In vitro diagnostic method for detecting presence of target by using stabilized membrane protein

Abstract

The present technology relates 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 at least a fragment of the isolated membrane protein comprising at least one epitope of the isolated membrane protein bound to the target.

Description

[0001] Description of the Invention

[0002] The present invention relates to a novel method and test device for detecting biological targets (such as viral receptors of host cells) using isolated stabilized membrane proteins as detection molecules for detecting viral infections. Background Art

[0003] Both biology and medicine require the analysis of biological samples to understand their characteristics and obtain information about various biological targets. A variety of methods can be used to analyze biological samples to detect the presence, absence, concentration and / or spatial distribution of biological targets. For example, histochemistry, immunohistochemistry (IHC) or immunofluorescence can be used to detect proteins in histological sections or cytological preparations. However, many existing technologies for detecting targets in biological samples have limitations in sensitivity, accuracy and / or multiplexing capabilities.

[0004] For example, clinical detection of viruses is usually done using 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, fecal materials, etc.). Retrospective diagnosis can be performed by serology. Although hemagglutination inhibition (HAI) and enzyme immunoassay (EIA) can be used to make specific types of diagnoses, complement fixation test (CFT) is the most widely used in this method. For faster diagnosis, antigen detection or RNA detection can be performed. Antigen detection can be performed by IFT or EIA, however, in order to achieve the highest level of sensitivity and specificity, RNA detection by reverse transcriptase polymerase chain reaction (RT-PCR) is required. However, the latter is expensive and technically demanding.

[0005] For example, approximately 2 / 3 of HBV infections are asymptomatic, with no overt clinical manifestations of hepatitis. Therefore, laboratory diagnosis is essential for reliably detecting HBV infection. In the early stages of HBV infection, HBV DNA (detected by PCR) is the first positive marker of infection (but usually only a few weeks after exposure). A few weeks later, viral antigens then serve as the first serological markers (HBsAg, HBcAg, HBeAg, detected by ELISA). With the onset of symptoms of acute hepatitis B, virus-specific antibodies can be detected in immunocompetent individuals (anti-HBs, anti-HBc, anti-HBe ELISA). Although antibodies are usually only detectable sometime after the onset of the disease, PCR provides a high degree of diagnostic certainty.

[0006] The disadvantages of existing detection methods are: (i) The viral components detected are different (viral proteins, viral genome). (ii) PCR is also time-consuming and material-intensive. (iii) Most tests are performed in laboratories and require at least one working day, i.e. the test results are available the next day at the earliest. (iv) Rapid tests are usually not sensitive and accurate enough.

[0007] Therefore, an object of the present disclosure is to provide an in vitro diagnostic method for detecting pathogen viruses, wherein the method shows high specificity, sensitivity and can be completed in a short time without the need for a laboratory. Summary of the invention

[0008] The present disclosure relates to novel methods and test devices for using isolated stabilized membrane proteins to detect biological targets (such as viral receptors of host cells) as detection molecules for detecting viral infections. For example, the binding of viruses to receptors enables the virus to infect, and therefore represents a highly conservative and invariant process. Rapid tests based on this method have the characteristics of high specificity, high sensitivity, rapid operation and no need for laboratories.

[0009] According to the present disclosure, the method for detecting a target in a biological sample generally comprises: (a) contacting the biological sample with an isolated membrane protein and with a polymer and / or copolymer that can solubilize and stabilize the membrane protein; and (b) detecting the formation of a target-protein complex comprising the target and the membrane protein or the fragment thereof in the biological sample.

[0010] In particular, the method disclosed herein uses the viral receptors of the host cell as detection agents. The binding of the virus to the receptor enables the virus to infect, and therefore represents a highly conserved and invariant process. Rapid tests based on this method have the characteristics of high specificity, high sensitivity, rapid operation and no laboratory required.

[0011] Typically, host cell receptors are water-insoluble membrane proteins. In order to separate them from the biological membrane and dissolve them, the most advanced solubilizing detergents (such as SDS, non-ionic glucoside or maltoside, Triton X-100, CHAPS) are used. Using these detergents, the lipid bilayer around the membrane protein is destroyed. However, without the lipid bilayer around the biological membrane, the protein will usually unfold, especially in human membrane proteins, resulting in irreversible functional damage. In this process, the specificity of binding to the virus will also be lost. For example, a non-aromatic, detergent-free polymer (DIBMA=diisobutylene / maleic acid copolymer) is used, which is combined with phospholipids to maintain the natural lipid bilayer in which the membrane protein is usually incorporated. For DIBMA, it has been demonstrated in the prior art that by wrapping the membrane protein in the so-called nanodisc with a polymer, the membrane protein can be functionally solubilized and stabilized without the need for detergent. Therefore, using DIBMA or a DIBMA variant, the host cell virus receptor in the nanodisc can be stabilized, and the specificity of binding to the virus is maintained in this way and used as.

[0012] 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, wherein the target binds to at least one epitope of an isolated membrane protein, or at least to a fragment of the isolated membrane protein comprising at least one epitope of the isolated membrane protein that binds to the target, wherein the method comprises:

[0013] (a) contacting the biological sample with the isolated membrane protein or the fragment thereof, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc;

[0014] (b) detecting the formation of a target-protein complex comprising the target and the membrane protein or the fragment thereof in the biological sample.

[0015] 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:

[0016] (a) at least one isolated membrane protein or fragment thereof, comprising at least one epitope binding to a target, wherein the isolated membrane protein or the fragment thereof is in the presence of a polymer and / or copolymer capable of solubilizing and stabilizing the membrane protein, in particular, the membrane protein is comprised in a polymer and / or copolymer nanodisc; and

[0017] (b) a reagent for detecting the formation of a target-protein complex between the target and the membrane protein or the fragment thereof in the biological sample, wherein the isolated membrane protein or the fragment thereof and the reagent are present in an amount sufficient to detect the formation of the target-protein complex.

[0018] In a third aspect, the present disclosure relates to a solubilized and stabilized isolated membrane protein or fragment thereof for use in treating a disease, in particular for use in treating a disease selected from the group consisting of: a viral-based disease, a malignant disease, or a chronic inflammatory disease 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic bowel disease, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc.

[0019] In a fourth aspect, the present disclosure relates to a test device for early and rapid detection of a target in a biological sample, wherein the device comprises a test strip, wherein the test strip comprises:

[0020] - at least one sample application location, in particular a sample pad;

[0021] - at least one test zone and one control zone, wherein the test zone comprises an immobilized isolated membrane protein or a fragment thereof, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by polymers and / or copolymers, in particular in polymer and / or copolymer nanodisks. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features, aspects and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout the several views, and in which:

[0023] Figure 1 is a schematic diagram showing that many pathogenic viruses utilize their integral membrane protein WNTCP in the hepatocyte membrane, which naturally functions as a bile acid transporter (left) and is used by the hepatitis B virus as a portal for binding and internalization via endocytosis (right).

[0024] Figure 2 is a schematic diagram of a test procedure, wherein the hepatitis B virus (HBV) specifically binds to the membrane protein NTCP, which is functionally stabilized via a polymer and bound via a linker to, for example, a rapid test membrane.

[0025] Figure 3Two designs are shown as embodiments of a test device for rapid detection of a target according to the present disclosure.

[0026] Figure 4 Schematic diagram of a typical surface plasmon resonance (SPR) assay, which is an optical-based, label-free detection technology used to monitor the binding interactions between two or more molecules in real time.

[0027] Figure 5 is a schematic diagram showing the kinetic curve of the analyte-target binding reaction.

[0028] Figure 6 It is a graph showing the kinetic data and formula of the SPR measurement.

[0029] Figure 7 is a graph showing the measurement results of Covid and ACE2 using LMNG.

[0030] Figure 8 Graph showing the results of measuring Covid and ACE2 using DIBMA.

[0031] Fig. 9 is a graph showing the measurement results of Covid and ACE2 in SMA.

[0032] Fig.10 A standard lateral flow test strip is shown.

[0033] Fig.11 A) shows a test device according to the present disclosure, wherein Fig.10 The difference between the standard device and the first antibody is that the first antibody is changed to a stabilized membrane protein; and B) a test device according to the present disclosure, wherein Fig.10 Unlike the standard setup, the primary antibody labeled with the nanoparticles is altered to target the stabilized membrane protein. DETAILED DESCRIPTION

[0034] 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 the target binds to at least one epitope of an isolated membrane protein, or at least to a fragment of the isolated membrane protein comprising at least one epitope of the isolated membrane protein that binds to the target, wherein the method comprises:

[0035] (a) contacting the biological sample with the isolated membrane protein or the fragment thereof, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc;

[0036] (b) detecting the formation of a target-protein complex comprising the target and the membrane protein or the fragment thereof in the biological sample.

[0037] For example, virus-receptor interactions play a key regulatory role in viral host range, tissue tropism, and viral pathogenesis. Viruses use sophisticated strategies to attach to one or more receptors, overcome the plasma membrane barrier, enter and access essential host cell machinery. Viral attachment proteins can be viewed as the "keys" that unlock host cells by interacting with "locks" on the cell surface - receptors, and the interactions of these locks and keys are essential for the successful invasion of host cells by viruses. Many common themes emerge in virus-receptor utilization within and across viral 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. The redundancy of receptor utilization suggests that viruses target specific receptors or "common locks" to exploit their cellular functions, and also indicates evolutionary conservation.

[0038] Virus receptors are mainly integral membrane proteins, which are a specific class of proteins that insert into biological membranes in vivo and penetrate their lipid bilayer. The surface of these proteins that naturally contacts the membrane (the transmembrane region) is particularly hydrophobic.

[0039] The operation of membrane proteins in aqueous solutions is usually a prerequisite for their purification and their structural and functional studies. It is necessary to avoid spontaneous aggregation of hydrophobic domains and maintain a relatively nonpolar environment around the transmembrane region. The standard preparation of such proteins in a water-soluble state contains surfactants at micellar concentrations. The success of this process is based on the high affinity of the transmembrane protein region for these amphipathic and dispersive compounds. However, this is a more complicated operation than the case of soluble proteins, particularly due to the presence of stabilizing polymers.

[0040] These stabilizing polymers must be added to all solutions containing the test protein at a concentration above its critical micelle concentration (cmc). In addition to any cost issues caused by the consumption of surfactant, experiments are often made difficult by the fact that membrane proteins are often fragile and sensitive to their environment. For example, in the presence of excess micelles, they may denature, while surfactant deficiencies often cause them to precipitate.

[0041] Several patents, among which WO-A-9,400,557; WO-A-9,115,505; EP-A-363106; DE-A-3527 139; JP-A-6,107,6500; U.S. Pat. No. 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. These proteins are either dispersed in micellar systems or inserted in lipid bilayers.

[0042] Schafmeister et al. Science, 262, pp. 734-738, 1993 also describes the formation of complexes between membrane proteins and amphiphilic peptide polymers. The amphiphilic polymers involved are small polypeptides called peptide detergents, which have a rigid structure (α-helix) in which one face is hydrophobic and the other face is hydrophilic. Peptide detergents maintain the solubility of bacteriorhodopsin. However, they were unsuccessful in the case of porins, undoubtedly because their rigidity limits their adaptability to various hydrophobic surfaces. The authors envisioned using peptide detergents to promote the crystallization of membrane proteins.

[0043] In the field of combinations of amphiphilic synthetic polymers and globular (water-soluble) proteins, mention will also be made of the following studies: F. Petit et al. Sci., 273, pp. 777-781, 1995 on modified amphiphilic polyacrylates with molecular weights between 150,000 and 200,000. The aim of these studies was to study protein / polymer combinations (particularly gel formation, complex dynamics and energetics) rather than to maintain membrane proteins as dispersed solutions.

[0044] As mentioned above, viral host cell receptors are water-insoluble membrane proteins. In order to separate them from the biological membrane and dissolve them, the most advanced solubilizing detergents (such as SDS, non-ionic glucoside or maltoside, TritonX-100, CHAPS) are used. Using these detergents, the lipid bilayer around the membrane protein is destroyed. However, without the lipid bilayer around the biological membrane, the protein will usually unfold, especially in human membrane proteins, resulting in irreversible functional damage. The specificity of virus binding will also be lost.

[0045] Surprisingly, it was found that the use of polymers and / or copolymers capable of solubilizing and stabilizing membrane proteins and / or GPCRs in combination with isolated membrane proteins, in particular in the absence of any detergents, can be used in an in vitro diagnostic method for detecting the presence of a target in a biological sample.

[0046] 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:

[0047] (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 the fragment thereof is in the presence of a polymer and / or copolymer that solubilizes and stabilizes the membrane protein, and

[0048] (b) a reagent for detecting the formation of a target-protein complex between the target and the membrane protein or the fragment thereof in the biological sample, wherein the isolated membrane protein or the fragment thereof and the reagent are present in an amount sufficient to detect the formation of the target-protein complex.

[0049] The terms "polypeptide", "peptide" or "protein" are used interchangeably herein to designate a linear series of amino acid residues interconnected by peptide bonds between the alpha 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 may replace any L-amino acid residue, provided that the polypeptide retains the desired functional properties. In addition, amino acids include modified and unusual amino acids in addition to the 20 "standard" amino acids.

[0050] In an advantageous embodiment, a membrane protein according to the present disclosure, in particular a water-insoluble membrane protein, is isolated. When used in relation to a nucleic acid or protein, the term "isolated" refers to a nucleic acid sequence or protein that is identified and separated from at least one contaminant (respectively a nucleic acid or protein) with which it is normally associated in its natural source.

[0051] The polymer used in the process may be a polymer, a homopolymer or a copolymer, as will be described in more detail below.

[0052] For example, the use of a non-aromatic, detergent-free copolymer (DIBMA = diisobutylene / maleic acid copolymer) was found to maintain the native lipid bilayer in which membrane proteins are normally incorporated, in combination with phospholipids.

[0053] Thus, by encapsulating membrane proteins with polymers in so-called nanodiscs, membrane proteins can be functionally solubilized and stabilized without detergents. Thus, using DIBMA or DIBMA variants, it should be possible to stabilize host cell virus receptors in nanodiscs, in this way maintaining specificity for binding to viruses and being used as capture in rapid tests.

[0054] The term "nanodisc" is well known in the art and is different from the nanodisc clathrate described herein. A nanodisc is a disc-shaped lipid bilayer surrounded by a protein scaffold. Certain exemplary protein scaffolds are derived from the carboxyl terminal tail of apolipoprotein AI and are amphipathic α-helical proteins interrupted by proline (Bayburt, et al., 2004). A mixture of lipid-free scaffold protein and lipids produces self-assembled nanoparticles comprising a lipid bilayer of about 10 nm in diameter, and two copies of the scaffold protein are wrapped around the periphery of the disk in an antiparallel manner. The hydrophobic face of the scaffold protein is used to isolate the hydrocarbon tail of the phospholipid from the solvent (Borhani, et al., 1997). The resulting particles are water-soluble and stable. The nanodisc according to the present disclosure can be a polymer-based lipid nanodisc (see, for example, US20190154698A1).

[0055] Detergents such as SDS, n-octyl-β- d -glucopyranoside (OG), n-dodecyl-β- d -maltoside (DDM) are widely used for membrane protein solubilization, although it is well known that different detergents have different weaknesses.

[0056] For example, short-chain nonionic detergents can affect the functional properties of membrane proteins. Obviously, removing the natural lipid bilayer from membrane proteins can interfere with the function of proteins. One way to simulate natural lipid membranes is MSP-nanodiscs or detergent-free polymer systems (styrene-maleic acid copolymer (SMA) (2), 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). Using the latter, you can extract membrane proteins directly from cells without the intermediate step of detergent solubilization. The synthetic polymer itself must carry styrene or maleic acid groups to solubilize the protein.

[0057] As mentioned above, the 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 (envelope or no envelope) of the viral shell affect the overall structure of the viral attachment protein. The attachment protein of enveloped viruses is usually spike-shaped, extending from the surface of the virion, making the attachment protein the first contact point with the receptor on the plasma membrane. Non-enveloped viruses can be essentially spherical without extension, such as polyoma viruses, or modified with viral proteins extending from the surface of the virion, such as reoviruses. Compared with the viral capsid proteins embedded in the surface of the spherical virus capsid, it seems quite obvious that the spike protein will be the first contact point between the virus and the host cell. However, although reovirus has a spike protein that engages with cell receptors, there are also additional receptor interactions mediated by the capsid component. Additionally, while the overall shape of the particle can affect the attachment protein structure and the mechanism by which the virion engages the cellular receptor, virus-receptor interactions have also been successfully modeled by pseudocoating the virion with glycoproteins from unrelated viruses. Pseudotyped virions have proven to be a powerful tool for functional analysis of virus-receptor interactions, tissue tropism, and immunity, particularly for human immunodeficiency virus (HIV) and highly pathogenic viruses such as Ebola virus (EBOV). Effectively pseudotyped virions, recapitulating the pattern of infectious natural virions, indicate that attachment protein structure and attachment protein-receptor stoichiometry are not always critical for viral localization and activation of the appropriate infection receptor.

[0058] The present disclosure also relates to an in vitro diagnostic method for detecting the presence of a target virus or a fragment thereof in a biological sample, wherein the virus or the 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 to a fragment of the membrane protein that comprises at least one epitope of the membrane protein to which the viral attachment protein binds, wherein the method comprises:

[0059] (a) contacting the biological sample with an isolated membrane protein or a fragment thereof, wherein the isolated membrane protein or a fragment thereof is included in a DIBMA / lipid particle or in particular in a styrene / maleic acid copolymer;

[0060] (b) detecting the formation of a virus-protein complex comprising the target virus or the fragment thereof and the membrane protein or the fragment thereof in a biological sample.

[0061] Stabilization of membrane proteins is understood as a transfer from the membrane environment to an aqueous solution. Preferably, the lipid environment of the membrane protein is not affected. The structure, binding properties and functions are substantially retained. An example can be found in: Marconnet,Baptiste Michon,Christel Le Bon,Fabrice Giusti,Christophe Tribet,etal..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.

[0062] The phrase "stabilized membrane protein" refers to a membrane protein that has been treated so that the protein is thermally stable, or so that the protein retains activity (e.g., activity of a specific receptor), or maintains a native conformation, e.g., when extracted from a membrane. For example, stabilizing a membrane protein with an amphiphilic molecule as described herein can increase its T50 value by about 5°C, about 10°C, about 15°C, about 20°C, or about 25°C compared to a standard detergent such as DDM. Increasing the stabilization of an isolated protein is very important for allowing researchers sufficient time to examine and characterize the protein.

[0063] In the following, a general scheme for the purification of membrane proteins stabilized in copolymers such as AASTY (copolymer from styrene and acrylic acid), Ultrasolute Amphipol (polyacrylic acid, partially coupled to amide functional groups via cycloalkylamines or cycloalkylalkylamines) is used as an example:

[0064] As explained above, the solubilization, stabilization and purification of membrane proteins from natural membrane environments depend on many parameters. Most parameters can be optimized during the purification process to improve efficiency. These parameters include buffer conditions (e.g., salt, pH), the selection of polymers, the ratio of protein to solubilizing agent, temperature and time. First, cell lysis and centrifugation are performed by, for example, using the following parameters: adding protease inhibitors (PI) to the buffer and readjusting the pH value, then destroying the cells (e.g., ultrasonic treatment, French press). Centrifuge at 9 000rcf for 30min at 4°C, discard the precipitate (cell debris), collect the supernatant, centrifuge the supernatant at 100 000rcf for 1h at 4°C, discard the supernatant and homogenize the precipitate. Then carry out the solubilization of membrane proteins: polymers form synthetic nanodisks around the protein, thereby maintaining the natural phospholipid environment and retaining the natural and therefore functional properties of the protein in a convenient one-step manner (solubilization and stabilization). On the other hand, detergents form micelles around the hydrophobic band, thereby removing the lipids around. For natural conditions, it is necessary to retain a unique lipid environment.

[0065] In one embodiment, the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins and transport proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, SLC transporters. That is, as a starting material for the method according to the present invention, a solution of free polymer is used, which solution is derived from the solubilization, stabilization and purification of the above-mentioned membrane proteins from their natural environment by applying a polymer.

[0066] In some advantageous embodiments, the membrane protein is a full-length membrane protein, particularly a water-insoluble membrane protein.

[0067] In some embodiments, the target detected using the method / kit according to the present disclosure includes a moiety, which is an affinity portion from an affinity substance or is an affinity substance in its entirety, selected from the group consisting of: antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptide allergens, recombinant allergens, allergen idiotypic antibodies, autoimmune triggering structures, tissue rejection inducing structures, immunoglobulin constant regions and derivatives, mutants or combinations thereof.

[0068] In some advantageous embodiments, the methods / kits of the present disclosure are used to diagnose a disease selected from the group consisting of: viral-based diseases such as COVID-19, malignant diseases, 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic intestinal disease.

[0069] Therefore, the target to be detected can be a target virus or a fragment thereof in a biological sample, and wherein the virus or the viral fragment comprises a viral attachment protein that binds to at least one epitope of the membrane protein or at least to a fragment of the membrane protein. Specifically, the virus to be detected is a SARS virus, in particular SARS-CoV-2 or a variant thereof.

[0070] In some advantageous embodiments, the methods / kits disclosed herein are used to diagnose coronavirus diseases, such as COVID-19. In some other 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 fluid comprising a sample.

[0071] As described above, the polymer may be a homopolymer or a copolymer. In one embodiment, the polymer may have a hydrophilic group such as COOH, maleimide, OH, amine, ammonium salt, zwitterion such as phosphorylcholine, and a hydrophobic group such as a polymerized styrene group, a polymerized diisobutylene group, or a linear C1 to C16 (such as methyl and ethyl) aliphatic group, a branched C1 to C16 (such as isopropyl or tert-butyl) aliphatic group and a cyclic C5 to C12 aliphatic or aromatic group.

[0072] The molecular weight of the polymer used according to the method of the invention may be from 1900 to 20000, for example from 2000 to 18000, or from 2000 to 15000, or from 4000 to 16000, or from 4000 to 13000 or from 5000 to 14000. The molecular weight may be determined by gel permeation chromatography or mass spectrometry.

[0073] Examples of polymers may be, 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, styrene / maleimide copolymers such as SMA 502. These materials may also be functionalized on the COOH groups, with amines such as ethanolamine or ethylenediamine as amides, or with alcohols such as glycerol as esters. The polymers may also be functionalized with polyethylene glycol as esters, and with aminated polyethylene glycol as amides.

[0074] The polymer may be a diisobutylene / maleic acid copolymer, such as DIBMA 10 and DIBMA 12 from Cube Biotech, a derivative of a diisobutylene / maleic acid copolymer, such as DIBMA Gly (diisobutylene / maleic acid copolymer, partially modified with 1-amino-glycerol), DIBMA Glu (diisobutylene / maleic acid copolymer, partially modified with glucosamine), Glyco DIBMA (diisobutylene / maleic acid copolymer, partially modified with N-methyl-D-glucosamine) and a diisobutylene / maleimide copolymer. The DIBMA copolymer may be functionalized with the same molecules as SMA.

[0075] Another polymer may be a copolymer from styrene and acrylic acid, in particular a copolymer with a molecular weight of 5,500 and 11,000 and an acrylic acid / styrene ratio of 45% / 55% to 55% / 45%, sold under the name "AASTY".

[0076] Modified polymers of polyacrylic acid may be used in which 10-90% of the carboxylic acid groups may be modified as amides with cyclooctylamine, 2-cyclohexyl-ethylamine, etc. These materials are sold under the name "Amphipol Ultrasolve".

[0077] In addition to the above disclosure of polymers, additional descriptions of polymers are given below.

[0078] Polymers with hydrophilic and hydrophobic functional groups:

[0079] Examples of hydrophilic groups can be, but are not limited to, the following polymers: acrylic acid and methacrylic acid, maleic acid, carboxylic acid groups in general, amides with α,ω-alkylenediamines, ω-hydroxyalkylamines and ω-aminoalkylthiols, trimethylammonium-alkylamines, amides from carboxylic acid groups with aminoglycerol, TRIS or Bis-Tris, amides with maltosamine, glucosamine, mannosamine and other amino-functionalized carbohydrates, or taurine.

[0080] Furthermore, esters of carboxylic acid groups with polyethylene glycols, diols, triols, polyols and carbohydrates may also be mentioned.

[0081] Other examples may be maleimides, where the nitrogen atom is functionalized with an alkyl chain with alcohols, thiols, amines, ammonium salts, and the like.

[0082] Alternatively, a zwitterionic molecule consisting of ammonium and phosphate groups can be attached to the carboxyl group as described in US2020281855A1 or US2021171673A1.

[0083] Examples of hydrophobic groups may be, but are not limited to, polymerized styrene and derivatives such as methylstyrene, diisobutylene and linear and branched olefins such as 2-propyl, hexyl, octyl or decyl, coupled to a carboxyl group via an ester or amide functional group. In addition, maleimide groups with alkyl or aryl groups on the amino functional group are also suitable examples.

[0084] Examples of the synthesis of styrene-maleic acid copolymers can be found in: Shintaro Sugai, Nobumichi Ohno, Conformational transitions of the hydrophobic polyacids, Biophysical Chemistry, Volume 11, Issues 3-4, June 1980, Pages 387-395.

[0085] The use of SMA to construct complexes with lipids is described in WO 2006 / 129127 and references therein. SMA can be purchased from Orbiscope or Cube Biotech as SMALP 140, SMALP 200 or SMALP 300.

[0086] BASF describes the synthesis of copolymers from diisobutylene and maleic anhydride in US Pat. No. 4,250,289. The hydrolysis of anhydride copolymers to diisobutylene-co-maleic acid is described in Lee, Nature Protocols Vol. 11, No. 7, 2016, 1149-1162, which describes SMA copolymers but can be applied to DIBMA without problems.

[0087] The synthesis of a DIBMA polymer with glucosamine functionalization on 50% of all carboxyl groups can be found below: Danielczak, Marie Rasche, Julia Lenz, Eugenio Pérez Patallo, Sophie Weyrauch, Florian Mahler, Michael Tope Agbadaola, Annette Meister, Jonathan Oyebamiji Babalola, Carolyn Vargas, Cenek Kolar and Sandro Keller, Abioinspired glycopolymer for capturing membrane proteins in native-likelipid-bilayer nanodiscs, DOI: 10.1039 / D1NR03811G (Paper) Nanoscale, 2022, 14, 1855-1867. DIBMA is available at Cube Biotech as DIBMA 10 and DIBMA 12.

[0088] 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.

[0089] Sometimes the copolymer is a copolymer of styrene and acrylic acid, or a copolymer of styrene and an acrylic acid derivative. Any copolymer derivative can be used for the subject copolymer. Examples of derivatives are acrylates, methacrylates, acrylates, acrylamides, and N-substituted acrylamides. In some cases, the acrylate or acrylamide is substituted with a zwitterionic species, as described in U.S. Patent Application 20190062469A1, the disclosure of which is incorporated herein by reference.

[0090] In certain embodiments, the copolymer comprises 30% to 70%, 35% to 65%, or 40% to 60% acrylic acid or acrylic acid derivative content.

[0091] The synthesis of Amphipol Ultrasolute-polyacrylic acid polymers partially modified by cycloalkylamines or cycloalkyl-alkylamines is described in WO 115083 and Marconnet, A., Michon, B., Le Bon, C., Giusti, F., Tribet, C., & Zooenns, M. (2020). Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules. doi: 10.1021 / acs.biomac.0c00929.

[0092] US2020 / 0383918 describes additional polyacrylates modified with alkyl groups such as pentyl, hexyl and tert-butyl and their use in forming complexes with membrane proteins.

[0093] Polymethacrylates, copolymers containing butyl methacrylate (BMA): ˜0.52, and copolymers containing acryoloxy choline (MAC): ˜0.48, degree of polymerization (DP): ˜39.00, 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 Fomation by Amphiphilic Polymethacrylate Copolymers. J Am Chem Soc. 139(51): 18657-18663.

[0094] Polyacrylate polymers modified with alkanes such as n-butyl, tert-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 US2020383918A1.

[0095] Alternatively, US2022093587A mentions linear carbohydrates with a degree of polymerization of less than 100 functionalized with hydrophobic groups. An example of a linear carbohydrate is inulin, and an example of a hydrophobic group is an alkyl, alkenyl, alkynyl, cycloalkyl or heteroalkyl having 1-3 heteroatoms. The hydrophobic group is bound to the carbohydrate via an ether, ester or amide group.

[0096] The present disclosure also relates to a solubilized and stabilized isolated membrane protein or fragment thereof for use in treating a disease, in particular for use in treating a disease selected from the group consisting of: a viral-based disease, a malignant disease, or a chronic inflammatory disease 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic bowel disease, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc.

[0097] The interaction of the copolymer-stabilized membrane protein and its interaction partner may be detected including, but not exclusively, via different analytical methods.

[0098] Examples of optical detection include SPR (surface plasmon resonance), RM (resonant mirror), GCI (grating coupled interferometry), ELISA (enzyme-linked immunosorbent assay) such as direct ELISA, sandwich ELISA, competitive ELISA or reverse ELISA and LFA (lateral flow assay).

[0099] Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between a negative dielectric constant material and a positive dielectric constant material in a particle stimulated by incident light. SPR is the basis for many standard tools for measuring the adsorption of materials on planar metal (usually gold or silver) surfaces or on the surfaces of metal nanoparticles. It is the basic principle behind many color-based biosensor applications and lab-on-a-chip sensors.

[0100] A good overview of this technique as well as RM, dual polarization interferometry and other methods is given 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."

[0101] ELISA assays use at least one antibody that is specific for a particular antigen. A sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) non-specifically (via adsorption to a surface) or specifically (via capture by another antibody specific for the same antigen in a "sandwich" ELISA). After antigen immobilization, a detection antibody is added to form a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or it can itself be detected by a second antibody linked to the enzyme by bioconjugation. Between each step, the plate is usually washed with a mild detergent solution to remove any protein or antibody that is non-specifically bound. After the final washing step, the plate is developed by adding an enzyme substrate to produce a visible signal that indicates the amount of antigen in the sample (Wikipedia, English).

[0102] The principle of the lateral flow assay is described below. Binding here is indicated by a visual signal that can be produced by almost any dye, but is primarily produced by gold nanoparticles or fluorescently or magnetically labeled particles.

[0103] Alternatively, binding can be detected using calorimetric methods, such as ITC (isothermal titration calorimetry). Isothermal titration calorimetry (ITC) is an analytical technique, a titration method for analyzing molecular interactions by calorimetric measurements. The titration is performed at constant pressure and temperature, which means that a single ITC experiment provides data on binding enthalpy, equilibrium binding constant and stoichiometry, from which binding entropy and Gibbs energy can be calculated. Therefore, a single ITC experiment can directly obtain the key thermodynamic potentials associated with the interaction process - Gibbs energy, enthalpy and entropy.

[0104] Test equipment

[0105] As described above, the present disclosure relates to a test device for early and rapid detection of a target in a biological sample, wherein the device comprises a test strip, wherein the test strip comprises:

[0106] - At least one sample application location, in particular a sample pad.

[0107] - at least one test zone and one control zone, wherein the test zone comprises an immobilized isolated membrane protein or a fragment thereof, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by polymers and / or copolymers, in particular in polymer and / or copolymer nanodisks.

[0108] Therefore, the present disclosure provides a simple and rapid diagnostic test system, which improves and simplifies diagnostic assays known in the art. According to the test device of the present disclosure, a housing may be included, which includes a test strip, wherein the test strip includes at least one sample application position (e.g., sample pad), at least one test area (test line) and a control area (control line). The housing may include an opening and / or a transparent material. The opening and / or the transparent material of the housing are preferably used to receive a test sample and to read the results from the test area and the control area.

[0109] The preferred transparent material of the shell will allow reading the results of the test area and the control from the test strip. The transparent material of the shell can also have a cover that can be opened at the reading time point. The shell can also be made of transparent material completely. The preferred material of the test strip is known in the art, such as nitrocellulose membrane, absorbent cellulose pad, blood filter or core (wick). The test strip can further include a backing layer, such as a polyethylene backing layer. The material of the test strip, such as nitrocellulose membrane and / or absorbent pad, can be assembled on the backing layer and connected to the backing layer by means of an adhesive (preferably a pressure-sensitive adhesive). In a preferred embodiment, the sample application position is a sample pad or a sample core.

[0110] The test strip of the device according to the present disclosure may include a test zone containing a target antibody (primary antibody). The antibody is preferably immobilized in the corresponding zone.

[0111] According to the present disclosure, there is a preferred device, wherein the test strip further comprises a zone containing a protein conjugate between the sample application position and the test zone. The protein conjugate can be a monoclonal or polyclonal antibody that binds to the target. When a liquid such as a biological sample flows through the zone, the protein conjugate in the zone is preferably released. In a preferred embodiment, the protein conjugate in the zone comprising the protein conjugate comprises a gold protein conjugate. A preferred device, wherein the device comprises a gold protein conjugate.

[0112] Furthermore, preferably, the test strip comprises said zone containing a protein conjugate, wherein the protein conjugate comprises a corresponding gold-protein conjugate, i.e., an anti-target antibody gold conjugate bound to a target and / or an immobilized isolated membrane protein-gold conjugate, wherein the isolated membrane protein or said fragment thereof is solubilized and stabilized by polymers and / or copolymers, in particular in polymer and / or copolymer nanodisks.

[0113] In a preferred embodiment, the region comprising the protein conjugate is a glass fiber gold release pad that releases the gold-protein conjugate when a liquid (eg, a sample) flows through it.

[0114] It is further preferred that the control zone of each of the two test strips includes a non-specific capture antibody, which is preferably immobilized. Preferred non-specific capture antibodies are antibodies that non-specifically capture the conjugated protein, anti-mouse antibodies in the case of using mouse clone conjugates, and anti-rabbit antibodies in the case of using rabbit clones. Other preferred capture antibodies are anti-(anti-human immunoglobulin) control antibodies. In addition, in the case of using mouse anti-human immunoglobulin colloidal gold conjugates, the control antibody will be anti-mouse immunoglobulin. Preferred embodiments of the test strips of the device of the present invention are as follows Figure 3 and 11 shown.

[0115] Preferred test strips include:

[0116] - a backing layer,

[0117] - a sample pad as a sample application site,

[0118] - a gold conjugate pad comprising a gold protein conjugate,

[0119] as a capture line of a test zone (test line), wherein the test zone comprises an immobilized isolated membrane protein or a fragment thereof bound to a target, wherein the isolated membrane protein or said fragment thereof is solubilized and stabilized by means of a polymer and / or copolymer, in particular in polymer and / or copolymer nanodisks,

[0120] - as a control line in the control area,

[0121] - the material of the test strip in the areas of the test zone and the control zone is a nitrocellulose membrane,

[0122] - The remaining materials of the test strip are the absorbent pad and the blood filter, and

[0123] - Pressure sensitive adhesive to assemble / attach the sample pad, nitrocellulose membrane, blood filter and absorbent pad to the backing layer.

[0124] Another preferred first test strip comprises

[0125] - a backing layer,

[0126] - a sample pad as a sample application site,

[0127] - a zone comprising a protein conjugate (conjugate pad), comprising a first antibody binding to a target and / or an immobilized isolated membrane protein or a fragment thereof, wherein said isolated membrane protein or said fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc.

[0128] - A test zone (test line) comprising an immobilized isolated membrane protein or a fragment thereof, wherein the isolated membrane protein or said fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc or a first antibody.

[0129] -Use the immobilized non-specific second antibody as the control line of the control area,

[0130] - The material of the test strips in the test area and control area is nitrocellulose membrane,

[0131] - The remaining materials of the test strip are the absorbent pad and the blood filter, and

[0132] - Pressure sensitive adhesive to assemble / attach the sample pad, nitrocellulose membrane and absorbent pad to the backing layer.

[0133] In a preferred embodiment, the gold conjugate in the zone (conjugate pad) is modified with a compound selected from the group comprising chitosan, oligochitosan, glucosamine, polylysine or other polymers or mixtures thereof.

[0134] In an advantageous embodiment, the test device is defined as a) said conjugate pad comprises a first antibody against a target labeled with nanoparticles, and said test zone comprises an immobilized isolated membrane protein or a fragment thereof bound to said target, wherein said isolated membrane protein or said fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc, or

[0135] b) the conjugate pad comprises an immobilized isolated membrane protein or a fragment thereof bound to the target, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc, and the test area comprises a first antibody against the target.

[0136] The effect of any of these compounds or their mixtures is the color intensity of colloidal gold. They are added during the preparation of colloidal gold, but before the colloidal gold is combined with the protein (i.e., antibody or antigen). Colloidal gold is conjugated with specific antibodies and / or antigens after modification with chitosan (and / or other modifiers). Chitosan and other modifiers can affect the color intensity of colloidal gold, and thus improve the ability of the human eye to recognize colors, and thus be able to detect very low concentrations. The signal amplification range is up to 10 times. Colloidal gold is preferably prepared by reducing an aqueous solution of 1% tetrachloroauric acid (HAuC14) with an aqueous solution of trisodium citrate to produce spherical gold particles. After the colloidal gold is prepared, an aqueous solution of chitosan (or any other modifier or mixture) of appropriate volume and concentration is added, and the color is converted from purple to violet according to the volume and concentration of the modified solution added.

[0137] Therefore, the present disclosure also relates to novel rapid testing methods and test devices that allow direct detection of the virus within 15 minutes of the first day of infection. Many pathogenic viruses use the integral membrane proteins of their host cells as receptors to attach to the cells and subsequently take up and replicate in the cells. Therefore, the part of the membrane protein on the surface of the host cell is "abused" in its function as a viral receptor, thereby determining the ability of the virus to infect and replicate in certain cell types. Although the virus itself is highly variable, binding to host cell receptors represents a highly conservative and invariant process.

[0138] Therefore, stabilized and solubilized host cell membrane proteins are integrated into rapid tests to serve as highly specific scavengers of pathogenic viruses and thus to construct innovative lateral flow rapid tests. For example, the HBV virus attaches itself to human liver cells via the membrane protein NTCP (= sodium taurocholate cotransporting polypeptide or bile acid transporter) anchored in the host cell membrane and multiplies therein, thereby causing hepatitis disease ( Figure 1 ).

[0139] The present invention is further described below with reference to examples and drawings. These examples should not be construed as limiting the present invention thereto.

[0140] Methods and Examples

[0141] It should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the present disclosure in any way.All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0142] Example 1: Purification of membrane proteins stabilized in copolymers such as poly(acrylic acid-co-styrene), ie AASTY, a super soluble amphiphilic polymer

[0143] The solubilization, stabilization and purification of membrane proteins from their native membrane environment depend on multiple parameters. Most parameters can be optimized during the purification process to improve efficiency. Parameters include: buffer conditions (salt, pH, etc.), selected polymer, protein to solubilizing agent ratio, temperature, time.

[0144] Cell lysis and centrifugation:

[0145] Add protease inhibitors (PI) to the buffer and readjust the pH, then disrupt the cells (e.g., sonication, French press). Centrifuge at 9,000 rcf for 30 min at 4°C, discard the pellet (cell debris), and collect the supernatant. Centrifuge the supernatant at 100,000 rcf for 1 h at 4°C, discard the supernatant, and homogenize the pellet.

[0146] Solubilization of membrane proteins:

[0147] Polymers form synthetic nanodisks around proteins, thereby maintaining the native phospholipid environment and preserving the native and functional properties of proteins in a convenient one-step manner (solubilization and stabilization). Detergents, on the other hand, form micelles around the hydrophobic band, thereby removing the surrounding lipids. For native conditions, the unique lipid environment needs to be preserved.

[0148] If the solubilization efficiency is low, it is recommended to screen for parameter changes to increase the yield of total solubilized protein. The standard protocol is described below:

[0149] - Add solubilizing agent to protein solution

[0150] - Ideal concentrations may vary, a good starting point is:

[0151] -0.5%-5% SMA / DIBMA

[0152] -0.1-2.5% super soluble amphiphilic polymer

[0153] -0.1-2.5% AASTY

[0154] -Solubilize at 4°C for 3 to 24 hours while stirring

[0155] -Higher temperatures can be screened for optimization

[0156] - Centrifuge at 100 000 rcf for 1 h at 4°C

[0157] -Discard the precipitate and collect the supernatant

[0158] - Affinity chromatography using solubilized membrane proteins in polymer nanodiscs (supernatant)

[0159] Example 2: Affinity chromatography

[0160] Polymers can interfere with the binding of proteins to the binding matrix, so it is advantageous to reduce the polymer concentration. For adequate binding, a polymer concentration of 0.25% is necessary. Further reductions to 0.025%-0.050% polymer may be beneficial to improve binding efficiency.

[0161] The selected protein is purified via a preferred protocol based on an affinity tag.

[0162] Example 3: Diagnosis using copolymer-supported membrane proteins:

[0163] Protocol for Standard ELISA Assays Using Copolymer-Solubilized Proteins

[0164] - The selected copolymer or copolymers stabilize the membrane protein in a purified state (multiple days at 4°C).

[0165] - One day before the ELISA assay, a sterile 96-well plate must be coated with protein (a concentration of 500 ng / well to 1 μg / well is a good starting point for optimization). 100 μL of protein solution is pipetted into each well, except for possible controls, such as blanks or a coating consisting of 1% BSA in TBS, to visualize any possible non-specific interactions.

[0166] - In addition, it is recommended to perform duplicate determinations for each test condition.

[0167] - Leave the plate at 4 °C for at least overnight, covered with parafilm or a plastic cover to avoid evaporation or drying.

[0168] - The next day, wash buffer [50 mM Tris, 100 mM NaCl; pH 7.0] was prepared by dissolving 0.05% Tween 20 in TBS buffer.

[0169] - Wash the coated plate three times [300 μL per well]: twice with TBS containing Tween 20 and once with TBS without additional detergent. Let the plate sit for approximately 5 minutes between buffer changes.

[0170] - After washing, add 300 μL of 3% milk powder in TBS or 3% BSA in TBS to each well and incubate 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 used in the assay.

[0171] - After this blocking step, the washing steps described previously were repeated in the same way.

[0172] - Dilute the selected ligand (can be a protein, antibody, etc.) to the selected concentration. It is recommended to perform serial dilutions in the same buffer as the stock protein.

[0173] - Pipette 100 μL of each ligand dilution into the corresponding wells and incubate for one hour at room temperature.

[0174] -After incubation repeat the wash step once more.

[0175] - Following these wash steps, add 100 μL of primary antibody targeting the ligand to each well and incubate for one hour at room temperature.

[0176] - The dilution should be chosen based on the manufacturer's recommendations. If this information is not available, a 1:1000 dilution using 3% milk powder or BSA is recommended.

[0177] - If the assay is performed using a conjugate of two antibodies, the incubation with the first antibody can be omitted.

[0178] - The same washing steps were performed again and then 100 μL of the now secondary antibody was incubated again for one hour at room temperature. Here, the antibody was always diluted to 1:1000 by using 3% milk powder in TBS.

[0179] - After the secondary antibody incubation, the plates were washed again. This time, twice with 0.05% Tween 20 in TBS buffer, twice with plain TBS, and finally a washing step with MilliQ water (300 μL per well in each washing step).

[0180] - As the visualization / development buffer must be as fresh as possible, it is recommended to mix the components of the solution immediately after the last washing step with water. For the visualization / development buffer, mix 9 mL of MilliQ water, 1 mL of 1 M sodium acetate [pH 6.0 adjusted by using 1 M citric acid], 62.5 μL of 3,3',5,5'-tetramethylbenzidine [abbreviation: TMB, 10 mg / mL in DMSO], and 15 μL of 3.5% HO. 2 O 2 Mix and use immediately by pipetting 100 μL of the solution into each well.

[0181] - Incubation in visualization / development buffer is flexible but should be at least 10 minutes but no longer than 90 minutes. However, the shorter the incubation time, the more obvious the differences between different samples will be.

[0182] -Terminate the enzymatic reaction by adding 100 μL of 10% sulfuric acid. Gently tap the frame of the plate to increase mixing of the solution in the wells. After five minutes, measure the plate at 450 nm using a standard photometer to obtain the result.

[0183] Example 4: Protocol for SPR Assay Using Membrane Protein (CoV Spike Protein) Solubilized with Copolymers

[0184] Surface plasmon resonance (SPR) is the resonant oscillation of conduction electrons at the interface between negative dielectric constant materials and positive dielectric constant materials in particles stimulated by incident light ( Figure 4 ). SPR is the basis of many standard tools for measuring the adsorption of materials on planar metal (usually gold or silver) surfaces or on the surfaces of metal nanoparticles. This is the basic principle behind many color-based biosensor applications and lab-on-a-chip sensors. It should be emphasized that SPP is not a resonance on a planar surface, but a polariton or surface wave-like phenomenon.

[0185] How SPR works:

[0186] -Optical methods for measuring mass changes of biomolecules

[0187] - Target biomolecules bind to the metal membrane in the flow chamber

[0188] - Analytes are directed through the flow chamber

[0189] -After binding to the analyte, the refractive index of the metal film changes

[0190] ->Change the reflection angle of light

[0191] "Surface plasmon resonance phenomenon"

[0192] - The more combinations, the greater the angle offset

[0193] ->The larger the measurement signal

[0194] How to use SPR?

[0195] -protein associated with a channel plated with gold

[0196] - For example, EDC / NHS coupling

[0197] -Ligand / protein partners are prepared in increasing concentrations

[0198] -The ligand is then directed through two channels to allow binding

[0199] - Binding is detected by increased signal

[0200] - After a defined time, also run buffer without ligand and measure dissociation

[0201] - Dissociation is detected by signal reduction ( Figure 5 ).

[0202] How to determine Kd and Ka values?

[0203] -The signal mainly depends on:

[0204] Binding rate (ka)

[0205] Dissociation rate (kd)

[0206] Amount of bound ligand

[0207] Analyte concentration

[0208] - Given a known ligand concentration and a number of different analyte concentrations, ka and kd can be calculated ( Figure 6 ).

[0209] - Example of fitting equation*:

[0210] ·

[0211] ·

[0212] *Simplified integral equation for reversible 1:1 binding of ligand and analyte

[0213] Example 5: Measurement using COVID and ACE

[0214] ACE receptor ectodomain fused to chip - Sars COV2 spike protein stabilized in LMNG, DIBMA or SMA was washed on ACE fusion chip. Binding and dissociation abilities were measured at different concentrations:

[0215] SPR biosensor SR7500DC (Reichert)

[0216] Chip: Indigo chip (Cube Biotech GmbH https: / / cube-biotech.com / ), prepared by chemically modifying a dextran chip (SCR 200R-DCM5, Xantec GmbH, Düsseldorf, Germany) according to WO2020 / 109162, the procedure of Example 7.

[0217] Wash the chip with ACE2 buffer until the baseline is stable

[0218] Immobilize the ACE2 extracellular domain on the left channel of the chip (approximately 600RU)

[0219] 60μg / mL, flow rate: 0.5ml / min

[0220] Wash the chip with ACE2 buffer until the baseline is stable

[0221] ·Change the buffer to COVID buffer and wash both channels until the baseline is stable

[0222] Analyte: COVID spike protein in DIBMA

[0223] COVID sample concentrations: 0nM; 0.488nM; 0.96nM; 1.9nM; 3.9nM; 7.8nM; 15.6nM; 31.25nM; 72.5nM 125nM; 250nM; 500nM;

[0224] Start measuring:

[0225] 25μL / min

[0226] Binding time: 6 minutes

[0227] Dissociation time: 11min

[0228] The experiment was cited twice: an empty channel without ACE2 and injected with running buffer (buffer blank)

[0229] The measurement results are as follows Figure 7 , 8 and 9.

[0230] Overview and Comparison

[0231] The results in Table 1 show:

[0232] - COVID in LMNG showed weaker dissociation than samples solubilized in polymer (lowest kd value)

[0233] -LMNG and COVID in SMA show comparable binding, so COVID in SMA is active

[0234] -COVID in DIMBA showed the weakest binding (lowest ka value)

[0235] - COVID in SMA showed the strongest dissociation (highest kd value)

[0236] Table 1

[0237]

[0238] The experiments showed that COVID in the SMA successfully stabilized the spike protein in a conformation that could bind to the ACE receptor on the chip.

[0239] Advantages of membrane proteins in polymers / copolymers / nanodiscs:

[0240] If two full-length membrane proteins are used for SPR, neither protein should be stabilized in detergent, as washing with detergent buffer may cause permanent dissociation of the coated Protein A from the chip. Copolymers stabilize membrane proteins in their native lipid environment without the need to add copolymers to each buffer.

Claims

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

2. The method according to 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 transport 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. The method according to any one of claims 1 to 4, wherein: The polymers / copolymers that solubilize and stabilize the membrane proteins have hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions such as phosphorylcholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, tert-butyl) and cyclic (C5 to C12) aliphatic or aromatic groups.

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

7. The process 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 acid, partially coupled to cycloalkylamines or cycloalkylalkylamines, in particular styrene / maleic acid copolymers.

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

9. The method according to claim 8, wherein: The DIBMA in the DIBMA / lipid particles is glucosamine and / or aminoglycerol-modified DIBMA.

10. The method according to any one of claims 1 to 9, wherein: The target comprises a portion, which is an affinity portion from an affinity substance or is an affinity substance in its entirety, selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptide allergens, recombinant allergens, allergen idiotypic antibodies, autoimmune triggering structures, tissue rejection inducing structures, immunoglobulin constant regions and derivatives, mutants or combinations thereof.

11. The method according to any one of claims 1 to 10, wherein: The method is used to diagnose a disease selected from the group consisting of: viral-based diseases such as COVID-19, malignant diseases, 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic intestinal disease.

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

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

14. The method according to any one of claims 1 to 13, wherein: The method is used to diagnose coronavirus diseases, such as 2019 coronavirus disease (COVID-19).

15. The method according to any one of claims 1 to 14, wherein: The biological sample is derived from humans or animals, such as blood, urine, tissue, organ, saliva, hair, nail clippings or any other cells or fluids including samples.

16. An 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 fragment thereof, comprising at least one epitope that binds to a target, wherein: The isolated membrane protein or the fragment thereof is in the presence of a polymer and / or copolymer capable of solubilizing and stabilizing the membrane protein, in particular, the membrane protein is included in a polymer and / or copolymer nanodisc; as well as (b) a reagent for detecting the formation of a target-protein complex between the target and the membrane protein or the fragment thereof in the biological sample, wherein the isolated membrane protein or the fragment thereof and the reagent are present in an amount sufficient to detect the formation of the target-protein complex.

17. The kit according to 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 transport 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 such as phosphorylcholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, tert-butyl) and cyclic (C5 to 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. 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 copolymers, styrene maleic acid copolymers, (acrylic acid-co-styrene) copolymers and amphiphilic polymers, in particular styrene / maleic acid copolymers.

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

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

25. The kit according to any one of claims 16 to 24, wherein The target comprises a portion, which is an affinity portion from an affinity substance or is an affinity substance in its entirety, selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptide allergens, recombinant allergens, allergen idiotypic antibodies, autoimmune triggering structures, tissue rejection inducing structures, immunoglobulin constant regions and derivatives, mutants or combinations thereof.

26. The kit according to any one of claims 16 to 25, wherein The method is used to diagnose a disease selected from the group consisting of: viral-based diseases such as COVID-19, malignant diseases, 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic intestinal disease.

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

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

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

30. The kit according to any one of claims 16 to 29, wherein The biological sample is derived from humans or animals, such as blood, urine, tissue, organ, saliva, hair, nail clippings or any other cells or fluids including samples.

31. A solubilized and stabilized isolated membrane protein or fragment thereof for use in treating a disease, in particular for use in treating a disease selected from the group consisting of: a viral-based disease, a malignant disease, or a chronic inflammatory disease 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic intestinal disease, wherein, The isolated membrane protein or the fragment thereof is solubilized and stabilized by polymers and / or copolymers, in particular in polymer and / or copolymer nanodiscs.

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 transport 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 34, 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. 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 such as phosphorylcholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, tert-butyl) and cyclic (C5 to C12) aliphatic or aromatic groups.

38. The membrane protein for use according to any one of claims 31 to 37, wherein the molecular weight of the polymer / copolymer is from 1900 to 20000.

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 copolymers, styrene maleic acid copolymers, (acrylic acid-co-styrene) copolymers and amphiphilic polymers, in particular styrene / maleic acid copolymers.

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

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

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

43. A membrane protein for use according to any one of claims 31 to 42, wherein The complex is used to treat a disease selected from the group consisting of: viral-based diseases such as COVID-19, malignant diseases, 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic intestinal disease.

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

45. A test device for early and rapid detection of a target in a biological sample, wherein: The device comprises a test strip, wherein the test strip comprises: - at least one sample application location, in particular a sample pad; - at least one test area and one control area, and - at least one zone serving as a conjugate pad comprising a gold protein conjugate, Wherein, the test area and / or the conjugate pad comprises an immobilized isolated membrane protein or a fragment thereof, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or a copolymer, in particular in a polymer and / or a copolymer nanodisc.

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

47. A test device according to any one of claims 45 to 46, wherein: a) the conjugate pad comprises a first antibody directed against a target labeled with nanoparticles, and the test zone comprises an immobilized isolated membrane protein or a fragment thereof bound to the target, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc, or b) the conjugate pad comprises an immobilized isolated membrane protein or a fragment thereof bound to the target, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or copolymer, in particular in a polymer and / or copolymer nanodisc, and the test area comprises a first antibody against the target.

48. A test device according to any one of claims 45 to 47, wherein: The test strip comprises an area containing a gold protein conjugate between the sample application site and the test area, wherein the protein in the gold protein conjugate is an antibody against the target and / or an immobilized isolated membrane protein or a fragment thereof bound to the target, wherein the isolated membrane protein or the fragment thereof is solubilized and stabilized by a polymer and / or a copolymer, in particular in a polymer and / or copolymer nanodisc.

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

50. A test device according to any one of claims 45 to 49, wherein: The test strip includes a nitrocellulose membrane.

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

52. A test device according to any one of claims 45 to 51, wherein: The control zone includes an immobilized non-specific capture antibody, such as an anti-gold control antibody.

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

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

55. A test device according to any one of claims 45 to 54, wherein: The polymers / copolymers that solubilize and stabilize the membrane proteins have hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions such as phosphorylcholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear (methyl, ethyl, up to C16), branched (isopropyl, tert-butyl) and cyclic (C5 to 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 from 1,900 to 20,000.

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 copolymers, styrene maleic acid copolymers, (acrylic acid-co-styrene) copolymers and polyacrylic acid, partially coupled to cycloalkylamines or cycloalkylalkylamines, in particular styrene / maleic acid copolymers.

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

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

60. A test device according to any one of claims 45 to 59, wherein: The target comprises a portion, which is an affinity portion from an affinity substance or is an affinity substance in its entirety, selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptide allergens, recombinant allergens, allergen idiotypic antibodies, autoimmune triggering structures, tissue rejection inducing structures, immunoglobulin constant regions and derivatives, mutants or combinations thereof.

61. A test device according to any one of claims 45 to 60, wherein: The method is used to diagnose a disease selected from the group consisting of: viral-based diseases such as COVID-19, malignant diseases, 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 disease, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases, including Crohn's disease and chronic intestinal disease.

62. 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 wherein the virus or the viral fragment comprises a viral attachment protein that binds to at least one epitope of the membrane protein or at least to 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. A test device according to any one of claims 45 to 63, wherein: The method is used to diagnose coronavirus diseases, such as 2019 coronavirus disease (COVID-19).

65. A test device according to any one of claims 45 to 64, wherein: The biological sample is derived from humans or animals, such as blood, urine, tissue, organ, saliva, hair, nail clippings or any other cells or fluids including samples.

66. A test device according to 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 transport proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane based ATPases and SLC transporters.

Images