CN113156119A - Method for detecting coronavirus by adopting angiotensin converting enzyme II (ACE2) - Google Patents

Abstract

The present invention relates to a method for the detection or quantification of coronavirus particles and their spike proteins or any fragment of the spike protein containing an RBD, using a ligand-receptor interaction, i.e. a strong binding of the Receptor Binding Domain (RBD) of the coronavirus spike protein (S protein) to angiotensin-converting enzyme II (ACE 2). As an alternative to antibodies necessary for conventional immunoassay methods, ACE2 may be used to coat solid phase surfaces to capture or enrich for the analyte, and it may also be conjugated to a label involved in signal generation. Depending on the nature of the analyte and the signal pattern, ACE2 may be used alone as an analyte-recognizing agent or in combination with an antibody.

Description

Method for detecting coronavirus by adopting angiotensin converting enzyme II (ACE2)

Technical Field

The invention belongs to the field of coronavirus detection methods.

Background

The envelope diameter of SARS-CoV-2 is about 85 nm, and the coronary Spike protein (Spike protein, abbreviated as S protein, see FIG. 1) is about 20 nm long. Numerous studies have demonstrated that SARS-CoV-2, like SARS-CoV, the causative agent of SARS, invades human cells by binding to the same receptor, angiotensin-converting enzyme II (ACE2), via the S protein, with the aid of a transmembrane serine protease (TMPRSS 2). The S protein is a trimer, consisting of two subunits, S1 and S2. The S1 subunit forms the top of the coronary spike, while the S2 subunit is anchored (anchored) to the viral envelope. After a Receptor Binding Domain (RBD) on a S1 subunit is combined with a human cell surface receptor ACE2, a specific site of an S protein is cut due to the action of a protease TMPRSS2, so that a virus envelope is fused with a cell membrane, and then the virus enters cells, wherein the RBD consists of 223 amino acids and has the molecular weight of 25 kDa.

ACE2 consists of 805 amino acids with a molecular weight of 91 kDa. It is expressed on endothelial cells and smooth muscle cells of almost all organs of the human body, such as oronasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph node, thymus, bone marrow, spleen, liver, kidney, brain, and testis, and is most abundantly expressed in lung tissue and small intestine tissue. ACE2 can be cleaved from the cell surface and exists in the human body as a soluble enzyme, but the physiological significance of soluble ACE2 is not clear at present. After SARS occurred in 2003, ACE2 was identified as a target for SARS-CoV to enter the body and attack other organs in the body. The gene sequence of SARS-CoV-2 has a high similarity to SARS-CoV, but SARS-CoV-2 and SARS-CoV differ in terms of ligand-receptor binding ability. The dynamic study of the binding of RBD of SARS-CoV-2 and ACE2 shows that the protein binds to ACE2Equilibrium dissociation constant of binding is K_D＝4.674×10^-9M or 14.7X 10^-9And M. Equilibrium dissociation constants of this magnitude indicate that SARS-CoV-2 binds to ACE2 approximately ten times more strongly than SARS-CoV binds to ACE 2. This superior ligand (RBD) -receptor (ACE2) binding capacity explains to some extent the faster spread and greater harm of SARS-CoV-2. On the other hand, the super ligand-receptor binding capacity also provides a new idea for virus detection and diagnosis and prognosis of COVID-19.

Since the outbreak of COVID-19, the technical means for diagnosing the etiology of COVID-19 was based on the detection of nucleic acids of viruses and antibodies generated by the immune reaction of the human body. The former uses pharyngeal swab or nasal swab to collect human respiratory tract (nasal cavity or pharyngeal) secretion as sample to carry out virus nucleic acid specific gene detection and virus genome sequencing, while the latter uses blood sample to detect antibodies (IgM, IgA and IgG) possibly generated due to virus infection in blood. Because of the limitations of sampling methods, sampling time and detection techniques on specificity and sensitivity, any single detection method cannot comprehensively and completely correctly judge whether the virus is infected or not, and the infected condition, and brings inconvenience to prevention and control of diseases and prognosis of patients. With the further rapid rise in the number of infections and patients and the heightened concern that SARS-CoV-2 may coexist with humans for a long period of time in the future, the development of various technical means for diagnosis and analysis is a matter of pressing and long-term value.

In contrast to long-term practice against infectious diseases caused by pathogens and current countermeasures facing the covi-19 pandemic, a significant technical shortage is the detection of viral antigen (antigen) substances, such as the S protein and fragments thereof, and the virion or virion (virion) itself. This situation is associated with the absence of specific antibodies against SARS-CoV-2 that have both high affinity and high selectivity.

Comparing the general antibody-antigen reaction binding constants (or equilibrium dissociation constants), we found that the binding ability of human ACE2 receptor to the S protein of SARS-CoV-2 is comparable to most antibody-antigen reactions, which can be used instead of antibodies as a measured recognition partner (recogmtion partner) in bioassays based on the principle of bioaffinity for capturing viral antigens or viral particles, or binding to viral antigens or viral particles that have been captured (or immobilized on a solid surface). Therefore, based on the principle of biological affinity and the high affinity of ACE2 with the S protein of SARS-CoV-2, the present invention discloses a method for establishing viral antigenic substances and the detection of virus particles or virions themselves using ACE2 instead of antibodies in conventional immunoassays.

Disclosure of Invention

Biological analysis methods based on the principle of biological affinity are widely applied to the fields of scientific research, clinical diagnosis, drug research and development, environmental and food monitoring and the like. The construction of the detection system and the pattern of the detection signal are many based on the difference in the properties of the system to be detected (from a specimen or sample prepared in human, animal, environmental, food or scientific research) and the substance to be detected (chemical substance, bioactive substance, genetic substance, microorganism, biological tissue, etc.). But the basic and common practice includes selecting one or more recognition objects (chemical substances, bioactive substances, genetic substances, microorganisms, biological tissues, etc.) capable of specifically binding to the tested object in the tested system, and forming the testing system by utilizing the specific binding of the tested object and the recognition objects under the condition of artificially set environment (liquid phase, gas phase or solid phase surface).

In various molecular diagnostic techniques applied to nucleic acid detection, a recognition entity is a nucleic acid that is complementary to a series (part or all) of nucleic acids to be detected, and in an immunoassay, a recognition entity is an antibody for detecting an antigen and an antigen for detecting an antibody. Bioassays with high sensitivity and specificity require a high binding capacity and high selectivity of the recognition entity for the analyte. If the binding capacity is high, the amount or concentration of the analyte that can be detected is low; if the selectivity is high, the possibility that other non-analyte in the sample is mistaken for the analyte is low. Therefore, in developing an analysis or detection method, selection of a recognition object of an object to be detected is an important factor in determining the analytical performance of the method.

After the identification object(s) of the test object are determined, detection methods based on a variety of signal patterns can be established. The signal in the "signal mode" may be derived from a label (label) labeled on the recognition entity (in some cases, an additional analyte added to the system by human) or a signal generated by the label participating in a physical, chemical or biochemical reaction in an artificially set environment. In some cases, the signal in the "signal pattern" may also be a change in a physicochemical signal directly from the event of specific binding of the analyte to the recognition entity. The former is known as label-based bioaffinity analysis and the latter as unlabeled bioaffinity analysis. FIG. 2 is a diagram of some label and non-label based detection methods commonly used in immunoassays.

The non-labeling bioaffinity analysis method (such as various methods based on signal detection principles of surface plasmon resonance, optical interference, quartz crystal surface quality change and the like) is mainly used in the fields of life science research and drug development (research on interaction between drug molecules and target proteins). Bioanalysis using a large amount of antibody and antigen combined and aggregated into a large particle to cause a change in light scattering effect can be considered as an example of the application of the unlabeled bioaffinity analysis method in the clinical medicine field (fig. 2).

However, in clinical immunoassays, the vast majority of the signals detected by the methods result from the markers or signals generated when the markers participate in the process of physical, chemical or biochemical reactions in an artificially defined environment. Where the label is a radioisotope (as in a radioimmunoassay), a gold nanoparticle (as in lateral flow immunochromatography), or a magnetic particle (as in lateral flow immunochromatography), the signal detected (radioactivity, color, or magnetic intensity) is derived from the label itself. In the case where the label is a fluorescent or phosphorescent substance (e.g., quantum dot, rare earth long-life phosphorescent molecule or microsphere, fluorescent molecule or microsphere, etc.), a chemiluminescent substance, an electrochemiluminescent substance, an enzyme, etc., the signal is generated when the label undergoes a physical process (e.g., external light excitation), a chemical reaction process (e.g., chemiluminescence without enzyme participation), an electrochemical reaction process (e.g., electrochemiluminescence), and a biochemical reaction process (e.g., chemiluminescence with enzyme participation). FIG. 3 lists markers and key substances involved in the luminescence process in a method using luminescence signals as a detection signal pattern, which is commonly used in clinical immunoassays (within the dashed box in FIG. 3).

All of the above analytical methods for clinical medicine, life science research and drug development (fig. 2 and 3) have been well established in the past and have been used to varying degrees in specific relevant fields. The invention is not concerned with the improvement of the signal generation mechanism or the signal detection principle in these methods or principles.

The invention focuses on changing the analyte-recognizing entity in clinical immunoassay and applying it to the above-mentioned detection method. Unlike the traditional detection system based on antibody-antigen reaction structure, the present invention adopts ACE2 receptor with high affinity to constitute SARS-CoV-2 virus and its antigen detecting system. Strictly speaking, when ACE2 (rather than an antibody) is used as the analyte (RBD of S protein) identifier in a sample, the assay method to which the present invention relates is not an immunoassay. However, in certain embodiments of the invention, ACE2 and antibodies may be used together as analyte-recognizing agents for the detection of antigenic material and viral particles of SARS-CoV-2.

As an S protein RBD recognition object of SARS-CoV-2 in an analysis method based on the principle of bioaffinity, the present invention discloses an example of using ACE2 as an analyte recognition object instead of an antibody in a conventional immunoassay method. These examples are not limited by the type of sample or specimen, and include both conventional clinical specimens such as nasal/pharyngeal swab samples, saliva, sputum, bronchial lavage, alveolar lavage, blood, urine, tears, and the like, as well as aerosols, air, dust, and samples collected or enriched from solid phase surfaces by wiping or rinsing.

The majority of immunoassays based on antibody-antigen reaction constructs traditionally are heterogeneous immunoassays (xenogeneous immunoassays). In a heterogeneous immunoassay system and a heterogeneous immunoassay program, a link that a solid phase surface (a hole bottom or an inner wall, a reactor inner wall, a magnetic particle surface and the like) participates in capturing a detected object or a complex formed by the detected object and an identification body is involved, and the link has multiple functions of capturing, enriching, separating and the like. Homogeneous immunoassays (homogeneous immunoassays) do not involve the involvement of a solid surface and are advantageous in certain applications. Immunoassays are generally limited to the detection of antigens and antibodies, and their use for the direct detection of coronavirus particles has not been disclosed. Unlike the common antigens (proteins, polypeptides or small molecules, etc.), the numerous spikes (S proteins) on the surface structure of SARS-CoV and SARS-CoV-2 provide multiple sites for the receptor ACE2 to bind to, and the presence of the RBD of these numerous S proteins makes it possible to use ACE2 alone as a recognition entity, both in heterogeneous and homogeneous situations.

In conventional immunoassays, a sandwich method (sandwich assay) and a competitive assay (competitive assay) have been developed depending on the size of a molecule to be measured and the antigen-antibody binding mode. Likewise, sandwich (sandwich assay) and competitive methods can be effectively established using ACE2 alone or in combination with ACE2 and antibodies. If the test substance is a viral particle, due to its numerous spike (S protein) sites on its surface, ACE2 can replace two antibodies against different antigen binding sites in conventional immunoassays (such as capture and signal antibodies in heterogeneous immunoassays).

In the first class of embodiments, ACE2 is immobilized (immobilized) or anchored (anchored) on the surface of the solid phase by chemical coupling or adsorption reaction to form an antigen capturing layer that has the ability to capture RBD-containing antigen (RBD-containing S protein or RBD-containing S protein fragment, etc.) and coronavirus particles in the sample. The captured antigen or SARS-CoV-2 virion can be detected by various signals by means of typical non-label bioaffinity assays, such as those shown in FIG. 2 but not limited thereto.

In the second class of embodiments (competitive antigen detection), ACE2 is immobilized (immobilized) or anchored (anchored) on the surface of a solid phase by chemical coupling or adsorption reaction to form an antigen capturing layer that has the ability to capture RBD-containing antigens (RBD-containing S protein or RBD-containing S protein fragment, etc.) in a sample. To the sample to be tested, RBD-containing S protein or its fragment labeled with a label (a substance capable of generating or participating in signal generation, such as nano-colloidal gold particles, latex particles, and those in fig. 3) is added, and when RBD-containing antigen (RBD-containing S protein or RBD-containing S protein fragment, etc.) is present in the sample to be tested, the artificially added, labeled antigen will compete with the test antigen present in the sample for binding to ACE2 on the solid phase surface. Finally, the quantitative information of the detected antigen can be obtained by detecting the signal generated or participated in the generation of the marker, the magnitude of the signal is in inverse proportion to the amount of the detected antigen, and the detected signal is strongest when no RBD-containing antigen substance exists in the detected sample. A schematic diagram of a competitive reaction and detection system constructed by using solid particles (such as magnetic particles) as solid phase and forming ACE2 on the surface thereof is shown in FIG. 4.

In the third class of embodiments (sandwich method for detecting antigen), ACE2 is immobilized (immobilized) or anchored (anchored) on the surface of the solid phase by chemical coupling or adsorption reaction to form an antigen capturing layer, which has the ability to capture RBD-containing antigen (RBD-containing S protein or RBD-containing S protein fragment, etc.) in the sample. An antibody is selected that does not coincide with the RBD and does not interfere with the binding of ACE2 to the RBD, and is labeled according to the pattern of the detected signal. The labeled antibody and ACE2 on the solid phase surface form a sandwich structure with the antigen substance to be detected (as shown in FIG. 5A). Also in the sandwich method, ACE2 can be labeled, and an antigen capture layer (as shown in fig. 5B) is formed on the solid phase surface by the antibody (the antibody does not affect the binding of ACE2 to RBD). Finally, the quantitative information of the detected antigen can be obtained by detecting the signal generated or participated in the generation of the marker, the signal size is in direct proportion to the amount of the detected antigen, and the detected signal is weakest when no RBD-containing antigen substance exists in the detected sample. This weak signal detected in the sample without the test substance is generated by non-specific adsorption.

In a fourth class of embodiments, the analyte is a viral particle. Due to the numerous spike (S protein) sites on the surface of the virion, ACE2 can act as both a recognition entity for capture of the virion and as a labeled recognition entity, which bind to different RBDs located on the surface of the virion, respectively (see fig. 6A). In addition, ACE2 may also be used in combination with antibodies (as shown in fig. 6B and C). In this application for the detection of viral particles, the choice of antibody is more flexible since the antibody used in combination with ACE2 does not bind to the same coronary spike.

In a fifth class of embodiments, the test agent viral particles are detected in a condition similar to a homogeneous immunoassay. It is called "similar to homogeneous immunoassay" because the diameter of coronavirus particles is about 100 nm, and the solution system when they are dispersed in liquid phase may not be strictly called homogeneous solution, but for the sake of simplifying the description, the invention still uses "homogeneous reaction" or "homogeneous detection" to describe the embodiment. FIG. 7 depicts three fluorescence resonance-based energy transfers using ACE2 (R) ((R))

A homogeneous detection mode of resonance energy transfer, or fluorescence resonance energy transfer, FRET) or a homogeneous detection mode based on singlet oxygen (1 Δ gO2) that achieves similarity to loci (luminal) detection. In both homogeneous detection methods, two recognizers are labeled with two labels. In FRET-based homogeneous assays, a fluorescent label that accepts incident light is called a donor, and another label that emits detected light (fluorescent phosphorescence) is called an acceptor; in homogeneous detection based on singlet oxygen (1 Δ gO2), the label that receives incident light is called a photosensitizer, and the other label that emits the fluorescence to be detected is called a luminophore. FIG. 7A depicts the use of ACE2 alone as the analyte-recognizing agent, one of which is donor (or photosensitizer) -labeled ACE2 and the other of which is acceptor (or luminophore) -labeled ACE 2. FIG. 7B depicts the use of ACE2 in combination with an antibody, where ACE2 is labeled with a donor (or photosensitizer) and the antibody is labeled with an acceptor (or luminophore). The labeling scheme shown in FIG. 7B may be reversed, i.e., ACE2 is labeled with an acceptor (or a luminophore) and the antibody is labeled with a donor (or a photosensitizer), as shown in FIG. 7C for homogeneous detection of S protein.

The above embodiments can be implemented on automated detection systems, as well as on rapid detection platform technologies (e.g., lateral flow immunochromatography). Fig. 8 and 9 show how ACE2 can be used in combination with antibodies in a lateral flow immunochromatographic method. Lateral flow immunochromatography is widely used for rapid detection, and the method provides convenience for directly and rapidly detecting virus particles.

The above examples of the use of ACE2 as analyte-recognizing agent are not all cases where ACE2 may be used, but the essence of the present invention is embodied by these examples.

It is worth noting that since ACE2 is a co-receptor for SARS-CoV and SARS-CoV-2, which is not highly selective or specific in recognizing antigens of pathogens or different pathogens, when more than one coronavirus is circulating, infection by non-SARS-CoV-2 or concurrent infection by more than one pathogen may make the detection result questionable, but this is not uncommon. Meanwhile, the detection method of virus particles and antigens (RBD-containing S protein or RBD-containing S protein fragment, etc.) using ACE2 as a recognition agent still has clinical significance and value for epidemiological investigation, and can be used as a means for assisting diagnosis at least when nucleic acid detection and antibody detection cannot be confirmed, firstly, infectious diseases which are epidemic or outbreak in a certain period are often related to a single pathogen, and secondly, SARS which occurs more than ten years ago has not occurred for several years; third, ACE2 binds weakly to SAR-CoV.

Drawings

FIG. 1 is a schematic representation of the simplified structure of coronaviruses and their spike proteins, together with the receptor binding domain of ACE 2.

FIG. 2 is a conventional immunoassay based on different detection signals.

FIG. 3 is a luminescent label and reaction system used in a conventional immunoassay method.

FIG. 4 shows the competitive method using ACE2 as the analyte-recognizing agent.

FIG. 5 is a sandwich method for detecting S protein using ACE2 in combination with an antibody.

Figure 6 is a method for detecting virions using ACE2 alone and ACE2 in combination with antibodies.

FIG. 7 shows the use of ACE2 in a process involving energy transfer (FRET) or singlet oxygen (F:)¹Δ_gO₂) The homogeneous reaction process of (1).

Figure 8 detection of S protein using ACE2 coated solid particles in a lateral flow immunochromatographic assay.

FIG. 9 detection of S protein on a detection line using ACE2 in a lateral flow immunochromatographic method

FIG. 10 shows the detection of S1 protein in a competition assay using ACE2 as the analyte-recognizing agent.

Detailed Description

1. Labeling recombinant humanized ACE2(rhACE2) with electrically Neutral Ruthenium Complex (NRC)

U.S. Pat. No. 4- (2,2 '-dipyridine-4-yl) butanic acid (NRC 16 for short) is a neutral electrically conductive ruthenium complex used as a luminescent marker in this example, Ru (2,2' -dipyridine) is selected as the marker, and ZL 201480045420.X discloses several markers which help to reduce non-specific adsorption and show stronger luminescent signals under electrochemiluminescent conditions.

Mu.g (1.1nmol) of rhACE2 were dissolved in 500. mu.L of 0.01M PBS (pH 7.4) buffer. Activation of the carboxyl group on NRC16 was performed using EDC (1-ethyl-3- (3-methylenepropyl) -carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysuccinimide sodium salt) in 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer) according to standard procedures. The activated MES solution of NRC16 (containing 5.5nmol of NRC16) was added to PBS buffer of rhACE2 at a predetermined labeling reaction ratio, i.e., a reaction concentration molar ratio of rhACE2 to NRC16 [ rhACE2]: NRC16] ═ 1: 5. After one hour of reaction, an ACE2 derivative (NRC 16-rhACE2) marked by NRC16 is obtained by gel chromatography column separation.

The actual labeling ratio, i.e.the molar ratio of rhACE2 to NRC16 in the labeled product, can be determined by quantitative protein analysis (BCA method) and absorption of NRC16 at 454nm (see ACS Omega 2020,5, 32591-32596).

2. Labelling of ACE2 with biotin

NHS-PEG4-Biotin (MW 588.67kDa) was used as a Biotin labeling reagent, and the molar ratio of the reaction concentrations of the labeling reaction [ rhACE2]: NHS-PEG4-Biotin ] ═ 1:10 was set. Mu.g (1.1nmol) of rhACE2 were dissolved in 500. mu.L of 0.01M PBS (pH 7.4) buffer. Then 11. mu.L of 1mM NHS-PEG4-biotin PBS solution was added. After one hour of reaction, biotin-labeled ACE2 derivatives (Bio-rhACE 2) were isolated by gel chromatography. The biotin-binding capacity of the product was evaluated using the method described in the literature (ACS Omega 2020,5, 32591-32596).

3. Marking of SARS-containing CoV-2RBD protein with NRC16

Mu.g (3.7nmol) of RBD protein (molecular weight 26.5kDa) was dissolved in 500. mu.L of 0.01M PBS (pH 7.4) buffer. Activation of the carboxyl group on NRC16 was performed using EDC (1-ethyl-3- (3-methylenepropyl) -carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysuccinimide sodium salt) in 2- (N-morpholine) ethanesulfonic acid buffer (MES buffer) according to standard procedures. The activated MES solution of NRC16 (containing 18.5nmol of NRC16) was added to PBS buffer of RBD protein at a set labeling reaction ratio, i.e., the reaction concentration molar ratio of RBD protein to NRC16 [ RBD ]: NRC16 ]: 1: 5. After one hour of reaction, the RBD protein marked by NRC16 (NRC 16-RBD for short) is obtained by gel chromatography column separation.

4. Detection of S1 protein by competition method

S1 protein (molecular weight 76.5kDa) in PBS was detected using NRC16-RBD, Bio-rhACE2 and streptavidin-coated magnetic beads (diameter 2.8 μm) in a fully automatic electrochemiluminescence immunoassay analyzer. First, solutions of NRC16-RBD (2.0. mu.g/mL), Bio-rhACE2 (3.0. mu.g/mL), magnetic beads (0.6mg/mL), S1 protein (1.0ng/mL,10ng/mL,100 ng/mL, 1.0. mu.g/mL) and various concentrations of the test substance S1 protein were prepared by dilution with 0.01M PBS (pH 7.4). Prepared NRC16-RBD, Bio-rhACE2 and 5.0mL of magnetic beads are respectively loaded on a preset reagent position on a full-automatic electrochemiluminescence immunoassay analyzer, and S1 protein solutions with different concentrations are loaded on a sample position.

The incubation reaction is carried out in two steps. First, the sampling needle of the instrument mixed the NRC16-RBD and Bio-rhACE2 (50. mu.L each) with 50. mu. L S1 protein samples in an incubation cup and reacted for 5 minutes at an incubation position of 37 ℃, then 50. mu.L of magnetic bead suspension was added to the reaction system, mixed and reacted for 10 minutes at an incubation position of 37 ℃. After the first incubation reaction is finished, the full-automatic analyzer automatically draws 150 mu L of solution from all reaction systems on the incubation positions in sequence, and injects the solution into the electrochemical reaction flow cell for electrochemical luminescence detection. The obtained dimensionless luminous intensity value is output by the instrument.

FIG. 10 is a graph showing the relationship between the concentration signals of S1 protein detected by the competition method. The dotted arrows in the figure indicate that the luminescence signal is increased when the concentration of NRC16-RBD competing with the sample S1 protein is increased, and the luminescence signal is decreased when the concentration of NRC16-RBD is decreased.

The detection of the S1 protein can also be used for detecting virus particles containing the S1 protein on the surface or pseudovirus (pseudovirus) particles (used in scientific research).

5. Detection of viral particles in saliva with biotin-labeled ACE2 and NRC 16-labeled ACE2

This example shows how, due to the numerous spike (S protein) sites on the surface of viral particles, biotin-labeled ACE2 derivative Bio-rhACE2 (3.0. mu.g/mL) and NRC 16-labeled ACE2 derivative NRC16-rhACE2 (2.0. mu.g/mL) can be used to detect viral particles as recognizers for capture of viral particles and labeled recognizers, respectively.

Prepared Bio-rhACE, NRC16-rhACE2 and magnetic beads (0.6mg/mL) are loaded to 5.0mL each of a reagent chamber preset on a full-automatic electrochemiluminescence immunoassay analyzer, and PBS buffer solution (as a known control without virus particles) and saliva sample solution are loaded to the sample chamber. The incubation reaction is carried out in two steps. First, the instrument sampling needle mixed 50. mu.L Bio-rhACE with 50. mu.L magnetic bead suspension in an incubation cup and reacted for 10 minutes at an incubation site of 37 degrees Celsius, then added 50. mu.L sample and NRC16-rhACE2 and continued to react for 10 minutes at an incubation site of 37 degrees Celsius. After the first PBS control sample is incubated, the full-automatic analyzer automatically draws 150 mu L of solution from all reaction systems on the incubation position in sequence, and injects the solution into the electrochemical reaction flow cell for electrochemical luminescence detection. The test results showed that the PBS control sample was very close to the five saliva samples, which is considered a weak signal from non-specific adsorption. Saliva samples can be considered negative samples.

6. Preparation of ACE 2-coated magnetic beads from magnetic beads with carboxyl groups on the surface

In the above embodiments 4 and 5, it is referred to that a biotin-labeled ACE2 derivative and streptavidin-coated magnetic beads are used as reaction components, respectively, to be added to the reaction system in the analysis procedure. Another embodiment involves the preparation of ACE2 coated magnetic beads in advance for direct use in the analysis procedure. The specific operation steps are as follows:

a. the magnetic beads (particle size range 1.0-4.5 μm, 30mg/mL) were suspended and mixed well, washed with 1mL of 25mM MES (pH 5.0) buffer, and spun for 10 minutes, followed by magnetic separation. The magnetic beads are washed repeatedly for 2-3 times.

b. Mu.g of ACE2 (concentration 1mg/mL, added 600. mu.L) was added to the beads (ACE 2/bead coating ratio 0.02mg:1mg), and the mixture was spun at low speed at room temperature for 30 minutes.

c. 100mg/mL EDC solution was prepared in 100mM MES, pH 5.0 buffer and used as is.

d. mu.L of EDC (i.e., 3mg) was added to the reaction solution of magnetic beads and ACE2 and mixed well, and 100. mu.L of 25mM MES was added, pH 5.0 buffer was added to a final volume of 1mL (to make the magnetic beads react at 30mg/mL), and the mixture was spun at low speed at 4 ℃ for 2 hours.

e. Closing and washing the coated beads: the ACE2 coated magnetic beads were spun down in 50mM Tris, pH 7.4 for 15 minutes at room temperature or 50mM ethanolamine in PBS, pH 8.0 for 60 minutes at room temperature to block excess reactive groups. The ACE2 coated magnetic beads were washed four times with 1mL PBS or 50mM Tris. During the washing process, 0.1% Tween-20 or Triton X-100 can be added to reduce non-specific binding, then 0.1% -0.5% BSA or skimmed milk powder is added, and finally the magnetic beads are resuspended in PBS or Tris buffer to the required concentration.

The method can also be used for preparing ACE2 coated solid particles (such as latex microparticles with particle size of 50 nM-10 μ M and quantum dots) made of other materials.

Claims (14)

1. A method for detecting coronavirus particles or its structural component proteins using angiotensin converting enzyme 2(ACE2), the method comprising the steps of:

a. contacting a sample (liquid, solid or gas phase) which may or may not be pretreated and which may contain coronavirus or its structural component proteins, with ACE2 in a liquid phase or on a solid surface comprising an ACE2 derivative;

b. detecting a physical signal generated when the substances are contacted or a signal generated by other substances in the system through physical, chemical or biochemical reaction after the substances are contacted;

c. by analyzing the signals, qualitative or quantitative information about whether or how much coronavirus or its structural protein is present in the sample is obtained.

Said structural component protein is a spike protein on the surface of a coronavirus particle, or a part of a spike protein structure comprising a Receptor Binding Domain (RBD) thereof;

the solid phase includes but is not limited to solid particles, containers, flat plates, fiber fabrics, optical fibers and other materials or objects made of various single or composite materials; the solid phase surface is the surface or end surface of the materials or the objects;

the ACE2 is biological macromolecule which is from human body, animal body, microorganism or obtained by biological engineering and can be combined with Receptor Binding Domain (RBD) of coronavirus spike protein, or ACE2 biological macromolecule complex with larger molecular weight formed by mutual crosslinking, polymerization or aggregation of the biological macromolecules;

the ACE2 derivative is a compound formed by ACE2 or an ACE2 biomacromolecule compound with larger molecular weight, other functional chemical substances (or groups), bioactive substances (such as enzymes, antibodies or structural fragments of the antibodies and the like) and solid particles; these substances include but are not limited to biological coupling groups (such as biotin, etc.), chromogenic substances (such as gold nanoparticles, graphite particles), fluorescent phosphorescent substances (such as quantum dots, rare earth long-life phosphorescent molecules or microspheres, fluorescent molecules or microspheres, etc.), substances capable of participating in chemiluminescent reactions, substances capable of participating in electrochemical reactions, substances capable of participating in energy transfer, enzymes capable of participating in catalytic reactions, etc.;

the ACE2 on the solid phase surface is an ACE2 formed on the solid phase surface or coated on the solid phase surface through covalent bond, adsorption, ligand-receptor interaction or antigen-antibody interaction or is an ACE2 biomacromolecule complex with larger molecular weight formed by mutual crosslinking, polymerization or aggregation on the solid phase surface; the solid phase surface can be completely or partially coated by ACE 2;

preferably, wherein said coronavirus is the COVID-9 pathogen SARS-CoV-2;

preferably, the physical signal generated by the contact or the signal generated by the physical, chemical or biochemical reaction of other substances in the system after the contact is a luminescence, reflection or scattering light signal, more preferably a luminescence (luminescence) signal or an electrical signal (current, voltage, impedance, complex impedance, frequency).

2. The method of claim 1, wherein the pretreatment of the liquid phase sample containing coronavirus or structural component protein thereof comprises artificially adding labeled spike protein, wherein the label is a bioconjugate group (such as biotin, etc.), a chromogenic substance (such as gold nanoparticle, graphite particle), a fluorescent phosphorescent substance (such as quantum dot, rare earth long-life phosphorescent molecule or microsphere, fluorescent molecule or microsphere, etc.), a substance capable of participating in chemiluminescent reaction, a substance capable of participating in electrochemical reaction, an enzyme, etc.; preferably, the label is a nano-gold particle, a chemiluminescent compound, a metal coordination compound or an organometallic compound, preferably a metal coordination compound or organometallic compound of rare earth, ruthenium and iridium.

3. The method of claim 1, wherein the pretreatment of the solid phase sample containing coronavirus or its structural component protein comprises immobilizing coronavirus or its structural component protein on the surface of the solid phase with ACE 2.

4. The method of claim 1, wherein the pretreatment of the solid phase sample containing the coronavirus or the structural component protein thereof comprises immobilizing the coronavirus or the structural component protein thereof on a surface of the solid phase using an antibody.

5. The method of claim 1, wherein the ACE2 derivative is ACE2 alone.

6. The method of claim 1, wherein the ACE2 derivative is also two or more different ACE2 derivatives that bind to coronavirus particles simultaneously or sequentially to form { ACE2 derivative (a)/virion/ACE 2 derivative (b) } complex.

7. The method of claim 1, wherein the ACE2 derivative is alone and after forming { ACE2 derivative/analyte } complex with the analyte, one or more ACE2 at the surface of the solid phase is used to form { ACE2 derivative/virion/ACE 2} complex at the surface of the solid phase.

8. The method of claim 1, wherein when the ACE2 derivative is a single species, one or more labeled antibodies against coronavirus or its structural component proteins are available to form an { ACE2 derivative/analyte/antibody } complex; the marker used for marking the antibody is a bioconjugate group (such as biotin and the like), a substance capable of developing color (such as gold nanoparticles and graphite particles), a fluorescent phosphorescent substance (such as quantum dots, rare earth long-life phosphorescent molecules or microspheres, fluorescent molecules or microspheres and the like), a substance capable of participating in a chemiluminescent reaction, a substance capable of participating in an electrochemical reaction, an enzyme and the like.

9. The method of claim 1, wherein when the ACE2 derivative is one, after it forms a { ACE2 derivative/analyte } complex with the analyte, one or more antibodies directed against coronavirus or its structural component proteins at the surface of the solid phase are used to form a { ACE2 derivative/analyte/antibody } complex at the surface of the solid phase.

10. The method of claim 1, wherein the solid phase surface partially coated with ACE2 is a linear or curved line with ACE2 formed on the solid phase surface) of 0.1-2 mm, or an area of 1-10mm²A spot or a circle.

11. Solid particles of single or composite material, totally or partially coated with ACE2, having a diameter comprised between 50nm and 10 μm.

12. A container, plate, fabric, fiber optic, or like material or article having a surface or end surface coated with all or part of ACE 2.

13. According to claim 11, the solid particles are magnetic particles.

14. According to claim 12, the fiber fabric is a membrane for lateral flow immunochromatography.

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