group consisting of viral-based diseases such as coronavirus disease 2019, malignant diseases, chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic asthma and extrinsic asthma; skin diseases including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic intestinal disease.

44. 44. The membrane protein for use according to any one of claims 31 to 43, wherein the complex is used for the treatment of a coronavirus disease, such as coronavirus disease 2019 (COVID-19).

45. 1. A test device for the early and rapid detection of a target in a biological sample, comprising a test strip, said test strip comprising: at least one sample application site, in particular a sample pad; at least one test zone and one control zone; and At least one zone as a conjugate pad containing a gold-protein conjugate. Including, the test zone and / or conjugate pad comprises an immobilized isolated membrane protein, or fragment thereof, which is solubilized and stabilized by a polymer and / or copolymer, particularly in nanodiscs of the polymer and copolymer; Test equipment.

46. 46. ​​The test device of claim 45, wherein the conjugate pad is located between the sample application site and the test zone, and in particular the protein conjugate is modified with a compound selected from chitosan, oligochitosan, glucosamine, and polylysine.

47. a) the conjugate pad comprises a primary antibody against a target labeled with nanoparticles and the test zone comprises an immobilized isolated membrane protein, or a fragment thereof, that binds to the target, the isolated membrane protein, or a fragment thereof, being solubilized and stabilized by polymers and / or copolymers, in particular in nanodiscs of polymers and copolymers, or b) the conjugate pad comprises an immobilized isolated membrane protein or a fragment thereof that binds to the target, the isolated membrane protein or fragment thereof being solubilized and stabilized by a polymer and / or copolymer, in particular in nanodiscs of polymers and copolymers, and the test zone comprises a primary antibody against the target, A test device according to any one of claims 45 to 46.

48. the test strip comprises a zone between the sample application site and the test zone containing a gold-protein conjugate, the protein in the gold-protein conjugate being an antibody against the target and / or an immobilized isolated membrane protein, or a fragment thereof, which binds to the target, and the isolated membrane protein, or a fragment thereof, is solubilized and stabilized by a polymer and / or a copolymer, in particular in nanodiscs of polymers and copolymers; A test device according to any one of claims 45 to 47.

49. A test device according to any one of claims 45 to 48, wherein the test strip further comprises an absorbent pad.

50. 50. A test device according to any one of claims 45 to 49, wherein the test strip comprises a nitrocellulose membrane.

51. 51. The test device according to any one of claims 45 to 50, wherein the protein conjugates in the conjugate pad are an HIV-1 antigen gold conjugate and a SARS virus, particularly SARS-CoV-2 or a variant thereof antibody gold conjugate.

52. A test device according to any one of claims 45 to 51, wherein the control zone comprises an immobilised non-specific capture antibody, such as an anti-gold control antibody.

53. 53. A test device according to any one of claims 45 to 52, wherein the membrane protein is a full-length membrane protein.

54. 54. The test device according to any one of claims 45 to 53, wherein the membrane protein is a water-insoluble membrane protein.

55. 55. The test device of any one of claims 45 to 54, wherein the polymer / copolymer that solubilizes and stabilizes the membrane protein has hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions like phosphocholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, t-butyl) and cyclic (C5-C12) aliphatic or aromatic groups.

56. A test device according to any one of claims 45 to 55, wherein the molecular weight of the polymer / copolymer is between 1900 and 20,000.

57. 57. A test device according to any one of claims 45 to 56, wherein the polymer / copolymer is selected from the group consisting of diisobutylene / maleic acid copolymer, styrene-maleic acid copolymer, (acrylic acid-co-styrene) copolymer and polyacrylic acid, in particular styrene / maleic acid copolymer, partially bonded to a cyclic alkylamine or cycloalkylalkylamine.

58. 58. A test device according to any one of claims 45 to 57, wherein the membrane protein or fragment thereof is comprised in a polymer / lipid particle, such as a DIBMA / lipid particle.

59. 59. The test device of claim 58, wherein the DIBMA in the DIBMA / lipid particles is glucosamine- and / or aminoglycerol-modified DIBMA.

60. 60. A test device according to any one of claims 45 to 59, wherein the target comprises an affinity substance in its entirety or in part which is an affinity moiety derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptidic allergen, a recombinant allergen, an allergen-idiotype antibody, an autoimmune inducing structure, a tissue rejection inducing structure, an immunoglobulin constant region and derivatives, variants or combinations thereof.

61. 61. A test device according to any one of claims 45 to 60, wherein the method is used for the diagnosis of a disease selected from the group consisting of viral based diseases such as coronavirus disease 2019, malignant diseases, chronic inflammatory diseases such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic asthma and extrinsic asthma; skin diseases including atopic dermatitis, polymorphous light eruption, SLE; autoimmune diseases including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic bowel disease.

62. 61. A test device according to any one of claims 45 to 60, wherein the target is a target virus or a fragment thereof in a biological sample, and the virus or a fragment thereof comprises a viral attachment protein that binds to at least one epitope of the membrane protein or to at least a fragment of the membrane protein.

63. A test device according to any one of claims 45 to 62, wherein the virus is a SARS virus, in particular SARS-CoV-2 or a variant thereof.

64. 64. A test device according to any one of claims 45 to 63, wherein the method is used for the diagnosis of a coronavirus disease, such as coronavirus disease 2019 (COVID-19).

65. 65. A test device according to any one of claims 45 to 64, wherein the biological sample is of human or animal origin, such as blood, urine, tissue, organ, saliva, hair, nail clippings, or any other cell or body fluid containing sample.

66. 66. The test device of any one of claims 45 to 65, wherein the membrane protein is selected from the group consisting of membrane receptor proteins such as G protein-coupled receptors (GPCRs), membrane enzymes, cell adhesion proteins, and transporter proteins such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases and SLC transporters.

Images