CN114295829B - Novel coronavirus antigen and total antibody combined detection kit and detection method - Google Patents

Abstract

The invention relates to a novel coronavirus antigen and total antibody joint detection kit, which consists of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment liquid, wherein the R1 magnetic beads consist of paramagnetic particles coated by an anti-new coronavirus N protein mouse monoclonal antibody and a new coronavirus spike protein S1 subunit recombinant protein, and magnetic bead diluent; the R2 acridine is acridine-labeled new coronavirus RBD (RBD) protein; r3 acridine is an acridine marked mouse-derived monoclonal antibody for resisting new coronavirus N protein. The kit can detect the novel coronavirus antigens and antibodies in serum and plasma of a detected person at one time, and can distinguish the detection results of the novel coronavirus antigens and the novel coronavirus antibodies. The method provides a novel auxiliary diagnosis method for coronavirus infection, can be used as a supplementary detection index for a suspected case of novel coronavirus nucleic acid detection or used together with nucleic acid detection in the diagnosis of the suspected case, and can prompt the morning and evening of the disease course.

Description

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

Technical Field

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

Background

The novel coronavirus (2019-nCoV, also known as SARS-CoV-2, HCoV-19) is a virus having a circular or elliptical shape with a diameter of 60-140nm and a typical coronavirus genome structure, and belongs to beta-CoV. The genome size is 29891 nucleotides, which comprises two end non-coding region sequences and a complete Open Reading Frame (ORF) and can code 9860 amino acids. Wherein ORF1a encodes a papain-like protease and a 3C-like protease, and ORF1b encodes a polyprotein. Papain-like and 3C-like proteases cleave polyproteinase to produce dependent RNA polymerase and helicase to participate in transcription and replication of viruses, structurally related proteins of which include spike (S), membrane (M), envelope (E) and nucleocapsid (N) proteins. The N protein gene is relatively conserved in the coronavirus and can be crossed with other virus genes, the ORF1ab gene has better specificity, and misdiagnosis can be effectively avoided by simultaneously detecting the two genes by using a fluorescence PCR method. The antigens of N protein, E protein, S protein and the like of the novel coronavirus can be used as immunogen to stimulate plasma cells to generate specific antibodies after the virus infects human bodies. The N protein and the virus genome RNA are intertwined to form the virus nucleocapsid, which plays an important role in the synthesis process of the virus RNA. Meanwhile, the N protein is relatively conservative and accounts for the largest proportion in the structural protein of the virus, an organism can generate a high-level antibody for resisting the N protein in the early infection period, and a method for quickly detecting the serum antibody of the new coronavirus can be established by utilizing the N protein.

Laboratory tests for the new coronavirus mainly include three categories, nucleic acid, antigen and antibody. The nucleic acid detection judges whether infection occurs by detecting the RNA sequence of the new coronavirus, is the 'gold standard' for determining new coronary pneumonia, and the most widely used technology at present is real-time fluorescent quantitative RT-PCR technology. The types of specimens are mainly respiratory tract samples, including nasal swabs, pharyngeal swabs, nasopharyngeal swabs, sputum, bronchial lavage, alveolar lavage, and the like. The antigen detection mainly uses a new coronavirus specific antibody to directly detect a new coronavirus antigen in a sample, the result can be used as direct evidence for early confirmation of infection, the detected antigen mainly comprises N protein and S protein of the new coronavirus, and the used sample types are respiratory tract samples generally, including nasopharyngeal swab, alveolar lavage fluid and the like. The antibody detection mainly uses N protein and S protein of the new coronavirus as capture antigens, and mainly detects the specific IgM and IgG antibodies of the new coronavirus in serum. It is generally accepted that 7 days after the onset of the novel coronavirus pneumonia, the infected person first presents IgM antibodies and then IgG antibodies. When IgG antibodies appear, their concentration will continue to rise, and will exist for a longer period of time; while IgM antibodies continue to decrease until they disappear. Common detection methods for antigens and antibodies include chemiluminescence, colloidal gold, and fluorescence immunochromatography. At present, no diagnostic product capable of simultaneously detecting the novel coronavirus antigen and the novel coronavirus antibody in serum is available on the market.

Since the occurrence of the epidemic situation of the novel coronavirus, the etiology diagnosis mainly adopts an RT-PCR method to detect the virus RNA in a respiratory tract sample, the detection result is positive, the infection of the novel coronavirus can be diagnosed, but the nucleic acid detection result is influenced by the factors of the disease course of a patient, the collection of a specimen, the quality of a detection reagent, the technology of a detection person and the like. The false negative of the nucleic acid detection result can be caused by improper sampling, improper specimen preservation, adoption of different types of specimens and use of reagents of different manufacturers, so that missed diagnosis occurs, and the positive detection rate is only 30-50%; meanwhile, when the specimen is collected, a sampling person can contact with pathogens, and the possibility of infection and virus transmission exists. Nucleic acid detection has higher standards in the aspects of sample preservation, transportation and the like, and cross contamination can be caused if the operation is improper in the experimental process, so that the requirements on the level of experimenters are also high, and the simple, convenient and automatic operation is difficult to realize.

The colloidal gold antibody detection method is an immune labeling technology which takes colloidal gold as a tracer marker and is applied to antigen-antibody detection, has the advantages of convenient and quick use, low cost, simple operation, less environmental condition requirements, capability of rapidly issuing results and breakthrough of the limitation of the existing detection technology on personnel and places; but its disadvantages are also evident: 1) only qualitative detection can be carried out, 2) the flux is low, 3) the accuracy of the detection result highly depends on the quality of the antibody, if the quality of the antibody is not good, cross reaction is easy to cause, and a false positive result is caused, 4) the detection of the antibody by the colloidal gold detection kit mainly comprises IgM and IgG antibodies, and is longer than the window period of nucleic acid detection.

Chemiluminescence immunoassay detection is also widely applied to the detection of novel coronavirus. The current chemiluminescent products on the market mainly aim at specific IgM antibodies and IgG antibodies of the new coronavirus, and no reagent for simultaneously detecting antigens and antibodies of the new coronavirus exists. Chemiluminescence immunoassay detection has the characteristics of high automation degree and high flux, and the main problems of the chemiluminescence immunoassay detection comprise: 1) The detection window of serum antibody is long, and the serum antibody cannot be used for early diagnosis. 2) It is impossible to judge whether the antibody in the sample to be tested is an immune response reaction caused by infection or a result of vaccination with a new corona vaccine. 3) False positives are prone to occur. The detection result is easily influenced by endogenous or exogenous interfering substances to cause false positive result; endogenous interfering substances generally comprise rheumatoid factors, heterophile antibodies, complements, lysozyme and the like; exogenous interfering substances generally include hemolysis of the specimen, contamination of the specimen, prolonged storage of the specimen, incomplete coagulation of the specimen, and the like. 4) The detection sensitivity of different kits is greatly different, and false negative results can occur.

The novel coronavirus antigen detection is generally to detect the novel coronavirus antigen in a respiratory tract sample, has the advantages of high speed and high flux, and usually adopts a double-antibody sandwich method to recognize and combine different epitopes of a target antigen by two antigen-specific antibodies, so that the probability of cross reaction can be reduced, and the specificity of the target antigen can be effectively improved. The disadvantages include 1) the patient is only detoxified through the respiratory tract within a period of time after infection, the antigen level is reduced after the infection time is longer, and the detection result is obviously influenced by the detection time. 2) The respiratory tract sample is unstable in toxin expelling, and because the novel coronavirus mainly invades lower respiratory tracts such as alveoli and the like, pathogens cannot be taken out from upper respiratory tracts such as nasopharynx, oropharynx and the like, or false negative can be caused due to the fact that the virus content in the sample is very low; 3) The toxicant elimination of asymptomatic patients is less, and the omission is easily caused.

In view of the above-mentioned shortcomings of the existing novel coronavirus detection kit and detection method, there is an urgent need to develop a novel detection kit and detection method which can overcome the shortcomings of the existing detection kit or can supplement the existing detection kit and detection method.

Disclosure of Invention

The invention provides a novel coronavirus antigen and total antibody combined detection kit and a detection method, wherein the kit and the detection method adopt a direct chemiluminescence one-step method to form a double-antibody sandwich immune complex, detect the novel coronavirus antigen and antibody in serum and plasma of a detected person at one time, and can distinguish the detection results of the novel coronavirus antigen and the novel coronavirus antibody. The method provides a novel auxiliary diagnosis method for coronavirus infection, can be used as a supplementary detection index for a suspected case of novel coronavirus nucleic acid detection or used together with nucleic acid detection in the diagnosis of the suspected case, can prompt the morning and evening of the disease course, but cannot be used as a basis for the confirmation and elimination of the novel coronavirus independently.

Specifically, the invention is realized by the following technical schemes:

in a first aspect, the invention provides a novel coronavirus antigen and total antibody joint detection kit, which consists of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment liquid, so that two targets of a novel coronavirus antigen and an antibody are respectively measured in one kit;

the R1 magnetic bead consists of an anti-new coronavirus N protein murine monoclonal antibody, paramagnetic particles coated by new coronavirus spike protein S1 subunit recombinant protein and a magnetic bead diluent;

the R2 acridine is a new coronavirus RBD protein marked by acridine;

the R3 acridine is an acridine marked mouse-derived monoclonal antibody for resisting new coronavirus N protein;

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,

the pH of the R4 sample treatment solution is 4.5;

wherein, the amino acid sequence and the nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;

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

the amino acid sequence and the nucleotide sequence of the new coronavirus RBD protein are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6.

Alternatively, in the kit, the murine monoclonal antibody against the novel coronavirus N protein is derived from mouse ascites extraction or hybridoma cell culture in vitro.

Alternatively, in the kit, the concentration of paramagnetic particles and magnetic beads coated by the anti-new coronavirus N protein murine monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein is preferably 0.2mg/mL, and/or the concentration of acridine is 5ug/mL-25ug/mL.

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

Alternatively, in the above-mentioned kit,

the target combination of the combined detection kit is selected from the following: anti-new coronavirus N protein murine monoclonal antibody epitope 74-105aa and anti-new coronavirus 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 the nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody surface position 74-105aa are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the amino acid sequence and the nucleotide sequence of the mouse-derived monoclonal antibody surface position Gly44-Glu174 for resisting the new coronavirus N protein are respectively shown as SEQ ID NO. 9 and SEQ ID NO. 10;

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

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

In a second aspect, the present invention provides a novel coronavirus antigen and total antibody combined detection method, using the kit of the first aspect, wherein the detection method is a direct chemiluminescence one-step method, two targets of a coronavirus antigen and an antibody are respectively detected in one kit, and detection results of the antigen and the antibody are respectively reported.

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

s1, sucking a sample, and respectively sucking 10-150 uL of sample into a first reaction cup and a second reaction cup for testing;

s2, adding magnetic beads and acridine in the step S1: automatically adding 50uL R1 reagent component and 50uL R2 reagent component to the first reaction cup, and simultaneously adding 50uL R1 reagent component and 50uL R3 reagent component to the second reaction cup according to a set program;

s3, incubating at 37 ℃ for 20 minutes, and in a first reaction cup, combining the total antibodies of the new coronavirus in the sample with an S1 protein antigen coated on magnetic beads and an acridine-labeled RBD protein antigen to form a sandwich immune complex if the total antibodies exist; in a second reaction cup, if the new coronavirus N protein antigen exists in the sample, combining the new coronavirus N protein antigen with an anti-N protein murine monoclonal antibody and an acridine-labeled anti-N protein murine monoclonal antibody coated on magnetic beads to form a sandwich immune complex;

s4, cleaning, namely adsorbing magnetic particles on the wall of the reaction cup under the magnetic force action of 3000Gs (magnetic field strength), and washing away unbound substances through a washing buffer solution;

s5, excitation and reading: the pre-excitation liquid and the excitation liquid are added into the reaction compound, and the test result is expressed by relative luminescence intensity (RLU).

Alternatively, in the above detection method, the concentration of paramagnetic microparticles and magnetic beads coated with the Anti-neocoronavirus N protein murine monoclonal antibody and the neocoronavirus spike protein S1 subunit recombinant protein is preferably 0.2mg/mL, and/or the concentration of acridine is 5ug/mL to 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 the composition of the acridine diluent is 30mM PBS, 0.15% BSA, 0.1% G-An-H IgG, 0.15% Tween-20 and 0.5% ProClin-300, and the pH is 7.0; the sample is serum, plasma, nasopharyngeal swab or pleural effusion of the subject.

Alternatively, in the above-described detection method,

the wash buffer comprises: 0.2% of Na ₂ HPO ₄ 、0.1％NaH ₂ PO ₄ And 0.5%Triton X-405，pH7.5；

The pre-excitation liquid comprises: 0.5% -1.32% ₂ O ₂ And 1% nitric acid, pH 1.10-2.0;

the exciting liquid comprises: 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 a kit according to the first aspect above for the preparation of a novel coronavirus detection kit.

Preferably, the novel coronavirus detection kit is used for auxiliary diagnosis of novel coronavirus infection, and can be used as a supplementary detection index of a suspected case of novel coronavirus nucleic acid detection or used together with nucleic acid detection in the diagnosis of the suspected case to prompt the morning and evening of the disease course.

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

(1) The invention provides a novel coronavirus antigen-antibody joint detection kit and a detection method thereof. The kit and the detection method adopt a one-step competitive immunoassay method of direct chemiluminescence immunoassay to detect the novel coronavirus antigens and antibodies in serum and plasma of a detected person at one time, and can distinguish the detection results of the novel coronavirus antigens and the antibodies.

(2) The method provides a novel auxiliary diagnosis method for coronavirus infection, can be used as a supplementary detection index for a suspected case of novel coronavirus nucleic acid detection or used together with nucleic acid detection in the diagnosis of the suspected case, can prompt the morning and evening of the disease course, but cannot be used as a basis for the confirmation and elimination of the novel coronavirus independently.

(3) The target detected in the invention respectively selects a new coronavirus N antigen and an antibody aiming at a Receptor Binding Domain (RBD) of the S1 subunit of the spike protein of the new coronavirus, and the combined application of the two can effectively avoid the cross reaction of the reagent in the detection process; the chemiluminescence method is adopted, and a matched full-automatic chemiluminescence tester is matched for use, so that the sensitivity of a detection result can be obviously improved, and the detection results of the antigen and the antibody can be respectively given by adopting separate cup detection of the antigen and the antibody in the detection process.

Detailed Description

For the purpose of promoting a clear understanding of the objects, solutions and advantages of the present invention, the following detailed description is given for clearness of understanding, and it is to be understood that changes and modifications may be made by those skilled in the art, without departing from the spirit and scope of the present invention, as those skilled in the art may recognize from the teachings of the present invention.

The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention. In addition, the same or similar numbered elements/components used in the embodiments are used to represent the same or similar parts.

As used herein, "first," "second," …, etc., are not specifically meant to be sequential or sequential, nor are they meant to limit the invention, but merely to distinguish between elements or operations described in the same technical language.

With respect to directional terminology used herein, for example: up, down, left, right, front or rear, and so on, and thus, the directional terms used are intended to be illustrative and not limiting.

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

As used herein, "and/or" includes any and all combinations of the described items.

As used herein, the terms "substantially", "about" and the like are used to modify any slight variation in quantity or error that does not alter the nature of the variation. Generally, the range of slight variations or errors modified by such terms may be 20% in some embodiments, 10% in some embodiments, 5% in some embodiments, or other values. It should be understood by those skilled in the art that the aforementioned values can be adjusted according to actual needs, and are not limited thereto.

Certain words used to describe the present application are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art in describing the present application.

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

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

Examples

Compositions of the kits of the invention

The kit consists of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment liquid, wherein the R1 magnetic beads consist of paramagnetic particles coated by an anti-new coronavirus N protein mouse monoclonal antibody, a new coronavirus spike protein S1 subunit recombinant protein and a magnetic bead diluent; r2 acridine is acridine marked new coronavirus RBD protein; r3 acridine is an acridine marked mouse-derived monoclonal antibody for resisting new coronavirus N protein; the composition of the R4 sample treatment solution was 80mM Trizma base, 50mM Trizma hydrochloride, 15mM urea, 10mM EDTA, 0.15% Tween-20, 0.15% ProClin-300, wherein the pH of the R4 sample treatment solution was 4.5.

Wherein, the amino acid sequence and the nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;

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

the amino acid sequence and the nucleotide sequence of the new coronavirus RBD protein are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6.

In a specific embodiment, the anti-new coronavirus N protein murine monoclonal antibody is derived from mouse ascites extraction or hybridoma cell culture in vitro.

The preparation method of the anti-new coronavirus N protein murine monoclonal antibody comprises the following steps: preparation of cell lines:

1) The purchased recombinant novel coronavirus N protein expressed by genetic engineering is mixed with Freund's adjuvant, ground into chyle, and injected into BALB/c mice by primary immunization abdominal cavity.

2) Two weeks after priming) aseptically collecting tail blood, and identifying the immune effect by ELISA method, and discarding the patients with extremely low antibody level as the failure of immunity.

3) And (3) carrying out secondary boosting immunization on the mice qualified by the primary immunization identification, wherein the dosage method is the same as the primary immunization, the adjuvant is Freund's incomplete adjuvant, and the antibody level is detected by adopting the same method three weeks after the secondary immunization.

4) If the antibody titer of the two booster immunizations is always increased, the booster immunizations are continued, and the dosage method is the same as that of the two booster immunizations; if the antibody titer does not rise, indicating that the antibody level has peaked, the boost must be terminated.

5) After the antibody titer stabilized at the highest level, the antigen was intraperitoneally injected, splenocytes from immunized mice were aseptically taken on the fourth day after booster immunization and fused with SP2/0 mouse myeloma cells in logarithmic growth phase by 50% PEG2000, and the fused cells were cultured in a feeder cells-containing 96-well cell culture plate under a certain environment.

6) After one week of fusion, ELISA is used for detecting the antibody level of the supernatant, positive holes are detected, the positive holes Kong Fujian are detected the next day, and the positive holes with the rising antibody level are subjected to monoclonality and subclonization for 3-4 times by a limiting dilution method until the cloning holes of the whole cell culture plate are positive, namely the screened monoclonal strains.

7) The monoclonal cell strain is subjected to amplification culture, is frozen and stored after being subjected to in vitro continuous passage for 4 weeks, and is selected to be recovered and cultured after being subjected to in vitro continuous passage culture for four weeks and frozen and stored for 3 months, so that the cell strain capable of stably secreting the antibody can be obtained.

2. Preparing monoclonal antibody ascites:

1) Injecting liquid paraffin into BALB/c mouse, injecting the cell strain meeting the conditions into the abdominal cavity of the mouse after one week, inoculating the mouse for 7-10 days to generate ascites, closely observing the health condition and ascites symptoms of the mouse, and collecting the ascites when the ascites is as much as possible and before the mouse is dying.

2) And (4) after ascites collection, centrifuging, removing upper-layer grease and lower-layer cells, and freezing and storing.

3. And (3) purification of the monoclonal antibody:

1) Adjusting the rotating speed of the ascites fluid to be high, centrifuging again to remove impurities, adding acetate buffer solution and caprylic acid with 3 times of volume, mixing uniformly, centrifuging and filtering to obtain supernatant.

2) Adjusting pH to 7.2 with sodium hydroxide, adding ammonium sulfate, mixing, centrifuging, removing supernatant, collecting precipitate, adding PBS buffer solution, dialyzing, centrifuging, and adding glycerol of the same volume into the supernatant.

The monoclonal antibody after the preliminary purification is further purified by a DEAE-cellulose chromatography to obtain the monoclonal antibody, and an ultraviolet spectrophotometer is adopted to determine the content of the purified protein.

The labeling method of acridine is as follows:

taking 100ug of a to-be-labeled substance and 10ug of acridinium ester, adding into 4M PBS buffer solution until the total volume of the mixed solution is 100ul, mixing uniformly, and reacting for 3-5h in a dark place; adding 100ul of 4M PBS buffer solution containing 0.05% -0.1% BSA, mixing well, and continuing to react for 3-5h in a dark place; storing at 2-8 deg.C.

The preparation method of the RBD is as follows:

1) Constructing a new coronavirus RBD fusion protein gene shown by a synthesized DNA sequence into an escherichia coli plasmid; 2) Transforming the new coronavirus RBD protein expression plasmid into escherichia coli competent cells for culture, selecting a single colony for induced expression, and centrifuging; 3) Ultrasonically crushing the obtained thalli in a buffer solution, centrifuging, rinsing twice in the buffer solution, and centrifuging to obtain a precipitate inclusion body; 4) Dissolving the precipitate inclusion body, carrying out ultrasonic crushing and centrifugation to obtain supernatant, purifying and eluting by using a chromatographic column, and collecting eluent; 5) Adding a reducing agent into the eluent, then filling the eluent into a dialysis bag for dialysis, and centrifuging the obtained dialysate to collect supernatant so as to obtain the novel recombinant coronavirus RBD protein.

Furthermore, the optimal concentration of paramagnetic particles and magnetic beads coated by the anti-new coronavirus N protein murine monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein is 0.2mg/mL.

In addition, the concentration of acridine is 5ug/mL-25ug/mL.

The preferred concentrations of the anti-neocoronavirus N protein murine monoclonal antibody and the paramagnetic microparticle bead coated with the recombinant protein of the S1 subunit of the neocoronavirus spike protein were determined to be 0.2mg/mL (as shown in Table I) by measuring the signal-to-noise ratio of the sample, the preferred concentrations of the paramagnetic microparticle beads were determined to be 0.2mg/mL (as shown in Table II), the preferred concentrations of the paramagnetic microparticle beads as diluted in Table II were determined to be a dilution containing 50mM MES, 0.15% BSA, 0.15% Tween-20, 0.5% ProClin-300 and a pH of 7.0 by measuring the signal-to-noise ratio of the sample.

The acridine concentration is 5ug/mL to 25ug/mL, with a final preferred result of 10ug/mL. The acridine diluent composition is shown in Table three, and the preferred results were determined by measuring the sample signal-to-noise ratio to contain 30mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20, 0.5% ProClin-300 composition diluent, pH 7.0.

Table one:

table two:

table three:

detection method of the invention

The kit needs to be matched with an IC-2000 and IC-6000 series chemiluminescence determinator for use, and the rest steps are finished by manual processing on the determinator except default cleaning solution, pre-excitation solution and excitation solution of the determinator and corresponding cleaning and luminescence reading steps.

Wherein the wash buffer comprises: 0.2% of Na ₂ HPO ₄ 、0.1％NaH ₂ PO ₄ And 0.5% Triton X-405, pH7.5;

the pre-excitation liquid comprises: 0.5% -1.32% of H ₂ O ₂ And 1% nitric acid, pH 1.10-2.0;

the exciting liquid comprises: 0.25M-0.35M NaOH and 1% ProClin-300, pH 12.5-13.5.

A novel coronavirus antigen and total antibody combined detection method comprises the following steps:

s1, sucking a sample, and respectively sucking 10-150 uL of sample into a first reaction cup and a second reaction cup for testing;

s2, adding magnetic beads and acridine in the step S1: automatically adding 50uL of R1 reagent component and 50uL of R2 reagent component to the first reaction cup, while simultaneously adding 50uL of R1 reagent component and 50uL of R3 reagent component to the second reaction cup according to the set program;

s3, incubating at 37 ℃ for 20 minutes, and in a first reaction cup, combining the total antibodies of the new coronavirus in the sample with an S1 protein antigen coated on magnetic beads and an acridine-labeled RBD protein antigen to form a sandwich immune complex if the total antibodies exist; in a second reaction cup, combining the new coronavirus N protein antigen in the sample with an anti-N protein murine monoclonal antibody and an acridine-labeled anti-N protein murine monoclonal antibody coated on magnetic beads to form a sandwich immune complex if the new coronavirus N protein antigen exists;

s4, cleaning, namely adsorbing the magnetic particles on the wall of the reaction cup under the magnetic force action of 3000Gs in magnetic field intensity, and washing away the unbound substances through a washing buffer solution;

s5, excitation and reading: adding a pre-excitation liquid containing hydrogen peroxide into the reaction combination, wherein the acid environment can prevent energy from being released prematurely and simultaneously separate the acridinium ester from the reaction combination; then adding an excitation liquid containing sodium hydroxide, carrying out oxidation reaction on the separated acridinium ester in an alkaline solution, forming N-methylacridone to release energy, and returning to a ground state; the optical path system reads its released energy, measures its luminescence intensity (RLU), and calculates the analyte concentration from the luminescence intensity.

In specific embodiments, the concentration of paramagnetic microparticles and magnetic beads coated with the Anti-neocoronavirus N protein murine monoclonal antibody and the neocoronavirus spike protein S1 subunit recombinant protein are preferably 0.2mg/mL, and/or the concentration of 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 is 7.0, and/or the composition of the acridine diluent is 30mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20, and 0.5% ProClin-300, pH is 7.0; the sample is serum, plasma, nasopharyngeal swab or pleural effusion of the subject.

The amount of the novel coronavirus antigen or antibody in the sample is directly related to the relative luminescence intensity (RLU) detected by the optical system of the detector. The result is determined by the calibration curve, the main curve is provided by the reagent two-dimensional code.

Test results of the invention

The dosage of various reagent components and various parameters related in the kit and the detection method are screened by taking the novel coronavirus antigen test signal-to-noise ratio and the novel coronavirus total antibody test signal-to-noise ratio as detection indexes.

Table four: TABLE I test results for different bead concentration combinations

Table five: test results with different concentrations of acridinium ester

Table six: test results of different magnetic bead diluent compositions in Table II

TABLE VII: test results of different acridine diluent components in table III

Table eight: test results of signal-to-noise ratio of different sample suction amounts

The results in the above tables show that: the optimal concentration of paramagnetic particle magnetic beads coated by the anti-new coronavirus N protein murine monoclonal antibody and the new coronavirus spike protein S1 recombinant protein is 0.2mg/mL; the preferable result of the concentration of the acridinium ester is 10ug/mL; the composition of the magnetic bead diluent was preferably a diluent containing 50mM MES, 0.15% BSA, 0.15% Tween-20, 0.5% ProClin-300, and had a pH of 7.0; the composition of acridine diluent preferably contains 30mM PBS, 0.15% BSA, 0.1% G-Anti-H IgG, 0.15% Tween-20, 0.5% ProClin-300, and has a pH of 7.0; the preferred result of the amount of the sample taken is that the test sample for the novel coronavirus antigen is 100uL, and the sample for the novel coronavirus total antibody is 20uL.

3 magnetic bead combinations and acridine combinations are selected to detect the novel coronavirus antigen and the total antibody, and the concentration of 5 samples is tested.

Wherein, the conventional anti-neocoronavirus N protein murine monoclonal antibody used as a contrast is purchased from Fenpeng biological corporation, and has a trade name of COV19-PS-MAb107 (marker); COV19-PS-MAb30 (coating).

Table nine: test results for different magnetic bead and acridine combinations

As shown in Table nine, the specific anti-new coronavirus N protein murine monoclonal antibody and the new coronavirus spike protein S1 subunit recombinant protein coated magnetic beads adopted by the invention, and the acridine respectively uses the specific anti-new coronavirus N protein murine monoclonal antibody and the new coronavirus RBD protein as the labeled antibodies, so that the detection result is more excellent.

And (3) taking the 5 samples which are not added with any interference substance as blank control, adding 1500IU/mL rheumatoid factors and 20mg/dL bilirubin into the other two groups respectively, and testing the samples by adopting the method to obtain a detection result.

TABLE Ten: test results for different interferents

As shown in Table ten, the test of the present invention was not affected by interfering substances, and had good specificity and excellent test results.

Preparing a positive sample for testing total antibodies of the new corona and a positive sample for testing antigens of the new corona respectively, and diluting the positive samples and the positive samples in proportion; meanwhile, an ELISA new crown total antibody detection kit and an ELISA new crown antigen detection kit are prepared to synchronously test samples.

The ELISA new corona total antigen detection kit is a novel coronavirus N protein detection kit and is 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 which is purchased from Beijing Boaosen Biotechnology Co.

Table eleven: comparison of the present invention with ELISA type reagent test

As shown in the eleventh table, the present invention can accurately detect samples with low concentration after the same sample is diluted in proportion, and obviously the sensitivity of ELISA at low end is inferior to that of the detection method of the present invention, and the present invention can improve the sensitivity of the test result.

The results are integrated to show that the detection method can simultaneously obtain the detection results of two target objects, namely the new coronavirus antigen and the total antibody, and avoid the situation that a patient collects a sample for multiple times; the method solves the problems that the antibody detection window is long, the specificity is not high, whether vaccination or virus infection cannot be distinguished, and the nucleic acid detection and the simple antigen detection result of a respiratory tract specimen are unstable.

The kit and the detection method have the advantages of high accuracy, good specificity, semi-quantitative property, simplicity, easy operation, short detection time, high automation degree and high-throughput detection; the method can break through the limitation of nucleic acid detection on the technology and the site conditions of personnel, and is more suitable for early screening, auxiliary diagnosis and monitoring of clinical patients of the new coronary pneumonia.

Notably, the nucleic acid and antibody/antigen detection of the novel coronaviruses are each heavily weighted and cannot be substituted for each other. The detection methods are combined and applied, complement each other, exert respective advantages, are beneficial to improving the sensitivity and specificity of the laboratory detection of the new coronavirus, can effectively shorten the detection window period, improve the positive detection rate, and provide laboratory support for diagnosis, disease monitoring, new coronary disease and situation prevention and control of infected persons.

In the invention, a chemiluminescence immunoassay method is adopted, two targets of a new coronavirus antigen and an antibody are respectively measured in a kit, and the design ideas of respectively reporting two detection results of the antigen and the antibody need to be protected; the selection of the combination of the new coronavirus N antigen and the specific target for anti-RBD antibody detection needs protection.

The foregoing is merely an illustrative embodiment of the present invention, and any equivalent changes and modifications made by those skilled in the art without departing from the spirit and principle of the present invention should fall within the protection scope of the present invention.

Sequence listing

<110> Shenyang medical science and technology Co Ltd of the first hospital affiliated to the university of medical science in China

<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 (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 (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 tttattttgc 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 (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 (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 (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 (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 (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 (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 (8)

1. A novel coronavirus antigen and total antibody joint detection kit is characterized by consisting of R1 magnetic beads, R2 acridine, R3 acridine and R4 sample treatment liquid, so that two targets of a new coronavirus antigen and an antibody are respectively measured in one kit;

the R1 magnetic bead consists of an anti-new coronavirus N protein murine monoclonal antibody, paramagnetic particles coated by new coronavirus spike protein S1 subunit recombinant protein and a magnetic bead diluent;

the R2 acridine is a new coronavirus RBD protein marked by acridine;

the R3 acridine is an acridine marked mouse-derived monoclonal antibody for resisting new coronavirus N protein;

the composition of the R4 sample treatment fluid is 80mM Trizma base, 50mM Trizma hydrochloride, 15mM urea, 10mM EDTA, 0.15%;

wherein, the amino acid sequence and the nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;

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

the amino acid sequence and the nucleotide sequence of the new coronavirus RBD protein are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6,

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

the amino acid sequence and the nucleotide sequence of the anti-new coronavirus N protein murine monoclonal antibody surface position 74-105aa are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the amino acid sequence and the nucleotide sequence of the mouse-derived monoclonal antibody surface position Gly44-Glu174 for resisting the new coronavirus N protein are respectively shown as SEQ ID NO. 9 and SEQ ID NO. 10;

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

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

2. The combined detection kit for the novel coronavirus antigen and the total antibody, according to claim 1, wherein the murine monoclonal antibody against the N protein of the novel coronavirus is derived from ascites extraction of a mouse or in vitro culture of hybridoma cells.

3. The combined assay kit for the novel coronavirus antigen and total antibody as claimed in claim 1, wherein the concentration of paramagnetic microparticles and beads coated with the N protein mouse-derived monoclonal antibody against the novel coronavirus and the S1 subunit recombinant protein against the novel coronavirus and the concentration of the new coronavirus spike protein S1 subunit are both 0.2mg/mL, the concentration of acridine is 5ug/mL-25ug/mL, the composition of the dilution of beads is 50mM MES, 0.15% BSA, 0.15% Tween-20, and 0.5% ProClin-300, the pH is 7.0, the composition of the dilution of acridine is 30mM PBS, 0.15% BSA, 0.1% G-AnH IgG, 0.15% Tween-20, and 0.5% ProCl in-300, the pH is 7.0.

4. The combined detection kit for coronavirus antigen and total antibody according to claim 1, wherein the sample is serum, plasma, nasopharyngeal swab or pleural effusion of the subject.

5. The combined detection kit for the novel coronavirus antigen and total antibody as claimed in claim 1, wherein the detection method using the kit is a direct chemiluminescence one-step method, two targets of the novel coronavirus antigen and the antibody are respectively determined in one kit, and the detection results of the antigen and the antibody are respectively reported.

6. The combined detection kit for the novel coronavirus antigen and total antibody according to claim 5, wherein the detection method comprises the following steps:

s1, sucking a sample, and respectively sucking 10-150 uL of sample into a first reaction cup and a second reaction cup for testing;

s2, adding magnetic beads and acridine in the step S1: automatically adding 50uL of R1 reagent component and 50uL of R2 reagent component to the first reaction cup, while simultaneously adding 50uL of R1 reagent component and 50uL of R3 reagent component to the second reaction cup according to the set program;

s3, incubating at 37 ℃ for 20 minutes, and in a first reaction cup, combining the total antibodies of the new coronavirus in the sample with an S1 protein antigen coated on magnetic beads and an acridine-labeled RBD protein antigen to form a sandwich immune complex if the total antibodies exist; in a second reaction cup, if the new coronavirus N protein antigen exists in the sample, combining the new coronavirus N protein antigen with an anti-N protein murine monoclonal antibody and an acridine-labeled anti-N protein murine monoclonal antibody coated on magnetic beads to form a sandwich immune complex;

s4, cleaning, namely adsorbing the magnetic particles on the wall of the reaction cup under the action of a magnetic field, and washing away the unbound substances through a washing buffer solution;

s5, excitation and reading: the pre-excitation liquid and the excitation liquid are added into the reaction compound, and the test result is expressed by relative luminescence intensity (RLU).

7. The novel coronavirus antigen and total antibody combined detection kit according to claim 6, wherein the kit comprises:

the wash buffer comprises: 0.2% of Na ₂ HPO ₄ 、0.1％NaH ₂ PO ₄ And 0.5% Triton X-405, pH7.5;

the pre-excitation liquid comprises: 0.5% -1.32% of H ₂ O ₂ And 1% nitric acid, pH 1.10-2.0;

the exciting liquid comprises: 0.25M-0.35M NaOH and 1% ProClin-300, pH 12.5-13.5.

8. Use of the kit of any one of claims 1 to 4 for the preparation of a novel coronavirus detection kit, wherein the novel coronavirus detection kit is used for the auxiliary diagnosis of a novel coronavirus infection and is used as a supplementary detection index for a suspected case of a novel coronavirus nucleic acid detection or used in conjunction with a nucleic acid detection in the diagnosis of a suspected case, which can indicate the morning and evening of the course of the disease.