CN114295829A - Novel coronavirus antigen and total antibody combined detection kit and detection method - Google Patents

Abstract

The present invention relates to a combined detection kit for novel coronavirus antigen and total antibody. The kit consists of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment solution, and the R1 magnetic beads are made of anti-new coronavirus N The protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein-coated paramagnetic particles and magnetic bead diluent are composed; the R2 acridine is acridine-labeled new coronavirus RBD (receptor binding domain, RBD). ) protein; R3 acridine is an acridine-labeled mouse-derived monoclonal antibody against the new coronavirus N protein. The kit can detect the novel coronavirus antigen and antibody in the serum and plasma of the subject at one time, and can distinguish the detection results of the novel coronavirus antigen and antibody. The method provides an auxiliary diagnostic method for novel coronavirus infection, which can be used as a supplementary detection index for novel coronavirus nucleic acid detection of suspected cases or used in conjunction with nucleic acid detection in the diagnosis of suspected cases, which can indicate the sooner or later course of the disease.

Description

Novel coronavirus antigen and total antibody combined detection kit and detection method

technical field

The invention belongs to the field of biomedicine, and particularly relates to a novel coronavirus antigen and total antibody combined detection kit and detection method.

Background technique

The new coronavirus (2019-nCoV, also known as SARS-CoV-2, HCoV-19) is a circular or oval shape with a diameter of 60-140nm, and has a typical coronavirus genome structure, belonging to β-CoV virus. Its genome size is 29891 nucleotides, including non-coding sequences at both ends and a complete open reading frame gene (Open reading frame, ORF), which can encode 9860 amino acids. Among them, ORF1a encodes papain-like protease and 3C-like protease, and ORF1b encodes polyprotease. Papain-like protease and 3C-like protease cleave polyprotease to produce dependent RNA polymerase and helicase to participate in the transcription and replication of virus. The structurally related proteins of virus include spike protein (S), membrane protein (M), Envelope protein (E) and nucleocapsid protein (N). The N protein gene is relatively conserved in coronaviruses and may cross with other viral genes, while the ORF1ab gene has better specificity, and the simultaneous detection of the two genes by fluorescent PCR can effectively avoid misdiagnosis. Antigens such as N protein, E protein and S protein of the new coronavirus can be used as immunogens to stimulate plasma cells to produce specific antibodies after the virus infects the human body. The N protein and the viral genome RNA are intertwined to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA. At the same time, the N protein is relatively conservative and accounts for the largest proportion of the structural proteins of the virus. The body can produce high-level antibodies against the N protein in the early stage of infection. The N protein can be used to establish a rapid detection method for new coronavirus serum antibodies.

Laboratory tests for the new coronavirus mainly include nucleic acid, antigen and antibody. Nucleic acid detection determines whether infection has occurred by detecting the RNA sequence of the new coronavirus, which is the "gold standard" for diagnosing new coronary pneumonia. At present, the most widely used real-time fluorescence quantitative RT-PCR technology. Specimen types are mainly respiratory samples, including nasal swabs, pharyngeal swabs, nasopharyngeal swabs, sputum, bronchial lavage fluid, bronchoalveolar lavage fluid, etc. Antigen detection mainly uses new coronavirus-specific antibodies to directly detect the new coronavirus antigen in the sample, and the results can be used as direct evidence for early confirmation of infection. The detected antigens are mainly the N protein and S protein of the new coronavirus, and the type of samples used is general For respiratory samples, including nasopharyngeal swabs and bronchoalveolar lavage fluid. Antibody detection mostly uses the N protein and S protein of the new coronavirus as the capture antigen, and mainly detects the new coronavirus-specific IgM and IgG antibodies in the serum. It is generally believed that 7 days after the onset of the new coronavirus pneumonia, the first infected person first developed IgM antibodies, and then IgG antibodies. When the IgG antibody appears, its concentration will continue to increase and exist for a long period of time; while the IgM antibody will continue to decrease until it disappears. Commonly used detection methods for antigens and antibodies include chemiluminescence, colloidal gold and fluorescence immunochromatography. At present, there is no diagnostic product that can simultaneously detect the novel coronavirus antigen and antibody in serum.

Since the outbreak of the new coronavirus, RT-PCR has been mainly used for etiological diagnosis to detect viral RNA in respiratory samples. A positive test result can confirm the diagnosis of new coronavirus infection. , the influence of factors such as testing personnel technology. Due to improper sampling, improper preservation of specimens, the use of different types of specimens and the use of reagents from different manufacturers, nucleic acid test results may be "false negative" and missed diagnosis, and the positive detection rate is only 30-50%; Persons come into contact with pathogens, and there is the potential for infection and transmission of the virus. Nucleic acid detection has high standards in the preservation and transportation of samples. Improper operation of the experimental process will lead to cross-contamination. Therefore, it also has high requirements on the level of experimental personnel, and it is difficult to achieve simple, convenient and automated operations.

The colloidal gold antibody detection method uses colloidal gold as a tracer marker and is an immunolabeling technology applied to the detection of antigen and antibody. It breaks through the limitations of the existing detection technology on personnel and places; but its shortcomings are also obvious: 1) only qualitative detection can be performed, 2) the throughput is low, and 3) the accuracy of the detection results is highly dependent on the quality of the antibody. Not good, it is easy to cause cross-reaction, resulting in false positive results. 4) The antibody detection for colloidal gold detection kits is mainly IgM and IgG antibodies, and the window period is longer than that of nucleic acid detection.

Chemiluminescence immunoassay detection is also widely used in the detection of new coronaviruses. At present, the chemiluminescence products on the market are mainly for 2019-nCoV-specific IgM antibodies and IgG antibodies, and there are no reagents for simultaneous detection of 2019-nCoV antigens and antibodies. Chemiluminescence immunoassay detection has the characteristics of high degree of automation and large throughput. The main problems include: 1) The detection window of serum antibodies is long and cannot be used for early diagnosis. 2) It is impossible to determine whether the antibodies in the tested samples are an immune response caused by infection or the result of vaccination against the new crown. 3) prone to false positives. Test results are easily affected by endogenous or exogenous interfering substances, resulting in false positive results; endogenous interfering substances generally include rheumatoid factor, heterophilic antibodies, complement, lysozyme, etc.; exogenous interfering substances generally include specimens Hemolysis, contamination of specimens, long storage time of specimens, incomplete coagulation of specimens, etc. 4) The detection sensitivity of different kits varies greatly, and false negative results may occur.

Novel coronavirus antigen detection is generally the detection of novel coronavirus antigens in respiratory samples. The advantages are rapid and high-throughput. Double-antibody sandwich method is often used, and two antigen-specific antibodies are used to identify and bind to different epitopes of a target antigen. , which can reduce the probability of cross-reaction and effectively improve its specificity. Disadvantages include: 1) After the patient is infected, it only detoxifies through the respiratory tract for a period of time, and the antigen level decreases for a long time after the infection, and the test results are obviously affected by the test timing. 2) The detoxification of respiratory samples is unstable. Since the new coronavirus mainly invades the lower respiratory tract such as the alveoli, sampling from the upper respiratory tract such as the nasopharynx and oropharynx may not be able to obtain pathogens, or the virus content in the sampling is very small, which may cause false negatives; 3 ) Asymptomatic patients have less detoxification and are prone to missed detection.

In view of the existing novel coronavirus detection kits and detection methods all have defects of one kind or another, there is an urgent need to develop new detection kits that can overcome the defects in existing detection kits, or can be used as existing detection kits and detection methods. Supplementary novel detection kits and detection methods.

SUMMARY OF THE INVENTION

The invention provides a novel coronavirus antigen and total antibody combined detection kit and detection method. The kit and the detection method adopt a direct chemiluminescence one-step method to form a double-antibody sandwich immune complex, and detect the serum of a subject at one time. , novel coronavirus antigens and antibodies in plasma, and can distinguish the test results of novel coronavirus antigens and antibodies. This method provides an auxiliary diagnostic method for novel coronavirus infection, which can be used as a supplementary detection index for the detection of suspected cases of novel coronavirus nucleic acid or used in conjunction with nucleic acid detection in the diagnosis of suspected cases. It is used alone as the basis for the confirmation and exclusion of the new coronavirus.

Specifically, the present invention is achieved through the technical solutions of the following aspects:

In a first aspect, the present invention provides a novel coronavirus antigen and total antibody combined detection kit, which is composed of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample processing solution, so that in one kit Determination of two targets of new coronavirus antigen and antibody respectively;

The R1 magnetic beads are composed of paramagnetic particles coated with anti-new coronavirus N protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein, and magnetic bead diluent;

The R2 acridine is acridine-labeled new coronavirus RBD protein;

The R3 acridine is an acridine-labeled anti-new coronavirus N protein mouse-derived monoclonal antibody;

The composition of the R4 sample treatment solution is 80mM Trizma base, 50mM Trizmahydrochloride, 15mM urea, 10mM EDTA, 0.15% Tween-20 and 0.15% ProClin-300,

The pH of the R4 sample treatment solution is 4.5;

Wherein, the amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein mouse-derived monoclonal antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;

The amino acid sequence and nucleotide sequence of the new coronavirus spike protein S1 subunit recombinant protein are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;

The amino acid sequence and nucleotide sequence of the novel coronavirus RBD protein are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

As an optional method, in the above kit, the anti-new coronavirus N protein mouse-derived monoclonal antibody is derived from mouse ascites extraction or hybridoma cell culture in vitro.

As an optional method, in the above kit, the preferred concentration of paramagnetic particles and magnetic beads coated with anti-new coronavirus N protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein are both 0.2 mg/ mL, and/or, the concentration of the acridine is 5ug/mL-25ug/mL.

Alternatively, in the above kit, the sample is serum, plasma, nasopharyngeal swab or pleural effusion of the subject.

As an optional way, in the above-mentioned kit,

The target combination of the combined detection kit is selected from the following: the anti-new coronavirus N protein murine monoclonal antibody epitope 74-105aa and the anti-new coronavirus N protein murine monoclonal antibody epitope Gly44-Glu174, S1 protein epitope Positions Val16-Arg685 and RBD protein epitopes 319-541aa;

Preferably, the amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody epitope 74-105aa are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

The amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody epitope Gly44-Glu174 are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively;

The amino acid sequence and nucleotide sequence of the S1 protein epitope Val16-Arg685 are respectively shown in SEQ ID NO:11 and SEQ ID NO:12;

The amino acid sequence and nucleotide sequence of the RBD protein epitope 319-541aa are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

In a second aspect, the present invention provides a combined detection method for novel coronavirus antigen and total antibody, using the kit described in the first aspect above, the detection method is a direct chemiluminescence one-step method, in a reagent The two targets of the novel coronavirus antigen and antibody are determined in the box, and the two detection results of the antigen and the antibody are reported respectively.

As an optional method, in the above detection method, the following steps are included:

S1. Draw samples, draw 10uL-150uL samples respectively in the first reaction cup and the second reaction cup for testing;

S2. Add magnetic beads and acridine in step S1: automatically add 50uL R1 reagent component and 50uL R2 reagent component to the first reaction cup according to the set procedure, and add 50uL R1 reagent component to the second reaction cup at the same time and 50uL R3 reagent components;

S3. Incubate at 37°C for 20 minutes. In the first reaction cup, the total antibody of 2019-nCoV in the sample, if present, binds to the S1 protein antigen coated on the magnetic beads and the acridine-labeled RBD protein antigen to form a sandwich immune system. The complex; in the second reaction cup, the SARS-CoV-2 N protein antigen in the sample, if present, is combined with the anti-N protein mouse monoclonal antibody and the acridine-labeled anti-N protein mouse monoclonal antibody coated on the magnetic beads combined to form a sandwich immune complex;

S4. Cleaning: Under the action of the magnetic field strength of 3000Gs, the magnetic particles are adsorbed on the wall of the reaction cup, and the unbound substances are washed away by the washing buffer;

S5. Excitation and reading: add pre-excitation solution and excitation solution to the reaction complex, and the test result is expressed as relative luminescence intensity (RLU).

As an optional method, in the above detection method, the preferred concentration of paramagnetic particles and magnetic beads coated with anti-new coronavirus N protein mouse-derived monoclonal antibody and new coronavirus spike protein S1 subunit recombinant protein are both 0.2 mg/ mL, and/or, the concentration of the acridine is 5ug/mL-25ug/mL, and/or, the magnetic bead diluent is composed of 50mM MES, 0.15% BSA, 0.15% Tween-20 and 0.5% ProClin- 300, pH 7.0, and/or acridine diluent consisting of 30 mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20 and 0.5% ProClin-300, pH 7.0; The above-mentioned sample is serum, plasma, nasopharyngeal swab or pleural effusion of the subject.

As an optional way, in the above detection method,

The washing buffer contains: 0.2% Na ₂ HPO ₄ , 0.1% NaH ₂ PO ₄ and 0.5% Triton X-405, pH 7.5;

The pre-excitation solution contains: 0.5%-1.32% H ₂ O ₂ and 1% nitric acid, pH 1.10-2.0;

The excitation solution contains: 0.25M-0.35M NaOH and 1% ProClin-300, pH 12.5-13.5.

In a third aspect, the present invention provides the use of the kit described in the first aspect above in preparing a novel coronavirus detection kit.

Preferably, the novel coronavirus detection kit is used for auxiliary diagnosis of novel coronavirus infection, and it is used as a supplementary detection indicator for novel coronavirus nucleic acid detection of suspected cases or used in conjunction with nucleic acid detection in the diagnosis of suspected cases, which can prompt The course of the disease sooner or later.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention provides a kit for the combined detection of novel coronavirus antigen and antibody and a detection method thereof. The kit and the detection method adopt a one-step competitive immunodetection method of direct chemiluminescence immunodetection, which can detect the novel coronavirus antigen and antibody in the serum and plasma of the subject at one time, and can distinguish the detection results of the novel coronavirus antigen and antibody. .

(2) This method provides an auxiliary diagnostic method for novel coronavirus infection, which can be used as a supplementary detection index for the detection of suspected cases of novel coronavirus nucleic acid or used in conjunction with nucleic acid detection in the diagnosis of suspected cases, which can indicate the sooner or later course of the disease. , but cannot be used alone as the basis for the confirmation and exclusion of the new coronavirus.

(3) The targets detected in the present invention are respectively the N-antigen of the new coronavirus and the antibody against the S1 subunit receptor binding domain (RBD) of the spike protein of the new coronavirus. The combined application of the two can effectively avoid the detection process of the reagents. Cross-reaction occurs in the detection process; the chemiluminescence method, together with the matching automatic chemiluminescence analyzer, can significantly improve the sensitivity of the detection results, and in the detection process, the antigen and antibody are used for cup detection, and the detection of the two can be given respectively. result.

Detailed ways

In order to make the implementation purpose, technical solutions and advantages of the present invention clearer, the following will describe the spirit of the disclosed content of the present invention in detail. Changes and modifications may be made to the techniques taught without departing from the spirit and scope of this disclosure.

The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention. In addition, elements/members using the same or similar reference numerals in the embodiments are intended to represent the same or similar parts.

Regarding the "first", "second", ... etc. used in this document, it does not specifically refer to the order or order, nor is it used to limit the present invention, it is only used to distinguish elements or operations described in the same technical terms. .

Regarding the directional terms used in this article, such as: up, down, left, right, front or back, etc., therefore, the directional terms used are used to illustrate rather than limit this creation.

As used herein, "comprising," "including," "having," "containing," and the like, are open-ended terms, meaning including but not limited to.

As used herein, "and/or" includes any and all combinations of the stated things.

As used herein, the terms "approximately", "about" and the like are used to modify any quantity or error that may vary slightly, but which does not alter its essence. In general, the range of minor variations or errors modified by such terms can be 20% in some embodiments, 10% in some embodiments, 5% in some embodiments, or other numerical values. Those skilled in the art should understand that the aforementioned values can be adjusted according to actual needs, and are not limited thereto.

Certain terms used to describe the application are discussed below or elsewhere in this specification to provide those skilled in the art with additional guidance in the description of the application.

The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, but not to limit the scope of the present invention.

If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased through formal channels.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Example

Composition of the kit of the present invention

The kit of the present invention is composed of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment solution. The R1 magnetic beads are composed of anti-new coronavirus N protein mouse-derived monoclonal antibody and new coronavirus spike protein S1 subunit recombinant protein package It consists of paramagnetic particles and magnetic bead dilution solution; R2 acridine is acridine-labeled new coronavirus RBD protein; R3 acridine is acridine-labeled anti-new coronavirus N protein mouse-derived monoclonal antibody; R4 sample treatment solution The composition was 80 mM Trizma base, 50 mM Trizma hydrochloride, 15 mM urea, 10 mM EDTA, 0.15% Tween-20, 0.15% ProClin-300, and the pH of the R4 sample treatment solution was 4.5.

Wherein, the amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein mouse-derived monoclonal antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;

The amino acid sequence and nucleotide sequence of the new coronavirus spike protein S1 subunit recombinant protein are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;

The amino acid sequence and nucleotide sequence of the novel coronavirus RBD protein are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

In a specific embodiment, the anti-COVID-19 N protein mouse-derived monoclonal antibody is derived from mouse ascites extraction or in vitro culture of hybridoma cells.

Wherein, the preparation method of the anti-new coronavirus N protein mouse-derived monoclonal antibody includes the following steps: 1. Preparation of cell strains:

1) Mix the purchased recombinant novel coronavirus N protein expressed by genetic engineering and Freund's adjuvant, grind it into a chyle, and inject it into BALB/c mice for the primary immunization.

2)) Aseptically collected rat tail blood two weeks after the initial immunization, ELISA method was used to identify the immune effect, and those with extremely low antibody levels were immune failures and were discarded.

3) The second booster immunization of the mice qualified for the first immunization, the dosage method is the same as that of the first immunization, the adjuvant is Freund's incomplete adjuvant, and the antibody level is detected by the same method three weeks after the second immunization.

4)) If the antibody titer of the two booster immunizations has been increasing, the booster immunization should be continued, and the dosage method is the same as that of the second immunization; if the antibody titer does not increase, it indicates that the antibody level has reached the highest point, and the booster immunization must be terminated. .

5) When the antibody titer stabilized at the highest level, the antigen was injected intraperitoneally, and the spleen cells of the immunized mice were aseptically collected on the fourth day after the booster immunization and the SP2/0 mouse myeloma cells in the logarithmic growth phase were treated with 50% PEG2000. Fusion cells were placed in a 96-well cell culture plate containing feeder cells and cultured in a certain environment.

6) After one week of fusion, the antibody level of the supernatant was detected by ELISA, and the positive wells were detected. The positive wells were re-examined the next day. For the positive wells whose antibody levels showed an upward trend, the limited dilution method was used for monoclonalization and 3-4 subcultures. Cloning until the cloning wells of the entire cell culture plate are positive, that is, the single clone is screened.

7) After the monoclonal cell line is expanded and cultured, continuously subcultured in vitro for 4 weeks and then cryopreserved, select cell lines that can stably secrete antibodies after continuous subculture in vitro for 4 weeks and after 3 months of cryopreservation.

2. Preparation of monoclonal antibody ascites:

1) Inject liquid paraffin into BALB/c mice intraperitoneally, and inject the above qualified cell lines into the abdominal cavity of mice one week later. Ascites can be produced 7-10 days after inoculation of mice. As many as possible, before the mice are dying, collect ascites.

2) After the ascites is collected, centrifuge, discard the upper layer of fat and the lower layer of cells, and cryopreserve.

3. Purification of monoclonal antibody:

1) Take the above-mentioned ascites fluid and increase the rotation speed again to remove impurities, add 3 times the volume of acetate buffer and caprylic acid, mix well, and centrifugally filter to leave the supernatant.

2) Adjust the pH to 7.2 with sodium hydroxide, add ammonium sulfate and mix well, then centrifuge, discard the supernatant, take the precipitate and add it to PBS buffer for dialysis, then centrifuge, retain the supernatant after centrifugation and add an equal volume of glycerol.

The primary purified monoclonal antibody is further purified by DEAE-cellulose chromatography to obtain the monoclonal antibody, and the purified protein content is determined by an ultraviolet spectrophotometer.

The labeling method of the acridine is as follows:

Take 100ug of the substance to be labeled and 10ug of acridine ester in 4M PBS buffer to mix evenly when the total volume of the mixture is 100ul, and react in the dark for 3-5h; then add 4M PBS buffer containing 0.05%-0.1% BSA. 100ul, mix well and continue to react in the dark for 3-5h; store at 2-8℃.

The preparation method of the RBD is as follows:

1) construct the new coronavirus RBD fusion protein gene shown in the synthesized DNA sequence into the E. coli plasmid; 2) transform the new coronavirus RBD protein expression plasmid into E. coli competent cells for cultivation, and select a single colony to induce expression after centrifugation; 3) ultrasonically disrupt the obtained bacterial cells in buffer solution, rinse twice in buffer solution after centrifugation, and centrifuge to obtain precipitated inclusion bodies; 4) dissolve the precipitated inclusion bodies, sonicate, and centrifuge The supernatant is purified and eluted using a chromatographic column, and the eluate is collected; 5) the eluate is added to a reducing agent and then loaded into a dialysis bag for dialysis, and the obtained dialysate is centrifuged to collect the supernatant to obtain recombinant novel coronavirus RBD protein.

Further, the preferred concentrations of paramagnetic particles and magnetic beads coated with anti-new coronavirus N protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein are both 0.2 mg/mL.

In addition, it should be noted that the concentration of acridine is 5ug/mL-25ug/mL.

By detecting the signal-to-noise ratio of the sample, it is determined that the preferred concentration of the paramagnetic microparticle magnetic beads coated with the anti-new coronavirus N protein mouse monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein is 0.2 mg/mL (as shown in Table 1). The composition of the magnetic bead diluent is shown in Table 2. The preferred result is determined by testing the signal-to-noise ratio of the sample to be a diluent containing 50 mM MES, 0.15% BSA, 0.15% Tween-20, 0.5% ProClin-300, pH is 7.0.

The concentration of acridine was 5ug/mL-25ug/mL, and the final optimal result was 10ug/mL. The composition of the acridine diluent is shown in Table 3, and the preferred result is determined by testing the signal-to-noise ratio of the sample to be composed of 30mM PBS, 0.15% BSA, 0.1% G-Anti-HIgG, 0.15% Tween-20, 0.5% ProClin-300 The diluent, pH 7.0.

Table I:

Table II:

Table 3:

The detection method of the present invention

The kit of the present invention needs to be used together with IC-2000 and IC-6000 series chemiluminescence analyzers. Except for the default cleaning solution, pre-excitation solution, excitation solution and the corresponding cleaning and luminescence reading steps of the analyzer, the rest of the steps are performed by This is done manually on the analyzer.

Wherein, the washing buffer comprises: 0.2% Na ₂ HPO ₄ , 0.1% NaH ₂ PO ₄ and 0.5% Triton X-405, pH 7.5;

The pre-excitation solution contains: 0.5%-1.32% H ₂ O ₂ and 1% nitric acid, pH 1.10-2.0;

The excitation solution contains: 0.25M-0.35M NaOH and 1% ProClin-300, pH 12.5-13.5.

A combined detection method for novel coronavirus antigen and total antibody, comprising the following steps:

S1. Draw samples, draw 10uL-150uL samples respectively in the first reaction cup and the second reaction cup for testing;

S2. Add magnetic beads and acridine in step S1: automatically add 50uL R1 reagent component and 50uL R2 reagent component to the first reaction cup according to the set procedure, and add 50uL R1 reagent component to the second reaction cup at the same time and 50uL R3 reagent components;

S3. Incubate at 37°C for 20 minutes. In the first reaction cup, the total antibody of 2019-nCoV in the sample, if present, binds to the S1 protein antigen coated on the magnetic beads and the acridine-labeled RBD protein antigen to form a sandwich immune system. The complex; in the second reaction cup, the SARS-CoV-2 N protein antigen in the sample, if present, is combined with the anti-N protein mouse monoclonal antibody and the acridine-labeled anti-N protein mouse monoclonal antibody coated on the magnetic beads combined to form a sandwich immune complex;

S4, cleaning: under the action of the magnetic field strength of 3000Gs, the magnetic particles are adsorbed on the wall of the reaction cup, and the unbound substances are washed away by the washing buffer;

S5. Excitation and reading: add a pre-excitation solution containing hydrogen peroxide to the reaction mixture, the acidic environment can prevent premature release of energy, and at the same time remove the acridine ester from the reaction complex; then add sodium hydroxide-containing solution The excitation solution of the acridine ester is oxidized in an alkaline solution, and N-methyl acridone is formed to release energy and return to the ground state; the optical system reads the released energy and measures its luminous intensity (RLU), Analyte concentration was calculated from luminescence intensity.

In a specific embodiment, the preferred concentration of the paramagnetic particles and magnetic beads coated with the anti-new coronavirus N protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein is 0.2 mg/mL, and/or, The concentration of the acridine is 5ug/mL-25ug/mL, and/or the composition of the magnetic bead diluent is 50mM MES, 0.15% BSA, 0.15% Tween-20 and 0.5% ProClin-300, and the pH is 7.0, and/or, acridine diluent consisting of 30 mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20 and 0.5% ProClin-300, pH 7.0; the sample was tested serum, plasma, nasopharyngeal swab or pleural effusion.

The amount of novel coronavirus antigen or antibody in a sample is directly related to the relative luminescence intensity (RLU) detected by the optical system of the analyzer. The results are determined by the calibration curve, and the master curve is provided by the reagent QR code.

Test Results of the Invention

Using the signal-to-noise ratio of the novel coronavirus antigen test and the signal-to-noise ratio of the novel coronavirus total antibody test as detection indicators, the amounts of various reagent components and various parameters involved in the kit and detection method of the present invention are screened.

Table 4: Test results of different combinations of magnetic bead concentrations in Table 1

Table 5: Test results using different concentrations of acridine esters

Table 6: Test results of different magnetic bead diluent components in Table 2

Table 7: Test results of different acridine diluent components in Table 3

Table 8: Signal-to-noise ratio test results for different aspiration volumes

From the results in the above tables, it can be known that the preferred concentration of paramagnetic microparticle magnetic beads coated with anti-new coronavirus N protein mouse monoclonal antibody and new coronavirus spike protein S1 recombinant protein is 0.2 mg/mL; acridine The preferred result of the ester concentration is 10ug/mL; the preferred result of the composition of the magnetic bead diluent is a diluent containing 50mM MES, 0.15%BSA, 0.15%Tween-20, 0.5%ProClin-300, pH is 7.0; acridine diluent The preferred result of the composition is a diluent containing 30mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20, and 0.5% ProClin-300, with a pH of 7.0; the preferred result of aspiration is the novel coronavirus The sample for virus antigen test is 100uL, while the sample for total antibody to 2019-nCoV is 20uL.

Three combinations of magnetic beads and acridine were selected to detect the novel coronavirus antigen and total antibody to test the concentration of five samples.

Among them, the conventional anti-COVID-19 N protein mouse-derived monoclonal antibody as a control was purchased from Fipeng Biological Co., Ltd. under the trade names of COV19-PS-MAb107 (labeled); COV19-PS-MAb30 (coated).

Table 9: Test results for different combinations of magnetic beads and acridine

The results are shown in Table 9. The specific anti-new coronavirus N protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein used in the present invention are coated with magnetic beads, and acridine uses specific anti-new coronavirus respectively. The N protein mouse-derived monoclonal antibody and the new coronavirus RBD protein are used as labeled antibodies, and the detection results are even better.

The 5 samples are 3 copies, and the sample without any interfering substances is used as a blank control, and the other two groups are respectively added with 1500IU/mL rheumatoid factor and 20mg/dL bilirubin, and the present invention is used to test them. Get test results.

Table 10: Test results of different interferents

The results are shown in Table 10. The test of the present invention is not affected by interfering substances, the specificity is good, and the test results are excellent.

Prepare a positive sample for testing new crown total antibody and a positive sample for testing new crown antigen respectively, and dilute them in proportion; at the same time, prepare ELISA new crown total antibody detection kit and ELISA new crown antigen detection kit and the present invention. Simultaneous test samples.

Among them, the ELISA new crown total antigen detection kit is the new coronavirus N protein detection kit, which was purchased from Haitai Biopharmaceutical Co., Ltd.

The ELISA new crown total antibody detection kit is a SARS-CoV-2 (2019-nCoV) total antibody detection ELISA kit, purchased from Beijing Boaosen Biotechnology Co., Ltd.

Table 11: Comparison results of the present invention and ELISA type reagent test

The results are shown in Table 11. After the same sample is diluted in proportion, the present invention can accurately detect low-end concentration samples. Obviously, the low-end ELISA is not as sensitive as the detection method of the present invention, and the present invention can improve the sensitivity of the test results.

Based on the above results, it is shown that the detection method of the present invention can obtain the detection results of the two targets of the novel coronavirus antigen and total antibody at the same time, which avoids the multiple collection of specimens by patients; makes up for the long antibody detection window period, low specificity, and inability to distinguish Vaccination is still a matter of virus infection, and the results of nucleic acid testing and simple antigen testing of respiratory specimens are unstable.

The kit and the detection method of the invention have the advantages of high accuracy, good specificity, semi-quantitative, simple and easy operation, short detection time, high degree of automation, and high-throughput detection; It is more suitable for early screening, auxiliary diagnosis and monitoring of clinical patients of new coronary pneumonia.

It is worth noting that the nucleic acid and antibody/antigen detection of the new coronavirus have their own focuses and cannot be substituted for each other. Through the combined application of multiple detection methods, they complement each other and give full play to their respective advantages, which will help to improve the sensitivity and specificity of laboratory detection of the new coronavirus, effectively shorten the detection window period, improve the positive detection rate, and help the infected person diagnose and diagnose the disease. Provide laboratory support for monitoring and new crown epidemic, situation prevention and control.

In the present invention, the chemiluminescence immunoassay method is used to measure the two targets of the novel coronavirus antigen and antibody respectively in one kit, and the design idea of reporting the two detection results of the antigen and the antibody respectively needs to be protected; the novel coronavirus N antigen and the The choice of anti-RBD antibodies to detect specific target combinations requires protection.

The above description is only an exemplary embodiment of the present invention, without departing from the concept and principle of the present invention, any equivalent changes and modifications made by those skilled in the art shall fall within the protection scope of the present invention .

sequence listing

<110> The First Affiliated Hospital of China Medical University Shenyang Yilu Biotechnology Co., Ltd.

<120> Novel coronavirus antigen and total antibody combined detection kit and detection method

<130> 5

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 178

<212> PRT

<213> Mus musculus

<400> 1

Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys

1 5 10 15

Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Ser Met His Trp Val Lys Gln

20 25 30

Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Gly Glu Pro Thr Tyr

35 40 45

Ala Asp Asp Phe Gln Gly Arg Phe Asp Phe Ser Leu Glu Thr Ser Ala

50 55 60

Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Ala Thr Ala

65 70 75 80

Thr Tyr Phe Cys Ser Arg Gly Gly Tyr Asp Ser Asp Gly Gly Tyr Tyr

85 90 95

Ala Leu Asp Tyr Trp Gly Gln Gly Thr Gln Arg Lys Gln Gly Lys Ser

100 105 110

Pro Gln Leu Leu Ile Tyr Gly Ala Ile Asn Leu Val Asp Gly Met Ser

115 120 125

Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Gln Tyr Ser Leu Lys Ile

130 135 140

Ser Ser Leu His Pro Asp Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val

145 150 155 160

Leu Ser Pro Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

165 170 175

Arg Ala

<210> 2

<211> 534

<212> DNA

<213> Mus musculus

<400> 2

ggacctgagc tgaagaagcc tggagagaca gtcaagatct cctgcaaggc ttctggttat 60

accttcacag actattcaat gcactgggtg aagcaggctc caggaaaggg tttaaagtgg 120

atgggctggg gtgagccaac atatgcagat gacttccagg gacggtttga cttctctttg 180

gaaacctctg ccagcactgc ctatttgcag atcaacaacc tcaaaaatga ggccacggct 240

acatatttct gttctagagg gggctatgat tccgacggag gttactatgc tttggactac 300

tggggtcaag gaacccagcg gaaacaggga aaatctcctc aactcctgat ctatggtgca 360

atcaacttgg tagatggcat gtcatcgagg ttcagtggca gtggatctgg tagacagtat 420

tctctcaaga tcagtagcct gcatcctgac gatgttgcaa cgtattactg tcaaaatgtg 480

ttaagtcctc cgttcacgtt cggggggggg accaagctgg aaataaaacg ggct 534

<210> 3

<211> 660

<212> PRT

<213> Artificial Sequence

<400> 3

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr

660

<210> 4

<211> 3822

<212> DNA

<213> Artificial Sequence

<400> 4

tctgagccag tgctcaaagg agtcaaatta cattacacat aaatgtttgt ttttcttgtt 60

ttattgccac tagtctctag tcagtgtgtt aatcttacaa ccagaactca attaccccct 120

gcatacacta attctttcac acgtggtgtt tattaccctg acaaagtttt cagatcctca 180

gttttacatt caactcagga cttgttctta cctttctttt ccaatgttac ttggttccat 240

gctatacatg tctctgggac caatggtact aagaggtttg ataaccctgt cctaccattt 300

aatgatggtg ttttattttgc ttccactgag aagtctaaca taataagagg ctggattttt 360

ggtactactt tagattcgaa gacccagtcc ctacttattg ttaataacgc tactaatgtt 420

gttattaaag tctgtgaatt tcaattttgt aatgatccat ttttgggtgt ttattaccac 480

aaaaacaaca aaagttggat ggaaagtgag ttcagagttt attctagtgc gaataattgc 540

acttttgaat atgtctctca gccttttctt atggaccttg aaggaaaaca gggtaatttc 600

aaaaatctta gggaatttgt gtttaagaat attgatggtt attttaaaat atattctaag 660

cacacgccta ttaatttagt gcgtgatctc cctcagggtt tttcggcttt agaaccattg 720

gtagatttgc caataggtat taacatcact aggtttcaaa ctttacttgc tttacataga 780

agttatttga ctcctggtga ttcttcttca ggttggacag ctggtgctgc agcttattat 840

gtgggttatc ttcaacctag gacttttcta ttaaaatata atgaaaatgg aaccattaca 900

gatgctgtag actgtgcact tgaccctctc tcagaaacaa agtgtacgtt gaaatccttc 960

actgtagaaa aaggaatcta tcaaacttct aactttagag tccaaccaac agaatctatt 1020

gttagatttc ctaatattac aaacttgtgc ccttttggtg aagtttttaa cgccaccaga 1080

tttgcatctg tttatgcttg gaacaggaag agaatcagca actgtgttgc tgattattct 1140

gtcctatata attccgcatc attttccact tttaagtgtt atggagtgtc tcctactaaa 1200

ttaaatgatc tctgctttac taatgtctat gcagattcat ttgtaattag aggtgatgaa 1260

gtcagacaaa tcgctccagg gcaaactgga aagattgctg attataatta taaattacca 1320

gatgatttta caggctgcgt tatagcttgg aattctaaca atcttgattc taaggttggt 1380

ggtaattata attacctgta tagattgttt aggaagtcta atctcaaacc ttttgagaga 1440

gatatttcaa ctgaaatcta tcaggccggt agcacacctt gtaatggtgt tgaaggtttt 1500

aattgttact ttcctttaca atcatatggt ttccaaccca ctaatggtgt tggttaccaa 1560

ccatacagag tagtagtact ttcttttgaa cttctacatg caccagcaac tgtttgtgga 1620

cctaaaaagt ctactaattt ggttaaaaac aaatgtgtca atttcaactt caatggttta 1680

acaggcacag gtgttcttac tgagtctaac aaaaagtttc tgcctttcca acaatttggc 1740

agagacattg ctgacactac tgatgctgtc cgtgatccac agacacttga gattcttgac 1800

attacaccat gttcttttgg tggtgtcagt gttataacac caggaacaaa tacttctaac 1860

caggttgctg ttctttatca ggatgttaac tgcacagaag tccctgttgc tattcatgca 1920

gatcaactta ctcctacttg gcgtgtttat tctacaggtt ctaatgtttt tcaaacacgt 1980

gcaggctgtt taataggggc tgaacatgtc aacaactcat atgagtgtga catacccatt 2040

ggtgcaggta tatgcgctag ttatcagact cagactaatt ctcctcggcg ggcacgtagt 2100

gtagctagtc aatccatcat tgcctacact atgtcacttg gtgcagaaaa ttcagttgct 2160

tactctaata actctattgc catacccaca aattttacta ttagtgttac cacagaaatt 2220

ctaccagtgt ctatgaccaa gacatcagta gattgtacaa tgtacatttg tggtgattca 2280

actgaatgca gcaatctttt gttgcaatat ggcagttttt gtacacaatt aaaccgtgct 2340

ttaactggaa tagctgttga acaagacaaa aacacccaag aagtttttgc acaagtcaaa 2400

caaatttaca aaacaccacc aattaaagat tttggtggtt ttaatttttc acaaatatta 2460

ccagatccat caaaaccaag caagaggtca tttattgaag atctactttt caacaaagtg 2520

acacttgcag atgctggctt catcaaacaa tatggtgatt gccttggtga tattgctgct 2580

agagacctca tttgtgcaca aaagtttaac ggccttactg ttttgccacc tttgctcaca 2640

gatgaaatga ttgctcaata cacttctgca ctgttagcgg gtacaatcac ttctggttgg 2700

acctttggtg caggtgctgc attacaaata ccatttgcta tgcaaatggc ttataggttt 2760

aatggtattg gagttacaca gaatgttctc tatgagaacc aaaaattgat tgccaaccaa 2820

tttaatagtg ctattggcaa aattcaagac tcactttctt ccacagcaag tgcacttgga 2880

aaacttcaag atgtggtcaa ccaaaatgca caagctttaa acacgcttgt taaacaactt 2940

agctccaatt ttggtgcaat ttcaagtgtt ttaaatgata tcctttcacg tcttgacaaa 3000

gttgaggctg aagtgcaaat tgataggttg atcacaggca gacttcaaag tttgcagaca 3060

tatgtgactc aacaattaat tagagctgca gaaatcagag cttctgctaa tcttgctgct 3120

actaaaatgt cagagtgtgt acttggacaa tcaaaaagag ttgatttttg tggaaagggc 3180

tatcatctta tgtccttccc tcagtcagca cctcatggtg tagtcttctt gcatgtgact 3240

tatgtccctg cacaagaaaa gaacttcaca actgctcctg ccatttgtca tgatggaaaa 3300

gcacactttc ctcgtgaagg tgtctttgtt tcaaatggca cacactggtt tgtaacacaa 3360

aggaattttt atgaaccaca aatcattact acagacaaca catttgtgtc tggtaactgt 3420

gatgttgtaa taggaattgt caacaacaca gtttatgatc ctttgcaacc tgaattagac 3480

tcattcaagg aggagttaga taaatatttt aagaatcata catcaccaga tgttgattta 3540

ggtgacatct ctggcattaa tgcttcagtt gtaaacattc aaaaagaaat tgaccgcctc 3600

aatgaggttg ccaagaattt aaatgaatct ctcatcgatc tccaagaact tggaaagtat 3660

gagcagtata taaaatggcc atggtacatt tggctaggtt ttatagctgg cttgattgcc 3720

atagtaatgg tgacaattat gctttgctgt atgaccagtt gctgtagttg tctcaagggc 3780

tgttgttctt gtggatcctg ctgcaaattt gatgaagacg ac 3822

<210> 5

<211> 223

<212> PRT

<213> Artificial Sequence

<400> 5

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 6

<211> 669

<212> DNA

<213> Artificial Sequence

<400> 6

agagtccaac caacagaatc tattgttaga tttcctaata ttacaaactt gtgccctttt 60

ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag gaagagaatc 120

agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc cacttttaag 180

tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt ctatgcagat 240

tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac tggaaagatt 300

gctgattata attataaatt accagatgat tttacaggct gcgttatagc ttggaattct 360

aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt gtttaggaag 420

tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc cggtagcaca 480

ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata tggtttccaa 540

cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt tgaacttcta 600

catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa aaacaaatgt 660

gtcaatttc 669

<210> 7

<211> 32

<212> PRT

<213> Mus musculus

<400> 7

Leu Lys Asn Glu Ala Thr Ala Thr Tyr Phe Cys Ser Arg Gly Gly Tyr

1 5 10 15

Asp Ser Asp Gly Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr

20 25 30

<210> 8

<211> 96

<212> DNA

<213> Mus musculus

<400> 8

ctcaaaaatg aggccacggc tacatatttc tgttctagag ggggctatga ttccgacgga 60

ggttactatg ctttggacta ctggggtcaa ggaacc 96

<210> 9

<211> 131

<212> PRT

<213> Mus musculus

<400> 9

Gly Glu Pro Thr Tyr Ala Asp Asp Phe Gln Gly Arg Phe Asp Phe Ser

1 5 10 15

Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys

20 25 30

Asn Glu Ala Thr Ala Thr Tyr Phe Cys Ser Arg Gly Gly Tyr Asp Ser

35 40 45

Asp Gly Gly Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Gln Arg

50 55 60

Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile Tyr Gly Ala Ile Asn Leu

65 70 75 80

Val Asp Gly Met Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Gln

85 90 95

Tyr Ser Leu Lys Ile Ser Ser Leu His Pro Asp Asp Val Ala Thr Tyr

100 105 110

Tyr Cys Gln Asn Val Leu Ser Pro Pro Phe Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu

130

<210> 10

<211> 393

<212> DNA

<213> Mus musculus

<400> 10

ggtgagccaa catatgcaga tgacttccag ggacggtttg acttctcttt ggaaacctct 60

gccagcactg cctatttgca gatcaacaac ctcaaaaatg aggccacggc tacatatttc 120

tgttctagag ggggctatga ttccgacgga ggttactatg ctttggacta ctggggtcaa 180

ggaacccagc ggaaacaggg aaaatctcct caactcctga tctatggtgc aatcaacttg 240

gtagatggca tgtcatcgag gttcagtggc agtggatctg gtagacagta ttctctcaag 300

atcagtagcc tgcatcctga cgatgttgca acgtattact gtcaaaatgt gttaagtcct 360

ccgttcacgt tcgggggggg gaccaagctg gaa 393

<210> 11

<211> 670

<212> PRT

<213> Artificial Sequence

<400> 11

Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser

1 5 10 15

Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val

20 25 30

Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr

35 40 45

Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe

50 55 60

Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr

65 70 75 80

Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp

85 90 95

Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val

100 105 110

Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val

115 120 125

Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val

130 135 140

Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe

145 150 155 160

Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu

165 170 175

Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His

180 185 190

Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu

195 200 205

Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln

210 215 220

Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser

225 230 235 240

Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln

245 250 255

Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp

260 265 270

Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu

275 280 285

Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg

290 295 300

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

305 310 315 320

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

325 330 335

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

340 345 350

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

355 360 365

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

370 375 380

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

385 390 395 400

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

405 410 415

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

420 425 430

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

435 440 445

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

450 455 460

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

465 470 475 480

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

485 490 495

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

500 505 510

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe

515 520 525

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

530 535 540

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

545 550 555 560

Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser

565 570 575

Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln

580 585 590

Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala

595 600 605

Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

610 615 620

Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His

625 630 635 640

Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys

645 650 655

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg

660 665 670

<210> 12

<211> 2010

<212> DNA

<213> Artificial Sequence

<400> 12

gttaatctta caaccagaac tcaattaccc cctgcataca ctaattcttt cacacgtggt 60

gtttattacc ctgacaaagt tttcagatcc tcagttttac attcaactca ggacttgttc 120

ttacctttct tttccaatgt tacttggttc catgctatac atgtctctgg gaccaatggt 180

actaagaggt ttgataaccc tgtcctacca tttaatgatg gtgtttattt tgcttccact 240

gagaagtcta acataataag aggctggatt tttggtacta ctttagattc gaagacccag 300

tccctactta ttgttaataa cgctactaat gttgttatta aagtctgtga atttcaattt 360

tgtaatgatc catttttggg tgtttattac cacaaaaaca acaaaagttg gatggaaagt 420

gagttcagag tttattctag tgcgaataat tgcacttttg aatatgtctc tcagcctttt 480

cttatggacc ttgaaggaaa acagggtaat ttcaaaaatc ttagggaatt tgtgtttaag 540

aatattgatg gttattttaa aatatattct aagcacacgc ctattaattt agtgcgtgat 600

ctccctcagg gtttttcggc tttagaacca ttggtagatt tgccaatagg tattaacatc 660

actaggtttc aaactttact tgctttacat agaagttatt tgactcctgg tgattcttct 720

tcaggttgga cagctggtgc tgcagcttat tatgtgggtt atcttcaacc taggactttt 780

ctattaaaat ataatgaaaa tggaaccatt acagatgctg tagactgtgc acttgaccct 840

ctctcagaaa caaagtgtac gttgaaatcc ttcactgtag aaaaaggaat ctatcaaact 900

tctaacttta gagtccaacc aacagaatct attgttagat ttcctaatat tacaaacttg 960

tgcccttttg gtgaagtttt taacgccacc agatttgcat ctgtttatgc ttggaacagg 1020

aagagaatca gcaactgtgt tgctgattat tctgtcctat ataattccgc atcattttcc 1080

acttttaagt gttatggagt gtctcctact aaattaaatg atctctgctt tactaatgtc 1140

tatgcagatt catttgtaat tagaggtgat gaagtcagac aaatcgctcc agggcaaact 1200

ggaaagattg ctgattataa ttataaatta ccagatgatt ttacaggctg cgttatagct 1260

tggaattcta acaatcttga ttctaaggtt ggtggtaatt ataattacct gtatagattg 1320

tttaggaagt ctaatctcaa accttttgag agagatattt caactgaaat ctatcaggcc 1380

ggtagcacac cttgtaatgg tgttgaaggt tttaattgtt actttccttt acaatcatat 1440

ggtttccaac ccactaatgg tgttggttac caaccataca gagtagtagt actttctttt 1500

gaacttctac atgcaccagc aactgtttgt ggacctaaaa agtctactaa tttggttaaa 1560

aacaaatgtg tcaatttcaa cttcaatggt ttaacaggca caggtgttct tactgagtct 1620

aacaaaaagt ttctgccttt ccaacaattt ggcagagaca ttgctgacac tactgatgct 1680

gtccgtgatc cacagacact tgagattctt gacattacac catgttcttt tggtggtgtc 1740

agtgttataa caccaggaac aaatacttct aaccaggttg ctgttcttta tcaggatgtt 1800

aactgcacag aagtccctgt tgctattcat gcagatcaac ttactcctac ttggcgtgtt 1860

tattctacag gttctaatgt ttttcaaaca cgtgcaggct gtttaatagg ggctgaacat 1920

gtcaacaact catatgagtg tgacataccc attggtgcag gtatatgcgc tagttatcag 1980

actcagacta attctcctcg gcgggcacgt 2010

<210> 13

<211> 223

<212> PRT

<213> Artificial Sequence

<400> 13

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 14

<211> 669

<212> DNA

<213> Artificial Sequence

<400> 14

agagtccaac caacagaatc tattgttaga tttcctaata ttacaaactt gtgccctttt 60

ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag gaagagaatc 120

agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc cacttttaag 180

tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt ctatgcagat 240

tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac tggaaagatt 300

gctgattata attataaatt accagatgat tttacaggct gcgttatagc ttggaattct 360

aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt gtttaggaag 420

tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc cggtagcaca 480

ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata tggtttccaa 540

cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt tgaacttcta 600

catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa aaacaaatgt 660

gtcaatttc 669

Claims (10)

1. A novel coronavirus antigen and total antibody combined detection kit is characterized in that, it is composed of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment liquid, so that the novel coronavirus antigen is determined respectively in a test kit and two targets of antibodies; The R1 magnetic beads are composed of paramagnetic particles coated with anti-new coronavirus N protein mouse-derived monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein, and magnetic bead diluent; The R2 acridine is acridine-labeled new coronavirus RBD protein; The R3 acridine is an acridine-labeled anti-new coronavirus N protein mouse-derived monoclonal antibody; The composition of the R4 sample treatment solution is 80mM Trizma base, 50mM Trizma hydrochloride, 15mM urea, 10mM EDTA, 0.15% Tween-20 and 0.15% ProClin-300, and the pH of the R4 sample treatment solution is 4.5; Wherein, the amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein mouse-derived monoclonal antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; The amino acid sequence and nucleotide sequence of the new coronavirus spike protein S1 subunit recombinant protein are respectively shown in SEQ ID NO:3 and SEQ ID NO:4; The amino acid sequence and nucleotide sequence of the novel coronavirus RBD protein are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. 2. The novel coronavirus antigen and total antibody combined detection kit according to claim 1, wherein the anti-new coronavirus N protein murine monoclonal antibody is derived from mouse ascites extraction or hybridoma cell culture in vitro . 3. The novel coronavirus antigen and total antibody combined detection kit according to claim 1, wherein the anti-coronavirus N protein mouse-derived monoclonal antibody and the novel coronavirus spike protein S1 subunit recombinant protein are coated. The preferred concentrations of paramagnetic particles and magnetic beads are both 0.2 mg/mL, and/or the concentration of the acridine is 5ug/mL-25ug/mL, and/or, the composition of the magnetic bead diluent is 50mM MES, 0.15 μg/mL %BSA, 0.15% Tween-20, and 0.5% ProClin-300, pH 7.0, and/or acridine diluent consisting of 30 mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween -20 and 0.5% ProClin-300, pH 7.0. 4. The novel coronavirus antigen and total antibody combined detection kit according to claim 1, wherein the sample is serum, plasma, nasopharyngeal swab or pleural effusion of the subject. 5. novel coronavirus antigen and total antibody joint detection kit according to claim 1, is characterized in that, the target combination of described joint detection kit is selected from following: anti-new coronavirus N protein murine monoclonal antibody table Positions 74-105aa and anti-COVID-19 N protein murine monoclonal antibody epitope Gly44-Glu174, S1 protein epitope Val16-Arg685 and RBD protein epitope 319-541aa; Preferably, the amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody epitope 74-105aa are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; The amino acid sequence and nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody epitope Gly44-Glu174 are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; The amino acid sequence and nucleotide sequence of the S1 protein epitope Val16-Arg685 are shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively; The amino acid sequence and nucleotide sequence of the RBD protein epitopes 319-541aa are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. 6. a novel coronavirus antigen and total antibody combined detection method, is characterized in that, using the test kit described in any one of claim 1 to 5, described detection method is direct chemiluminescence one-step method, in a test kit The two targets of the new coronavirus antigen and antibody were measured in the 2019-nCoV, and the two detection results of the antigen and the antibody were reported respectively. 7. novel coronavirus antigen and total antibody combined detection method according to claim 6, is characterized in that, comprises the following steps: S1, draw samples, draw 10uL-150uL samples respectively in the first reaction cup and the second reaction cup for testing; S2. Add magnetic beads and acridine in step S1: automatically add 50uL R1 reagent component and 50uL R2 reagent component to the first reaction cup according to the set procedure, and add 50uL R1 reagent component to the second reaction cup at the same time and 50uL R3 reagent components; S3. Incubate at 37°C for 20 minutes. In the first reaction cup, the total antibody of 2019-nCoV in the sample, if present, binds to the S1 protein antigen coated on the magnetic beads and the acridine-labeled RBD protein antigen to form a sandwich immune system. The complex; in the second reaction cup, the SARS-CoV-2 N protein antigen in the sample, if present, is combined with the anti-N protein mouse monoclonal antibody and the acridine-labeled anti-N protein mouse monoclonal antibody coated on the magnetic beads combined to form a sandwich immune complex; S4, cleaning: under the action of the magnetic field, the magnetic particles are adsorbed on the wall of the reaction cup, and the unbound substances are washed away by the washing buffer; S5. Excitation and reading: add pre-excitation solution and excitation solution to the reaction complex, and the test result is expressed as relative luminescence intensity (RLU). 8. The method for combined detection of novel coronavirus antigen and total antibody according to claim 7, characterized in that: the paramagnetic anti-coronavirus N protein mouse-derived monoclonal antibody and the novel coronavirus spike protein S1 subunit are recombinantly coated. The preferred concentration of microparticles and magnetic beads is 0.2mg/mL, and/or the concentration of the acridine is 5ug/mL-25ug/mL, and/or, the composition of the magnetic bead diluent is 50mM MES, 0.15%BSA , 0.15% Tween-20 and 0.5% ProClin-300, pH 7.0, and/or acridine diluent consisting of 30 mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20 and 0.5% ProClin-300, pH 7.0; the sample is the subject's serum, plasma, nasopharyngeal swab or pleural effusion. 9. the novel coronavirus antigen and total antibody combined detection method according to claim 7 or claim 8, is characterized in that: The washing buffer contains: 0.2% Na ₂ HPO ₄ , 0.1% NaH ₂ PO ₄ and 0.5% Triton X-405, pH 7.5; The pre-excitation solution contains: 0.5%-1.32% H ₂ O ₂ and 1% nitric acid, pH 1.10-2.0; The excitation solution contains: 0.25M-0.35M NaOH and 1% ProClin-300, pH 12.5-13.5. 10. Use of the test kit according to any one of claims 1 to 5 in the preparation of a novel coronavirus detection kit, characterized in that, preferably, the novel coronavirus detection kit is used for the detection of novel coronavirus infection. Auxiliary diagnosis, and its use as a supplementary detection indicator for new coronavirus nucleic acid detection of suspected cases or in conjunction with nucleic acid detection in the diagnosis of suspected cases, can indicate the sooner or later course of the disease.