CN114544947A - Reagent kit for detecting SARS-CoV-2 antigen, method and use thereof - Google Patents
Reagent kit for detecting SARS-CoV-2 antigen, method and use thereof Download PDFInfo
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Abstract
The application relates to a kit, a method and application for detecting SARS-CoV-2 antigen. The application utilizes the characteristic that SARS-CoV-2 antigen has high affinity with human angiotensin converting enzyme II (ACE2) protein, a solid phase carrier coated with the ACE2 protein, polypeptide or protein fragment is used as a capture component, the capture component is combined with SARS-CoV-2 antigen in a sample, and SARS-CoV-2 spike protein S1 subunit protein marked with a detectable signal marker is used as a detection component, and the detection component competes with SARS-CoV-2 antigen in the sample to be detected for a combination site on ACE 2; or the solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein is used as a capture component, ACE2 protein, polypeptide or protein fragment marked with detectable signal marker is used as a detection component, SARS-CoV-2 spike protein S1 subunit protein coated on the solid phase carrier competes with SARS-CoV-2 antigen in a sample to be detected for binding sites on ACE2, and finally the content of SARS-CoV-2 antigen in the sample to be detected is determined through the signal value generated by the signal marker.
Description
Technical Field
The application relates to a kit for detecting SARS-CoV-2 antigen, a method for detecting SARS-CoV-2 antigen and the use of the kit in detecting SARS-CoV-2 antigen.
Background
In 7/1/2020, the pathogenic agent was identified as a novel coronavirus, followed by gene sequence analysis and development of a detection method. On 11/2/2020, the World Health Organization (WHO) formally named the disease caused by the novel coronavirus as COVID-19, while the International Committee for Classification of viruses formally named the novel coronavirus as SARS-CoV-2.
The novel coronavirus (SARS-CoV-2) is a single-stranded positive-strand RNA virus, belongs to a coronavirus of beta genus, has an envelope, is round or oval in particle shape, is usually polymorphic, and has a diameter of 60-140 nm. The common signs of human infection with the novel coronavirus include respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. On 10/1/2020, the first genomic sequence data of SARS-CoV-2 was published, followed by the release of multiple viral genomic sequences isolated from the patient. Xuxinqian et al (Xintian Xu et al, SCIENCE CHINA LIFE SCIENCES, 2020) speculates that the natural host of SARS-CoV-2 is bat like SARS by genetic evolution analysis, and obtains that SARS-CoV-2 is a molecular mechanism for infecting human respiratory epithelial cells by the interaction of Receptor Binding Domain (RBD) on S1 subunit of Spike Protein (S Protein) and human angiotensin converting enzyme II (ACE2) by molecular structure simulation calculation. In vitro experiments of the Wuhan institute of Virus, Stone and Zhengli team of the Chinese academy of sciences (Zhou P et al, Nature, 2020) found that SARS-CoV-2 can infect non-sensitive cells overexpressing human ACE2, indicating that SARS-CoV-2 can invade cells with the same receptor of SARS-CoV, ACE 2. The American researcher (Daniel Wrapp et al, Science, 2020) further showed that SARS-CoV-2 binds ACE2 with higher affinity than SARS-CoV by structural studies of the S protein trimer frozen EM of SARS-CoV-2.
The in vitro detection and confirmation of the novel coronavirus mainly adopts a real-time fluorescent polymerase chain reaction (RT-PCR) method to detect nucleic acid at present. With the continuous rising of the number of people who can be diagnosed, the situation that a large number of highly suspected cases cannot be diagnosed due to negative continuous nucleic acid detection occurs because the positive rate of the nucleic acid detection reagent is too low; a large number of suspected patients cannot be diagnosed in time, and high-risk carrying groups can become infection sources again, so that great adverse effects are brought to epidemic prevention and control.
An effective method recommended by the national clinical test center of Weijian Commission is to supplement the detection of SARS-CoV-2 specific antibodies (IgM or IgG). Antibodies are the product of the immune response of the body following infection with the virus, usually IgM antibodies appear early in the infection and IgG antibodies appear late in the infection. The detection of SARS-CoV-2 antibody can determine whether the patient is infected with the novel coronavirus recently or previously, and can help to confirm the diagnosis of the patient which is negative in nucleic acid detection and clinically suspected. The operation requirement of antibody detection on a clinical laboratory is lower than that of nucleic acid detection, and the antibody detection can be rapidly carried out in a large amount. However, SARS-CoV-2 specific antibody detection has a "window phase" problem: after the novel coronavirus pneumonia occurs for 3-5 days, serum specific antibodies are gradually generated. The first occurring immunoglobulin IgM antibodies were produced in about 5 to 7 days; IgG antibody then appeared, and was produced in about 10-15 days. Usually, the IgM antibody is produced early, is produced rapidly once infected, is maintained for a short time, disappears rapidly, and the detection positive in blood can be used as an indicator of early infection. IgG antibody production is late, maintenance time is long, disappearance is slow, and positive detection in blood can be used as an index of infection and past infection. Because the SARS-CoV-2 antigen needs to generate IgM and IgG after entering the organism through a certain incubation period, and IgM and IgG cannot be detected by serum during this period, the infection of a patient in the window period cannot be detected.
Another rapid, high throughput method is to perform the detection of SARS-CoV-2 antigen. The traditional serological detection of antigen generally adopts a double antibody sandwich method, namely two specific anti-SARS-CoV-2 antibodies are respectively used, wherein one antibody is used as a capture antibody on a solid phase, and the other antibody is used as a detection antibody; when SARS-CoV-2 antigen exists in the sample, the detection reagent is mixed with the sample and forms a sandwich compound of 'antibody-antigen-antibody'; finally, the content of the antigen in the serum or the plasma is determined by detecting the signal value. Since such SARS-CoV-2 antigen detection requires the use of two specific anti-SARS-CoV-2 antibodies, its cost is relatively high.
With the spread of global epidemic situation and the increasing number of infected people, the development of a rapid and high-flux in-vitro diagnosis tool is urgently needed, which can be used for detecting suspected and confirmed cases and screening high-risk groups in an epidemic area, and is beneficial to the overall control of the epidemic situation.
Disclosure of Invention
The object of the present application is to provide a novel kit for detecting SARS-CoV-2 antigen and a method for detecting SARS-CoV-2 antigen.
The present application relates in a first aspect to a kit for the detection of SARS-CoV-2 antigen, comprising at least a capture component and a detection component, wherein the capture component is a solid phase carrier coated with a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is a SARS-CoV-2 spike protein S1 subunit protein labelled with a detectable signal marker, or the capture component is a solid phase carrier coated with a SARS-CoV-2 spike protein S1 subunit protein and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labelled with a detectable signal marker.
Specifically, the kit of the present application does not need to use a specific anti-SARS-CoV-2 antibody, but utilizes the property that the SARS-CoV-2 antigen has high affinity with human angiotensin-converting enzyme II (ACE2) protein, and the solid phase carrier coated with the human angiotensin-converting enzyme II (ACE2) protein, polypeptide or protein fragment is used as a capture component, which binds to SARS-CoV-2 antigen in a sample, while the SARS-CoV-2 spike protein S1 subunit protein labeled with a detectable signal marker is used as a detection component, which competes with the SARS-CoV-2 antigen in a sample to be tested for a binding site on ACE 2; or, the solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein is used as a capture component, simultaneously the human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment which is marked with a detectable signal marker is used as a detection component, the SARS-CoV-2 spike protein S1 subunit protein coated on the solid phase carrier competes with SARS-CoV-2 antigen in a sample to be detected for a binding site on ACE2, finally the content of SARS-CoV-2 antigen in the sample to be detected is determined according to the height of a signal value generated by the signal marker, and the content of SARS-CoV-2 antigen in the sample to be detected is inversely proportional to the height of the signal value.
A second aspect of the present application relates to a method for detecting a SARS-CoV-2 antigen, comprising:
a sample to be tested and a capture component are used;
washing the obtained mixture to wash away unbound substances;
adding a marked detection component into the cleaned mixture, and uniformly mixing to ensure that the detection component and the S1 protein of the SARS-CoV-2 antigen in the sample to be detected compete for the binding site on the human angiotensin converting enzyme II (ACE 2);
washing away unbound substances after the reaction is completed;
adding a detection substrate to the washed reaction mixture to perform detection;
wherein the content of the first and second substances,
the capture component is a solid phase carrier coated with the human angiotensin II (ACE2) protein, polypeptide or protein fragment and
the detection component is SARS-CoV-2 spike protein S1 subunit protein marked with detectable signal marker,
alternatively, the first and second electrodes may be,
the capture component is a solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein, and the detection component is human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment marked with detectable signal marker.
In addition, the application also relates to the use of a kit for detecting SARS-CoV-2 antigen, said kit comprising at least a capture component and a detection component, wherein the capture component is a solid phase carrier coated with a human angiotensin-converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is a SARS-CoV-2 spike protein S1 subunit protein labeled with a detectable signal marker, or the capture component is a solid phase carrier coated with a SARS-CoV-2 spike protein S1 subunit protein and the detection component is a human angiotensin-converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker.
Drawings
Hereinafter, some embodiments of the present application are described with reference to the drawings. Objects and advantages of the present application will be further understood from the following detailed description and the accompanying drawings. The figures show:
FIG. 1 shows a calibration curve of the novel coronavirus (SARS-CoV-2) antigen detection kit 1 of the present application;
FIG. 2 shows a calibration curve of a novel coronavirus (SARS-CoV-2) antigen detection kit 2 of a comparative example;
FIG. 3 shows a calibration curve of the novel coronavirus (SARS-CoV-2) antigen detection kit 3 of the present application.
Detailed Description
Hereinafter, the present application will be described in more detail by specific examples. However, these examples are merely representative and the present application will in no way be construed as being limited by these examples.
When such expressions, features, values, or ranges are referred to in the context of this application in combination with an expression, for example, "about, substantially, generally, at least, minimum," or the like, this application also includes the exact or precise expression, feature, value, or range, and the like.
In the context of the present application, an antibody may also be referred to as an antibody or an antigen-binding antibody fragment thereof, and an antigen-binding antibody fragment refers to a fragment having the same antigen-binding property as that of the original antibody, among partial fragments of an antibody that specifically recognizes an epitope composed of an amino acid sequence.
In the context of the present application, the term detection may be used interchangeably with the terms "determination", "quantitative", "analysis", and the like, and is intended to encompass both quantitative and qualitative determinations. The detection in the present application is preferably performed in vitro.
In the context of the present application, the term Cut-off value refers to a positive determination value and is a criterion for determining whether the detection result is negative or positive. The Cut-off value is generally determined by the analysis of ROC curve.
In the context of the present application, the term ROC (receiver operating characteristic curve) is a curve plotted with the false positive rate (1-specificity) as abscissa and the true positive rate (sensitivity) as ordinate, which is a comprehensive indicator of the reaction sensitivity and the continuous variable of specificity. To comprehensively evaluate the specificity and sensitivity of the kit, the concept of joden index (Youden index) can be used: the jotan index is specificity% + sensitivity% -1, and the Cut-off point corresponding value corresponding to the maximum jotan index can be used as the Cut-off value to ensure that the accuracy of the kit is optimal.
In the context of the present application, the SARS-CoV-2 genome encodes four major structural proteins: spike Protein (S Protein), Nucleocapsid Protein (N Protein), Membrane Protein (M Protein), and Envelope Protein (E Protein); these proteins are essential components of viral particles. As with all coronaviruses, SARS-CoV-2 facilitates entry into the host cell using the S protein, which is the most important surface membrane protein of coronaviruses and contains two subunits (subbunit), namely S1 and S2. Wherein S1 mainly comprises a Receptor Binding Domain (RBD) responsible for recognizing a receptor of a cell; s2 contains essential elements required for the membrane fusion process. However, exposure of RBDs in subunits of the S1 protein results in unstable subunit conformations, and during binding, the subunit undergoes conformational rearrangements in two states, referred to as the up and down conformations, respectively. The downward state temporarily conceals the RBD, while the upward state exposes the RBD, but temporarily destabilizes the protein subunit. In the trimeric S protein, only one of the three RBDs binds to the human angiotensin 2(ACE2) host cell receptor in accessible conformation.
The present application relates in a first aspect to a kit for the detection of SARS-CoV-2 antigen, comprising at least a capture component and a detection component, wherein the capture component is a solid phase carrier coated with a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is a SARS-CoV-2 spike protein S1 subunit protein labeled with a detectable signal marker, or the capture component is a solid phase carrier coated with a SARS-CoV-2 spike protein S1 subunit protein and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker.
In some embodiments of the first aspect of the present application, when the detection component is a SARS-CoV-2 spike protein S1 subunit protein labeled with a detectable signal marker, the detection component competes with SARS-CoV-2 antigen in the sample to be tested for binding sites on the human angiotensin II (ACE2) protein, polypeptide, or protein fragment; when the capture component is a solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein, the capture component competes with SARS-CoV-2 antigen in the sample to be tested for binding sites on the human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment.
In some embodiments of the first aspect of the present application, the human angiotensin-converting enzyme II (ACE2) protein, polypeptide, or protein fragment is a recombinant protein or a native protein.
In some embodiments of the first aspect of the present application, the solid support may be selected from magnetic beads, chips, test strips, and the like, preferably magnetic beads, more preferably superparamagnetic magnetic beads.
In some embodiments of the first aspect of the present application, the kits for detecting SARS-CoV-2 antigen of the present application may further comprise other reagent components, including but not limited to a primary antibody, a labeled secondary antibody, a labeled enzyme, and other labeling substances; a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, a specific binding substance such as biotin-streptavidin, an insoluble carrier, a blocking agent, a diluent, a washing solution, a standard substance and the like.
For example, a kit for use in a chemiluminescent enzyme immunoassay (CLEIA) may include an antibody coated on a solid phase, an enzyme-labeled antibody, a chemiluminescent substrate, a diluent, a washing solution, and the like. Of course, in other embodiments, the antibodies of the embodiments of the present application can also be applied to the preparation of enzyme immunoassay kits, electrochemiluminescence immunoassay kits, immunochromatography kits, and the like based on a double antibody sandwich method or a competition method.
In some embodiments of the first aspect of the present application, the signal label in the detection component can be, for example, a chemiluminescent label (e.g., alkaline phosphatase, luminol, isoluminol, acridinium ester, horseradish peroxidase), an electrochemiluminescent label (e.g., ruthenium terpyridyl), a quantum dot (e.g., gold quantum dot, CdSe quantum dot, ZnCdSe quantum dot, etc.), a fluorescent microsphere, or the like, or a combination thereof.
In some embodiments of the first aspect of the present application, the concentration of the capture component is between 0.1mg/mL and 1.0 mg/mL.
In some embodiments of the first aspect of the present application, the concentration of the detection component is between 0.1 μ g/mL and 2.0 μ g/mL.
A second aspect of the present application relates to a method for detecting a SARS-CoV-2 antigen, comprising:
a sample to be tested and a capture component are used;
washing the obtained mixture to wash away unbound substances;
adding a marked detection component into the cleaned mixture, and uniformly mixing to ensure that the detection component and the S1 protein of the SARS-CoV-2 antigen in the sample to be detected compete for the binding site on the human angiotensin converting enzyme II (ACE 2);
washing away unbound substances after the reaction is completed;
adding a detection substrate to the washed reaction mixture to perform detection;
wherein the content of the first and second substances,
the capture component is a solid phase carrier coated with the human angiotensin II (ACE2) protein, polypeptide or protein fragment and
the detection component is SARS-CoV-2 spike protein S1 subunit protein marked with detectable signal marker,
alternatively, the first and second electrodes may be,
the capture component is a solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein, and the detection component is human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment marked with detectable signal marker.
Examples of the method for immunological measurement using the kit of the present invention include enzyme-linked immunosorbent assay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), fluorescent antibody method (FAT), Fluorescence Enzyme Immunoassay (FEIA), electrochemiluminescence immunoassay (ECLIA), Radioimmunoassay (RIA), immunochromatography, agglutination, competition, and the like, but are not limited thereto. The embodiments of the present application are particularly suited for use in chemiluminescent enzyme immunoassays.
ELISA (enzyme-linked immunological assay) is one of immunoassay methods using an enzyme-labeled antibody, and includes direct methods, indirect methods, and the like, such as competitive ELISA. By competition ELISA is meant: the sample antigen competes with the reference antigen for binding to a specific amount of labeled antibody, and the concentration of the antigen is measured by detecting signal interference, i.e., the more antigen in the sample, the less reference antigen detected, and the weaker the signal. In the context of this application, however, the labeled antibody in a conventional competitive ELISA is replaced with an angiotensin II converting enzyme (ACE2) protein, polypeptide or protein fragment such that the sample antigen competes with the reference antigen for binding to a specific amount of angiotensin II converting enzyme (ACE2) protein, polypeptide or protein fragment.
The chemiluminescence enzyme immunoassay comprises two parts of immunoreaction and chemiluminescence, and the principle is as follows: the antigen in the sample is reacted with the antibody immobilized on the solid phase, then reacted with an enzyme-labeled antibody, washed, then added with a chemiluminescent substrate for an enzyme reaction, and then the luminescence intensity is measured.
In some embodiments of the second aspect of the present application, the SARS-CoV-2 spike protein S1 subunit protein labeled with a detectable signal marker competes with the S1 protein of SARS-CoV-2 in the test sample for a binding site on human angiotensin converting enzyme II (ACE2) when the capture component, the detection component, and the test sample are mixed.
In some embodiments of the second aspect of the present application, when determining the amount of SARS-CoV-2 antigen in the test sample by the level of the signal value generated by the signal marker, the amount of SARS-CoV-2 antigen in the test sample is inversely proportional to the level of the signal value.
In some embodiments of the second aspect of the present application, the sample to be tested may be a nasal swab, a pharyngeal swab, an anal swab, sputum, alveolar lavage, blood, a blood component such as whole blood, serum or plasma, preferably a pharyngeal swab. However, the sample of the present application is not limited thereto, and may be serosal fluid, lymph fluid, or urine.
In some embodiments of the second aspect of the present application, the sample size of the sample to be tested is between 5 μ L and 50 μ L.
In some embodiments of the second aspect of the present application, two different sample-adding methods can be adopted, for example, a sample to be tested can be added first, a capture component can be added for incubation, and then a detection component can be added for incubation (sample-adding method 1); or adding the sample to be detected, the capture component and the detection component, and then incubating (sample adding mode 2). In some embodiments of the second aspect of the present application, it is preferred to employ loading format 1.
In some embodiments of the second aspect of the present application, the incubation time is between 2min and 20 min.
Furthermore, the application relates to the use of a kit for the detection of a SARS-CoV-2 antigen, comprising at least a capture component and a detection component, wherein the capture component is a solid phase carrier coated with a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is a SARS-CoV-2 spike protein S1 subunit protein labelled with a detectable signal marker, wherein the capture component is used to capture the SARS-CoV-2 antigen in a sample to be tested.
The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
EXAMPLE 1 preparation of novel coronavirus (SARS-CoV-2) antigen detection kit 1
Step 1: preparation of Capture Components
Human angiotensin converting enzyme II (ACE2) (10108-H02H, Beijing Yiqiao Shenzhou science and technology Co., Ltd.) was coated on the surface of magnetic beads (Thermo Fisher). The corresponding coating method is described in detail below:
firstly, human angiotensin converting enzyme II (ACE2) (limited Provisions of science and technology of Beijing Yi Qiao Shenzhou, 10108-H02H) is pretreated, and protective components in a buffer matrix of the human angiotensin converting enzyme II are removed through dialysis. The coating is carried out in a ratio of 0.5. mu.g to 40. mu.g of antibody per mg of magnetic beads. Carboxyl on the surface of the magnetic beads is coupled with human angiotensin II (ACE2) under the catalysis of EDC/NHS during the reaction. The typical preparation process is as follows: and (2) taking 20mg of magnetic beads modified with carboxyl on the surfaces, ultrasonically dispersing the magnetic beads in 10mM MES buffer solution, adding 80mg of EDC and 120mg of NHS, ultrasonically mixing the mixture uniformly, and placing the mixture in a shaking table at 37 ℃ for 15 min. Then adding human angiotensin converting enzyme II (ACE2) into the treated magnetic beads according to the proportion, mixing uniformly, and placing the mixture in a shaking table at 37 ℃ for reaction for 10 to 18 hours. After washing and sealing, a magnetic bead coating coated with angiotensin II (ACE2) is prepared. The magnetic bead coating was diluted in 50mM MES buffer (0.5M NaCl, 0.5% BSA, 0.05% tween 20, pH 6.0) at a magnetic bead coating concentration of 0.5 mg/mL.
Step 2: preparation of assay Components
The SARS-CoV-2 spike protein S1-RBD protein (limited Provisions of Science and technology of Beijing Yi Qian Shen, 40592-V05H) is coupled with alkaline phosphatase (Roche Life Science) to prepare the enzyme marker. The enzyme label was diluted in 50mM MES buffer (0.5M NaCl, 0.5% BSA, 0.05% tween 20, pH 6.0) at an enzyme label concentration of 1.0 μ g/mL.
And step 3: sample detection
In the first step, pharyngeal swab samples (10 μ L) taken from confirmed cases of new coronary pneumonia (positive for nucleic acid detection) and magnetic beads coated with human angiotensin-converting enzyme II (ACE2) were added to reaction tubes for incubation. After the reaction is completed, the magnetic beads are attracted by the magnetic field, and unbound substances are washed away.
In the second step, SARS-CoV-2 spike protein S1-RBD protein labeled with alkaline phosphatase is added to the reaction tube and incubated, and SARS-CoV-2 antigen in the sample competes with or prevents SARS-CoV-2 spike protein S1-RBD protein or protein fragment labeled with alkaline phosphatase from reacting with human angiotensin converting enzyme II (ACE2) site on the surface of magnetic beads. After the reaction is completed, the magnetic beads are attracted by the magnetic field, and unbound substances are washed away.
Adding chemiluminescent substrate solution into reaction tube, decomposing luminescent substrate (3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxane, AMPPD) by alkaline phosphatase, removing a phosphate group to generate unstable intermediate product, generating m-oxybenzoic acid methyl ester anion by intramolecular electron transfer, and generating chemiluminescence when the excited m-oxybenzoic acid methyl ester anion returns to ground state from excited state. And measuring the number of photons generated in the reaction by a photomultiplier, wherein the amount of the generated photons is inversely proportional to the content of SARS-CoV-2 antigen in the sample. The calibration curve is shown in fig. 1.
And 4, step 4: determination of Positive judgment value
As positive samples, 83 pharyngeal swab samples from confirmed cases of new coronary pneumonia (positive nucleic acid detection) and 116 pharyngeal swab samples from excluded cases of new coronary pneumonia (negative nucleic acid detection) were collected. And (3) carrying out centrifugal treatment (relative centrifugal force is 1000g, centrifugal time is 5min) before sample testing, transferring the supernatant into a clean sample tube, and testing according to the step 3 of sample detection to obtain the concentration value of SARS-CoV-2 antigen in each sample. A sample test result is subjected to statistical analysis by adopting a Receiver Operating Curve (ROC) method, and the positive judgment value (Cut-off value) of the kit is 5.3 pg/mL.
Comparative example preparation of novel coronavirus (SARS-CoV-2) antigen detection kit 2
Step 1: preparation of Capture Components
Specific anti-SARS-CoV-2 antibody A (Sanyou biomedicine (Shanghai) Co., Ltd., AHA001) was coated on the surface of magnetic beads (Thermo Fisher). The corresponding coating method is described in detail below:
the specific anti-SARS-CoV-2 antibody is first pre-treated and the protective components in its buffering matrix are removed by dialysis. The coating is carried out in a proportion of 0.5. mu.g to 40. mu.g of antibody per mg of magnetic beads. During the reaction, carboxyl on the surface of the magnetic bead is coupled with amino of the specific anti-SARS-CoV-2 antibody under the catalysis of EDC/NHS. The typical preparation process is as follows: and (2) ultrasonically dispersing 20mg of magnetic beads modified with carboxyl on the surface into 10mM MES buffer solution, adding 80mg of EDC and 120mg of NHS, ultrasonically mixing uniformly, and placing in a shaking table at 37 ℃ for 15 min. Then adding a specific anti-SARS-CoV-2 antibody into the treated magnetic beads according to the proportion, mixing uniformly, and placing the mixture in a shaking table at 37 ℃ for reaction for 10 to 18 hours. After cleaning and sealing, preparing the magnetic bead coating material coated with the specific anti-SARS-CoV-2 antibody A. The magnetic bead coating was diluted in 50mM MES buffer (0.5M NaCl, 0.5% BSA, 0.05% tween 20, pH 6.0) at a magnetic bead coating concentration of 0.5 mg/mL.
And 2, step: preparation of assay Components
An enzyme label was prepared by coupling specific anti-SARS-CoV-2 antibody B (Sanyou biomedical (Shanghai) Co., Ltd., AHA003) with alkaline phosphatase (Roche Life Science). The enzyme label was diluted in 50mM MES buffer (0.5M NaCl, 0.5% BSA, 0.05% tween 20, pH 6.0) at an enzyme label concentration of 1.0 μ g/mL.
And step 3: sample detection
In the first step, a pharyngeal swab sample (10. mu.L) collected from a confirmed case of new coronary pneumonia (positive in nucleic acid detection) and a magnetic bead coated with a specific anti-SARS-CoV-2 antibody A were added to a reaction tube and incubated, and SARS-CoV-2 antigen in the sample was bound to the specific anti-SARS-CoV-2 antibody on the surface of the magnetic bead. After the reaction is completed, the magnetic beads are attracted by the magnetic field, and unbound substances are washed away.
In the second step, the detection component, i.e., specific anti-SARS-CoV-2 antibody B labeled with alkaline phosphatase, is added into the reaction tube for incubation, and combined with SARS-CoV-2 antigen on the conjugate formed in the first step to form a sandwich complex. After the reaction is completed, the magnetic beads are attracted by the magnetic field, and unbound substances are washed away.
And thirdly, adding chemiluminescent substrate liquid into the reaction tube, decomposing a luminescent substrate (3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxane, AMPPD) by alkaline phosphatase, removing a phosphate group to generate an unstable intermediate product, generating a methyl meta-oxybenzoate anion through intramolecular electron transfer, and generating chemiluminescence when the methyl meta-oxybenzoate anion in an excited state returns to a ground state from the excited state. The number of photons generated in the reaction is measured by a photomultiplier tube, and the amount of the generated photons is in direct proportion to the content of SARS-CoV-2 antigen in the sample. The calibration curve is shown in fig. 2.
And 4, step 4: determination of Positive judgment value
As positive samples, 83 pharyngeal swab samples from confirmed cases of new coronary pneumonia (positive nucleic acid detection) and 116 pharyngeal swab samples from excluded cases of new coronary pneumonia (negative nucleic acid detection) were collected. And (3) carrying out centrifugal treatment (relative centrifugal force is 1000g, centrifugal time is 5min) before sample testing, transferring the supernatant into a clean sample tube, and testing according to the step 3 of sample detection to obtain the concentration value of SARS-CoV-2 antigen in each sample. A sample test result is subjected to statistical analysis by adopting a Receiver Operating Curve (ROC) method, and the positive judgment value (Cut-off value) of the kit is 6.2 pg/mL.
Example 2 examination of the consistency of the neo-corona antigen assay kit prepared in example 1 and comparative example with the clinical diagnosis result of neo-corona pneumonia
The collected clinical throat swab samples were tested in the "detection method" in example 1 using the antigen assay kits prepared in example 1 and comparative example, respectively, and the test results of the neo-corona antigen assay kits prepared in example 1 and comparative example were compared with the sample nucleic acid detection results for consistency.
The results are shown in Table 1, the positive coincidence rate (sensitivity) of the detection result of the kit 1 of example 1 and the nucleic acid detection result is 88.0%, and the negative coincidence rate (specificity) is 90.6%; the consistency of the test kit 2 of the comparative example and the nucleic acid test results is shown in Table 2, the positive coincidence rate is 88.0%, and the negative coincidence rate is 92.2%.
TABLE 1
TABLE 2
Example 3 examination of the influence of the sample application method on the test results
In order to study the influence of the sample adding mode on the consistency of the detection result of the kit and the detection result of the sample nucleic acid in the sample detection in example 1. The following two sample adding modes are respectively adopted:
sample addition method 1: after incubation with the sample and capture components, the assay components are added (i.e., the steps in example 1).
Sample addition method 2: the sample, capture component and detection component are added first and then incubated.
The remaining test procedures were the same as in example 1.
The consistency of the two sample application methods and the detection result of the sample nucleic acid is studied. As shown in table 3, the test result of the kit 1 of example 1 using the sample application method 2 has a positive coincidence rate of 84.0% and a negative coincidence rate of 89.1% with the sample nucleic acid detection result, which are significantly lower than the result of the sample application method 1 (see table 1). Therefore, the sample addition system 1 is preferable for the kit 1 of example 1.
TABLE 3
Example 4 examination of the influence of the amount of sample on the test results
In order to study the influence of the sample amount on the detection result in the sample detection in example 1, different sample amounts are respectively adopted to test the signal values of the samples 1-4 and calculate the signal-to-noise ratio, and the rest steps are unchanged. Wherein, the sample 1 is a negative control which does not contain SARS-CoV-2 antigen; the antigen concentrations in samples 2 to 4 increased in sequence;
the results are shown in table 4, where the signal-to-noise ratio increases with increasing sample size. When the sample size is 5-10 mu L, the signal-to-noise ratio is obviously improved along with the increase of the sample size, the acceptable sample size (10-50 mu L) is continuously improved, and the signal-to-noise ratio is not obviously improved. Therefore, the sample size is acceptable in the range of 5. mu.L to 50. mu.L, and the sample size is preferably 10. mu.L.
TABLE 4
Example 5 examination of the Effect of incubation time on assay results
In order to study the influence of the incubation time on the detection result in the sample detection in example 1, different incubation times were used to test the signal values of samples 1-4 and calculate the signal-to-noise ratio, and the rest steps were unchanged.
The results are shown in Table 5. The signal to noise ratio increases with increasing incubation time. When the incubation time is 2-5 min, the signal-to-noise ratio is obviously increased along with the increase of the incubation time, the acceptable incubation time (5-20 min) is continuously increased, and the signal-to-noise ratio is not obviously increased. Therefore, the incubation time is acceptable between 2min and 20min, preferably 5 min.
TABLE 5
Example 6 determination of magnetic bead coating of human angiotensin-converting enzyme II (ACE2) and concentration of SARS-CoV-2 spike protein S1-RBD protease marker
The capture fraction was prepared according to the method of example 1, in which the amount of human angiotensin-converting enzyme II (ACE2) coating was fixed at 10. mu.g/mg, and the effect of the concentration of the human angiotensin-converting enzyme II (ACE2) magnetic bead coating and SARS-CoV-2 spike protein S1-RBD protease marker on the signal value and the signal-to-noise ratio was determined.
In one aspect, the concentration of the immobilized SARS-CoV-2 spike protein S1-RBD protease label was 1. mu.g/mL, and the signal values of samples 1-4 were measured and the signal to noise ratio was calculated using different concentrations of the magnetic bead coating of human angiotensin converting enzyme II (ACE2) according to the "sample assay" method of example 1.
As shown in Table 6, as the concentration of the magnetic bead coating increases, the signal-to-noise ratio of the magnetic bead coating at the stage of 0.1mg/mL to 0.5mg/mL is remarkably improved; further increasing the concentration of the magnetic bead coating (0.5 mg/mL-1.0 mg/mL), and the signal-to-noise ratio is not remarkably improved; therefore, the concentration of the magnetic bead coating material is acceptable within the range of 0.1 mg/mL-1.0 mg/mL, and the signal to noise ratio is better when the concentration of the magnetic bead coating material is 0.5 mg/mL.
TABLE 6
On the other hand, the concentration of the magnetic bead coating material for immobilizing human angiotensin-converting enzyme II (ACE2) is 0.5mg/mL, different SARS-CoV-2 spike protein S1-RBD protease marker concentrations are adopted, the signal values of samples 1-4 are tested according to the sample detection method in the embodiment 1, and the signal to noise ratio is calculated.
The results are shown in Table 7. Along with the increase of the concentration of the enzyme marker, the signal-to-noise ratio of the enzyme marker is obviously improved at the stage of 0.1-0.5 mug/mL; (ii) a Further increasing the concentration of an enzyme marker (0.5-2.0 mug/mL), and the signal-to-noise ratio is not remarkably improved; therefore, the concentration of the enzyme marker is acceptable within the range of 0.1 to 2.0 mug/mL, and the signal to noise ratio is better when the concentration of the enzyme marker is 0.5 mug/mL.
TABLE 7
EXAMPLE 7 preparation of novel coronavirus (SARS-CoV-2) antigen detection kit 3
Step 1: preparation of Capture Components
The capture component was prepared as in example 1.
Step 2: preparation of assay Components
The SARS-CoV-2 spike protein S1 subunit protein (Shenzhen Meyer biomedical electronics limited company) and alkaline phosphatase (Roche Life Science) are coupled to prepare the enzyme label. The enzyme label was diluted in 50mM MES buffer (0.5M NaCl, 0.5% BSA, 0.05% Tween 20, pH 6.0) and the concentration of the enzyme label was 1.0. mu.g/mL.
And step 3: sample detection
In the first step, a sample (10 μ L) and magnetic beads coated with human angiotensin-converting enzyme II (ACE2) were added to a reaction tube and incubated. After the reaction is completed, the magnetic beads are attracted by the magnetic field, and unbound substances are washed away.
In the second step, SARS-CoV-2 spike protein S1 labeled with alkaline phosphatase is added to the reaction tube and incubated, and SARS-CoV-2 antigen in the sample competes with or prevents SARS-CoV-2 spike protein S1 labeled with alkaline phosphatase or protein fragment from reacting with the human angiotensin converting enzyme II (ACE2) site on the surface of the magnetic beads. After the reaction is completed, the magnetic beads are attracted by the magnetic field, and unbound substances are washed away.
Adding chemiluminescent substrate solution into reaction tube, decomposing luminescent substrate (3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxane, AMPPD) by alkaline phosphatase, removing a phosphate group to generate unstable intermediate product, generating m-oxybenzoic acid methyl ester anion by intramolecular electron transfer, and generating chemiluminescence when the excited m-oxybenzoic acid methyl ester anion returns to ground state from excited state. And measuring the number of photons generated in the reaction by a photomultiplier, wherein the amount of the generated photons is inversely proportional to the content of SARS-CoV-2 antigen in the sample. The calibration curve is shown in fig. 3.
And 4, step 4: determination of Positive judgment value
As positive samples, 83 pharyngeal swab samples from confirmed cases of new coronary pneumonia (positive nucleic acid detection) and 116 pharyngeal swab samples from excluded cases of new coronary pneumonia (negative nucleic acid detection) were collected. And (3) carrying out centrifugal treatment (relative centrifugal force is 1000g, centrifugal time is 5min) before sample testing, transferring the supernatant into a clean sample tube, and testing according to the step 3 of sample detection to obtain the concentration value of SARS-CoV-2 antigen in each sample. A sample test result is subjected to statistical analysis by adopting a Receiver Operating Curve (ROC) method, and the positive judgment value (Cut-off value) of the kit is 6.9 pg/mL.
Example 8 examination of the consistency of the detection results of the antigen measurement kit prepared in example 7 and the new corona in the sample
The collected clinical pharyngeal swab samples (samples were the same as in example 2) were tested by the "detection method" in example 7 using the neo-corona antigen assay kit prepared in example 7, and the consistency of the detection result of the neo-corona antigen assay kit prepared in example 7 and the detection result of the neo-corona nucleic acid of the samples was compared.
The results are shown in Table 8, and the test results of the kit in example 7 and the nucleic acid detection results of the samples showed a positive coincidence rate of 86.0% and a negative coincidence rate of 92.2%.
TABLE 8
Example 9 examination of the influence of the sample application method on the test results
In order to study the influence of the sample adding mode on the consistency of the detection result of the kit and the detection result of the sample nucleic acid in the sample detection in example 7. The following two sample adding modes are respectively adopted:
sample addition method 1: after the sample, capture and detection components are added, incubation is performed (i.e., the procedure of example 1).
Sample addition method 2: the sample and the capture component are added for incubation, and then the detection component is added for incubation.
The rest of the detection procedure was the same as in example 1.
The consistency of the two sample application methods and the detection result of the sample nucleic acid is studied. As shown in Table 9, the test result of sample application method 2 showed a positive match rate of 82.0% and a negative match rate of 89.1% with the nucleic acid test result, which was lower than that of sample application method 1 (see Table 8), and thus sample application method 1 was preferred for the kit 3 of example 7.
TABLE 9
Example 10 examination of the influence of the amount of sample on the test results
In order to study the influence of the sample amount on the detection result in the sample detection in example 7, different sample amounts are respectively adopted to test the signal values of the samples 1-4 and calculate the signal-to-noise ratio, and the rest steps are unchanged.
The results are shown in table 10, where the signal-to-noise ratio increases with increasing sample size. When the sample size is 5-10 mu L, the signal-to-noise ratio is obviously improved along with the increase of the sample size, the acceptable sample size (10-50 mu L) is continuously improved, and the signal-to-noise ratio is not obviously improved. Therefore, a sample size of 5. mu.L to 50. mu.L is acceptable, and a sample size of 10. mu.L is preferred.
Watch 10
Example 11 examination of the Effect of incubation time on the results of measurement
In order to study the influence of the incubation time on the detection result in the sample detection in example 7, different incubation times were used to test the signal values of samples 1-4 and calculate the signal-to-noise ratio, and the rest steps were unchanged.
The results are shown in table 11, where the signal to noise ratio increases with increasing incubation time. When the incubation time is 2-5 min, the signal-to-noise ratio is obviously increased along with the increase of the incubation time, the acceptable incubation time (5-20 min) is continuously increased, and the signal-to-noise ratio is not obviously increased. Therefore, an incubation time of 2min to 20min is acceptable, preferably an incubation time of 5 min.
TABLE 11
Example 12 determination of magnetic bead coating of human angiotensin-converting enzyme II (ACE2) and concentration of SARS-CoV-2 spike protein S1 protease marker
The capture fraction was prepared according to the method of example 1, in which the amount of human angiotensin-converting enzyme II (ACE2) coating was fixed at 10. mu.g/mg, and the effect of the concentration of human angiotensin-converting enzyme II (ACE2) magnetic bead coating and SARS-CoV-2 spike protein S1 protease marker on the signal value and signal-to-noise ratio was determined.
In one aspect, the concentration of the protease marker of SARS-CoV-2 spike protein S1 was fixed at 1. mu.g/mL, samples 1-4 were tested for signal values and the signal to noise ratio calculated using different concentrations of magnetic bead coatings of human angiotensin converting enzyme II (ACE2) according to the "sample detection" method of example 1.
The results are shown in table 12, and as the concentration of the magnetic bead coating increases, the signal-to-noise ratio of the magnetic bead coating at the stage of 0.1mg/mL to 0.5mg/mL is remarkably improved; further increasing the concentration of the magnetic bead coating (0.5 mg/mL-1.0 mg/mL), and the signal-to-noise ratio is gradually improved; therefore, the concentration of the coating material on the magnetic beads is acceptable in the range of 0.1mg/mL to 1.0mg/mL, and the concentration of the coating material on the magnetic beads is preferably 0.5 mg/mL.
TABLE 12
On the other hand, the concentration of the immobilized human angiotensin-converting enzyme II (ACE2) magnetic bead coating was 0.5mg/mL, and the signal values of samples 1 to 4 were tested and the signal-to-noise ratio was calculated according to the "sample detection" method of example 1 using different SARS-CoV-2 spike protein S1 protease marker concentrations.
The results are shown in Table 13, and the signal-to-noise ratio of the enzyme marker concentration is remarkably improved at the stage of 0.1-0.5 mu g/mL along with the increase of the enzyme marker concentration; further increasing the concentration of the enzyme marker (0.5-2.0 mug/mL), and the signal-to-noise ratio is gradually improved; therefore, an enzyme label concentration in the range of 0.1. mu.g/mL to 2.0. mu.g/mL is acceptable, and an enzyme label concentration of 0.5. mu.g/mL is preferred.
Watch 13
From the various embodiments described above, it follows that: the kit of the embodiment of the application has high specificity and sensitivity and is suitable for detecting the novel coronavirus (SARS-CoV-2) antigen in a sample to be detected, and meanwhile, the kit for detecting the novel coronavirus (SARS-CoV-2) antigen can realize the detection of the SARS-CoV-2 antigen without adopting a specific antibody, thereby greatly reducing the detection cost and improving the quality and the precision of the detection of the SARS-CoV-2 antigen.
The present application is not limited thereto by the description according to the embodiments. Rather, the application encompasses any novel feature and any combination of features, which in particular includes any combination of features in the patent claims, even if this feature or this combination itself is not specified in detail in the patent claims or exemplary embodiments.
Claims (12)
1. A kit for detecting SARS-CoV-2 antigen, comprising at least a capture component and a detection component, wherein,
the capture component is a solid phase carrier coated with human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is SARS-CoV-2 spike protein S1 subunit protein marked with a detectable signal marker,
alternatively, the first and second electrodes may be,
the capture component is a solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein and the detection component is human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker.
2. The kit according to claim 1, wherein,
the protein, polypeptide or protein fragment of the human angiotensin converting enzyme II (ACE2) is recombinant protein or natural protein.
3. The kit according to claim 1, wherein,
the solid phase carrier is a magnetic microbead.
4. The kit according to claim 1, wherein,
the concentration of the capture component is between 0.1mg/mL to 1.0 mg/mL.
5. The kit according to claim 1, wherein,
the concentration of the detection component is between 0.1 μ g/mL and 2.0 μ g/mL.
6. A method for detecting a SARS-CoV-2 antigen, comprising:
mixing and incubating a sample to be detected, a capture component and a detection component to detect SARS-CoV-2 antigen;
wherein the content of the first and second substances,
the capture component is a solid phase carrier coated with human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is SARS-CoV-2 spike protein S1 subunit protein marked with a detectable signal marker,
alternatively, the first and second electrodes may be,
the capture component is a solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein and the detection component is marked with detectable signal human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment.
7. The method of claim 6, wherein the mixing and incubating the test sample, the capture component, and the detection component to detect the SARS-CoV-2 antigen comprises:
mixing the sample to be tested and the capture component;
washing the obtained mixture to wash away unbound substances;
adding the marked detection component into the cleaned mixture and uniformly mixing;
washing away unbound substances after the reaction is completed;
a detection substrate is added to the washed reaction mixture and incubated to perform detection.
8. The method of claim 6, wherein,
the solid phase carrier is magnetic microbeads, and the magnetic microbeads are adsorbed by a magnetic field for cleaning.
9. The method of claim 6, wherein,
the sample to be detected is selected from nasal swab, throat swab, anal swab, sputum, alveolar lavage fluid, blood, serum, plasma and urine.
10. The method of claim 6, wherein,
the sample size of the sample to be detected is between 5 and 50 mu L.
11. The method of claim 6, wherein,
the incubation time is between 2min and 20 min.
12. An application of the reagent kit in detecting SARS-CoV-2 antigen,
the kit comprises at least a capture component and a detection component, wherein,
the capture component is a solid phase carrier coated with human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is SARS-CoV-2 spike protein S1 subunit protein marked with a detectable signal marker,
alternatively, the first and second electrodes may be,
the capture component is a solid phase carrier coated with SARS-CoV-2 spike protein S1 subunit protein and the detection component is human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker.
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