CN114544958A - 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 for detecting SARS-CoV-2 antigen, a method for detecting SARS-CoV-2 antigen and the use of a kit in detecting SARS-CoV-2 antigen. The kit for detecting SARS-CoV-2 antigen of the invention can realize the detection of SARS-CoV-2 antigen only by adopting a specific antibody, wherein the kit for detecting SARS-CoV-2 antigen at least comprises a capture component and a detection component, and the capture component and the detection component are respectively selected from specific anti-SARS-CoV-2 antibody or human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment. After the capture component and the detection component in the kit are mixed and incubated with a sample to be detected, a sandwich compound of 'antibody-antigen-ACE 2' is formed, and finally the content of SARS-CoV-2 antigen in the sample to be detected is determined according to the signal value generated on 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 a kit in detecting SARS-CoV-2 antigen.
Background
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. After people are infected with the novel coronavirus, the common signs of people 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. Xuxinbi et al (Xintian Xu et al, SCIENCE CHINA LIFE SCIENCES, 2020) speculate that the natural host of SARS-CoV-2 is bat like SARS by genetic evolution analysis, and the molecular structure simulation calculation shows that SARS-CoV-2 is a molecular mechanism for infecting human respiratory epithelial cells by the interaction of the Receptor Binding Domain (RBD) on the S1 subunit of Spike Protein (S Protein) and human angiotensin converting enzyme II (ACE 2). The inventor of the Chinese academy of sciences Shizhen team (Zhou P et al, Nature, 2020) found that SARS-CoV-2 can infect non-sensitive cells overexpressing human ACE2 by in vitro experiments, indicating that SARS-CoV-2 can invade cells with the same receptor ACE2 of SARS-CoV. The American researcher (Daniel Wrapp et al, Science, 2020) further showed that SARS-CoV-2 binds ACE2 with higher affinity than SARS-CoV by cryo-EM structural studies of S protein trimer of SARS-CoV-2.
Currently, the in vitro detection and diagnosis of the novel coronavirus mainly adopts a real-time fluorescent polymerase chain reaction (RT-PCR) method to carry out nucleic acid detection. 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 prevention and control of epidemic situations.
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 a patient has been infected with a novel coronavirus recently or in the past, and can help to confirm the diagnosis of a patient who is negative in nucleic acid detection but 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. Firstly, immunoglobulin IgM antibodies appear, which are produced in about 5 to 7 days; IgG antibody then appeared, and was produced in about 10-15 days. Usually, IgM antibodies are produced early, rapidly produced upon infection, maintained for a short time, and disappeared rapidly, and detection of positivity in blood can be used as an indicator of early infection. The IgG antibody is produced late, maintained for a long time, disappeared slowly, and the positive detection in blood can be used as the 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 antigens 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 incubated to form 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.
At present, a rapid and high-flux in-vitro diagnosis tool is urgently needed to be developed, can be used for detecting suspected and confirmed cases, and can also be used for screening high-risk groups in an epidemic area, so that the epidemic situation can be comprehensively controlled.
Disclosure of Invention
The purpose of the application is to provide 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. The kit for detecting SARS-CoV-2 antigen can realize the detection of SARS-CoV-2 antigen by only adopting one specific antibody.
The present application relates in a first aspect to 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 support coated with a specific anti-SARS-CoV-2 antibody and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker, or the capture component is a solid support coated with a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is a specific anti-SARS-CoV-2 antibody labeled with a detectable signal marker, wherein the capture component is used to capture SARS-CoV-2 antigen in a sample to be tested.
A second aspect of the present application relates to a method for detecting a SARS-CoV-2 antigen, the method comprising:
mixing the sample to be tested with the capture component;
washing the obtained mixture to remove unbound material;
adding a labeled detection component into the washed mixture, and uniformly mixing to enable the detection component to be combined with the mixture to form a sandwich compound;
washing the sandwich complex to remove unbound material;
a detection substrate is added to the washed sandwich complex for detection.
Specifically, in the method for detecting SARS-CoV-2 antigen of the present application, a solid phase carrier coated with a specific anti-SARS-CoV-2 antibody is used as a capture component, while taking advantage of the property of SARS-CoV-2 antigen having high affinity with human angiotensin converting enzyme II (ACE2) protein, ACE2 protein, polypeptide or protein fragment labeled with a detectable signal marker is used as a detection component; alternatively, a solid support coated with ACE2 protein, polypeptide or protein fragment is used as the capture component and a specific anti-SARS-CoV-2 antibody labelled with a detectable signal label is used as the detection component. After the capture component, the detection component and the detection substrate are mixed and incubated with a sample to be detected, a sandwich compound of anti-SARS-CoV-2 antibody-SARS-CoV-2 antigen-ACE 2 is formed, and finally the content of SARS-CoV-2 antigen in the sample is determined by the signal value generated on the signal marker.
Furthermore, the application relates to the use of a kit for the detection of a SARS-CoV-2 antigen, the kit comprising at least a capture component and a detection component, wherein the capture component is a solid phase carrier coated with a specific anti-SARS-CoV-2 antibody and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker, or 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 specific anti-SARS-CoV-2 antibody labeled with a detectable signal marker, wherein the capture component is used to capture the SARS-CoV-2 antigen in a sample to be tested.
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, which is a criterion for determining whether the detection result is positive or negative. The Cut-off value is generally determined by the analysis of ROC curve.
In the context of the present application, the term ROC curve (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 specificity continuous variable. 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 accuracy of the kit can be guaranteed to be optimal by taking the corresponding value of the interception point corresponding to the maximum jotan index as the Cut-off value.
The first aspect of the present application relates to 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 support coated with a specific anti-SARS-CoV-2 antibody and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker, or the capture component is a solid support coated with a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment and the detection component is a specific anti-SARS-CoV-2 antibody labeled with a detectable signal marker, wherein the capture component is used to capture SARS-CoV-2 antigen in a sample to be tested.
In some embodiments of the first aspect of the present application, the anti-SARS-CoV-2 antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a single chain antibody, or an antigen-binding fragment of a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, or a single chain antibody.
In some embodiments of the first aspect of the present application, the anti-SARS-CoV-2 antibody specifically binds to any epitope on SARS-CoV-2; preferably, Spike Protein (S Protein) that specifically binds SARS-CoV-2; more preferably, specifically binds to the S1 subunit of the spike protein of SARS-CoV-2; most preferably, it specifically binds to the non "Receptor Binding Domain (RBD) of the S1 subunit of SARS-CoV-2.
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, primary antibodies, labeled antibodies or ACE2, labeled enzymes, and like 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, the method comprising:
mixing the sample to be tested with the capture component;
washing the obtained mixture to remove unbound material;
adding a labeled detection component into the washed mixture, and uniformly mixing to enable the detection component to be combined with the mixture to form a sandwich compound;
washing the sandwich complex to remove unbound material;
adding a detection substrate to the washed sandwich complex to perform detection;
wherein the content of the first and second substances,
when the capture component is a solid phase carrier coated with a specific anti-SARS-CoV-2 antibody, the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker, or, when the capture component is a solid phase carrier coated with a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment, the detection component is a specific anti-SARS-CoV-2 antibody labeled with a detectable signal marker, wherein the capture component is used for capturing SARS-CoV-2 antigen in a sample to be tested.
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 method, competition method, 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 immunoassays using an enzyme-labeled antibody, and includes direct methods, indirect methods, and the like, such as sandwich ELISA. By sandwich ELISA is meant: antibodies having different antigen recognition sites are used, one of which is coated on a solid phase, and the other is used to hold an antigen to be detected, thereby forming an antibody-antigen-antibody complex.
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 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 application methods can be used, for example, after the sample to be detected is added and the capture component is incubated, the magnetic beads are attracted by the magnetic field, the unbound substances are washed away, and the detection component is added for incubation (sample application 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 present application relates to the use of a kit according to the first aspect of the present application for the detection of SARS-CoV-2 antigen.
Furthermore, the present application relates to the use of a kit for the detection of a 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 specific anti-SARS-CoV-2 antibody and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker, or 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 specific anti-SARS-CoV-2 antibody labeled with a detectable signal marker, wherein the capture component is used to capture the SARS-CoV-2 antigen in a sample to be tested.
Some examples of the present application are described below, which are not intended to limit the present application but to better illustrate the present application.
EXAMPLE 1 preparation of novel coronavirus (SARS-CoV-2) antigen detection kit 1
Step 1: preparation of Capture Components
A specific anti-SARS-CoV-2 antibody (AHA 001, Sanyou biological medicine, Inc.) is coated on the surface of a magnetic microbead (magnetic bead for short) (Thermo Fisher). The corresponding coating method is described in detail below:
the specific anti-SARS-CoV-2 antibody is first pre-treated and its protective components in the buffer 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) 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 specific anti-SARS-CoV-2 antibody into the treated magnetic beads according to a certain proportion, uniformly mixing them, placing them into a shaker at 37 deg.C and making them react for 10-18 h. After washing and sealing, the magnetic bead coating material coated with the specific anti-SARS-CoV-2 antibody 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 enzyme label was prepared by coupling human angiotensin converting enzyme II (ACE2) (10108-H02H, Beijing Yi Qiao Shenzhou Science, Inc.) 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 (20. 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 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, namely the protein of human angiotensin-converting enzyme II (ACE2) marked with alkaline phosphatase, is added into a reaction tube for incubation, and is combined with the 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 by intramolecular electron transfer of the intermediate product, 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 quantity 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. 1.
And 4, step 4: determination of Positive judgment value
As positive samples, 83 pharyngeal swab samples (nucleic acid detection was positive) collected from confirmed cases of new coronary pneumonia and 116 pharyngeal swab samples (nucleic acid detection was negative) collected from excluded cases of new coronary pneumonia were collected. Centrifuging (relative centrifugal force 1000g, centrifuging time 5min) before sample testing, transferring supernatant into clean sample tube, and testing according to step 3 "sample detection" to obtain 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 5.9 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 its protective components in the buffer 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) 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 specific anti-SARS-CoV-2 antibody into the treated magnetic beads according to a certain proportion, uniformly mixing them, placing them into a shaker at 37 deg.C and making them react for 10-18 h. After washing 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.
Step 2: 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 (20. 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 by intramolecular electron transfer of the intermediate product, 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 quantity 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 (nucleic acid detection was positive) collected from confirmed cases of new coronary pneumonia and 116 pharyngeal swab samples (nucleic acid detection was negative) collected from excluded cases of new coronary pneumonia were collected. Centrifuging (relative centrifugal force 1000g, centrifuging time 5min) before sample testing, transferring supernatant into clean sample tube, and testing according to step 3 "sample detection" to obtain 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 SARS-CoV-2 antigen detection kit prepared in example 1 and comparative example with the results of nucleic acid detection of a sample
The collected clinical throat swab specimens (centrifuged) were tested by the "detection method" in example 1 using the SARS-CoV-2 antigen detection kits prepared in example 1 and comparative example, respectively, and the results of the SARS-CoV-2 antigen detection kits prepared in example 1 and comparative example were compared with the results of nucleic acid detection of the specimens, respectively. The results of 114 clinical pharyngeal swab samples were as follows:
the consistency of the detection result of the kit 1 and the detection result of the nucleic acid in the embodiment 1 is shown in table 1, wherein the positive coincidence rate is 90.0 percent, and the negative coincidence rate is 95.3 percent; 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 magnetic beads are attracted to the magnetic field, unbound material is washed away, and the detection components are added for incubation (i.e., the steps in example 1).
Sample addition method 2: after the sample, capture component and detection component are added, incubation is performed.
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 88.0% and a negative coincidence rate of 92.2% 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 of the present application.
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 were sequentially increased;
the results are shown in table 4, where the signal-to-noise ratio increases with increasing sample size. When the sample size is 5-20 mu L, the signal-to-noise ratio is obviously improved along with the increase of the sample size, the acceptable sample size (20-50 mu L) is continuously improved, the signal-to-noise ratio is not obviously improved, and the signal-to-noise ratio is acceptable. Therefore, a sample size of 5. mu.L to 50. mu.L is acceptable, and a sample size of 20. mu.L is preferred.
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 2-10 min, the signal-to-noise ratio is obviously increased along with the increase of the incubation time, the acceptable incubation time (10-20 min) is continuously increased, the signal-to-noise ratio is not obviously increased, and the incubation time is acceptable. Therefore, the incubation time is acceptable from 2min to 20min, preferably 10 min.
TABLE 5
EXAMPLE 6 magnetic bead coating of specific anti-SARS-CoV-2 antibody and determination of the concentration of the enzyme marker for human angiotensin-converting enzyme II (ACE2)
A capture fraction was prepared according to the method in example 1, in which the amount of the specific anti-SARS-CoV-2 antibody coating was fixed at 10. mu.g/mg, and the influence of the specific anti-SARS-CoV-2 antibody magnetic bead coating and the concentration of the human angiotensin-converting enzyme II (ACE2) enzyme label on the signal value and the signal-to-noise ratio was determined.
In one aspect, the concentration of the enzyme label of immobilized angiotensin-converting enzyme II (ACE2) was 1. mu.g/mL, and the signal values of samples 1-4 were tested and the signal to noise ratio calculated using different specific anti-SARS-CoV-2 antibody magnetic bead envelope concentrations according to the "sample detection" 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 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 6
On the other hand, the concentration of the immobilized specific anti-SARS-CoV-2 antibody magnetic bead coating is 0.5mg/mL, different concentrations of the angiotensin-converting enzyme II (ACE2) enzyme label of human are adopted, the signal values of the samples 1 to 4 are tested according to the method of sample detection 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; 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.
TABLE 7
EXAMPLE 7 preparation of novel coronavirus (SARS-CoV-2) antigen detection kit 3
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 the magnetic beads (Thermo Fisher).
The specific coating method comprises the following steps: human angiotensin converting enzyme II (ACE2) was first pre-treated and its protective components in the buffer matrix were 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. Carboxyl on the surface of the magnetic beads is coupled with amino of angiotensin-converting enzyme 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 shaker 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
An enzyme label was prepared by coupling a specific anti-SARS-CoV-2 antibody (Sanyou biomedical (Shanghai) Co., Ltd., AHA001) 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 (20. mu.L) obtained from a diagnosed case of neocoronary pneumonia (positive in nucleic acid detection) and magnetic beads coated with human angiotensin-converting enzyme II (ACE2) were added to a reaction tube and incubated, and SARS-CoV-2 antigen in the sample was bound to human angiotensin-converting enzyme II (ACE2) 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.
In the second step, the detection component, i.e., the specific anti-SARS-CoV-2 antibody labeled with alkaline phosphatase, is added to the reaction tube and incubated, and binds to the SARS-CoV-2 antigen on the conjugate formed in the first step, forming 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 by intramolecular electron transfer of the intermediate product, 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 quantity 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. 3.
And 4, step 4: determination of Positive judgment value
As positive samples, 83 pharyngeal swab samples (nucleic acid detection was positive) collected from confirmed cases of new coronary pneumonia and 116 pharyngeal swab samples (nucleic acid detection was negative) collected from excluded cases of new coronary pneumonia were collected. Centrifuging (relative centrifugal force 1000g, centrifuging time 5min) before sample testing, transferring supernatant into clean sample tube, and testing according to step 3 "sample detection" to obtain 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 5.8 pg/mL.
Example 8 examination of the consistency of the SARS-CoV-2 antigen detection kit 3 prepared in example 7 with the detection result of the sample neocoronal nucleic acid
Using the SARS-CoV-2 antigen detection kit 3 prepared in example 7, a collected clinical throat swab sample (sample was the same as in example 2) was tested by the "detection method" in example 7, and the consistency between the detection result of the SARS-CoV-2 antigen detection kit 3 prepared in example 7 and the detection result of the nucleic acid in the sample was compared.
The results are shown in Table 8, and the test result of the kit 3 of example 7 has a positive coincidence rate of 88.0% and a negative coincidence rate of 93.8% with the nucleic acid detection result of the sample.
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 coincidence rate of the detection result and the clinical diagnosis result of the kit in the sample detection in example 7. The following two sample adding modes are respectively adopted:
sample addition method 1: after incubation with the sample and capture components, the magnetic beads are attracted to the magnetic field, unbound material is washed away, and the detection components are added for incubation (i.e., the procedure of example 7).
Sample addition mode 2: after the sample, capture component and detection component are added, incubation is performed.
The remaining test procedures were the same as in example 7.
The consistency of the detection results of the two sample adding methods and the detection result of the sample nucleic acid is researched. As shown in Table 9, the test result of sample application method 2 has a positive coincidence rate of 86.0% and a negative coincidence rate of 89.1% with the nucleic acid test result, which is slightly lower than that of sample application method 1 (see Table 8), and thus, sample application method 1 is preferable for the kit 3 of example 7 of the present application.
TABLE 9
Example 10 examination of the Effect of sample amount 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 5-20 mu L, the signal-to-noise ratio is obviously improved along with the increase of the sample size, the sample size (20-50 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 20. mu.L is preferred.
Watch 10
Example 11 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 7, the signal values of the samples 1 to 4 were tested with different incubation times and the signal-to-noise ratio was calculated, 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 2-10 min, the signal-to-noise ratio is obviously increased along with the increase of the incubation time, the incubation time is continuously increased (10-20 min), and the signal-to-noise ratio is not obviously increased. Therefore, the incubation time is acceptable in the range of 2min to 20min, preferably 10 min.
TABLE 11
EXAMPLE 12 determination of the concentration of the magnetic bead coating of angiotensin-converting enzyme II (ACE2) and the enzyme marker of the specific anti-SARS-CoV-2 antibody
A capture fraction was prepared according to the method in example 7, 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 the specific anti-SARS-CoV-2 antibody enzyme label on the signal value and the signal-to-noise ratio was determined.
In one aspect, the enzyme label concentration of the immobilized specific anti-SARS-CoV-2 antibody is 1. mu.g/mL, samples 1-4 are tested for signal value and the signal to noise ratio is calculated using different concentrations of the magnetic bead coating of human angiotensin converting enzyme II (ACE2) according to the "sample detection" method of example 7.
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) antibody magnetic bead coating is 0.5mg/mL, different specific anti-SARS-CoV-2 antibody enzyme label concentrations are adopted, the signal values of the samples 1-4 are tested according to the method of sample detection in example 7, and the signal to noise ratio is calculated.
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 SARS-CoV-2 antigen in a sample to be detected, and meanwhile, the kit for detecting SARS-CoV-2 antigen of the application can realize the detection of SARS-CoV-2 antigen only by adopting one specific antibody, thereby improving the quality and the precision of the detection of SARS-CoV-2 antigen and greatly reducing the detection cost.
The present application is not limited thereto by the description according to the embodiments. Rather, the application includes every novel feature and every combination of features, which is contained in the claims, in particular, even if this feature or this combination itself is not specified in the claims or exemplary embodiments.
Claims (13)
1. A kit for detecting SARS-CoV-2 antigen, which at least comprises a capture component, a detection component,
wherein the capture component is a solid phase carrier coated with a specific anti-SARS-CoV-2 antibody and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker,
alternatively, the first and second electrodes may be,
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 a specific anti-SARS-CoV-2 antibody marked with a detectable signal marker,
wherein the capture component is used for capturing SARS-CoV-2 antigen in a sample to be tested.
2. The kit according to claim 1, wherein,
the anti-SARS-CoV-2 antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a single chain antibody, or an antigen-binding fragment of a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, or a single chain antibody.
3. The kit according to claim 1 or 2, wherein,
the anti-SARS-CoV-2 antibody specifically binds to the non-receptor binding region of the S1 subunit of SARS-CoV-2.
4. 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.
5. The kit according to claim 1, wherein,
the solid phase carrier is a magnetic microbead.
6. The kit according to claim 1, wherein,
the concentration of the capture component is between 0.1mg/mL to 1.0 mg/mL.
7. The kit according to claim 1, wherein,
the concentration of the detection component is between 0.1 μ g/mL and 2 μ g/mL.
8. 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 first and the second end of the pipe are connected with each other,
the capture component is a solid phase carrier coated with a specific anti-SARS-CoV-2 antibody, and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment marked with a detectable signal marker,
alternatively, the first and second electrodes may be,
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 a specific anti-SARS-CoV-2 antibody labeled with a detectable signal marker.
9. The method of claim 8, 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 remove unbound material;
adding the labeled detection component into the washed mixture, and uniformly mixing to enable the detection component to be combined with the mixture to form a sandwich compound;
washing the sandwich complex to remove unbound material;
a detection substrate is added to the washed sandwich complex and incubated to perform detection.
10. The method of claim 8, wherein,
the sample to be detected is selected from nasal swab, pharyngeal swab, anal swab, sputum, alveolar lavage fluid, blood, serum, plasma and urine.
11. The method of claim 8, wherein,
the sample size of the sample to be detected is between 5 and 50 mu L.
12. The method of claim 8, wherein,
the incubation time is between 2min and 20 min.
13. An application of the reagent kit in detecting SARS-CoV-2 antigen,
the kit at least comprises a capture component and a detection component,
wherein the capture component is a solid phase carrier coated with a specific anti-SARS-CoV-2 antibody and the detection component is a human angiotensin converting enzyme II (ACE2) protein, polypeptide or protein fragment labeled with a detectable signal marker,
alternatively, the first and second electrodes may be,
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 a specific anti-SARS-CoV-2 antibody marked with a detectable signal marker,
wherein the capture component is used for capturing SARS-CoV-2 antigen in a sample to be tested.
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