CN112903996A - nCoV-N protein detection kit and nCoV-N protein detection method - Google Patents
nCoV-N protein detection kit and nCoV-N protein detection method Download PDFInfo
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Abstract
The invention relates to the technical field of immunological detection, in particular to an nCoV-N protein detection kit and an nCoV-N protein detection method. The invention discloses an nCoV-N protein detection kit, which comprises: bonding a board; the adhesive plate is sequentially provided with a sample pad, a combination pad, a reaction film and an absorption pad, and the reaction film is provided with a detection line and a control line; the combination pad is coated with a first nCoV-N protein monoclonal antibody solution marked by gold nanoparticles, the control line is coated with a second nCoV-N protein antibody solution, and the control line is coated with a goat anti-mouse IgG solution; the first nCoV-N protein monoclonal antibody and the second nCoV-N protein monoclonal antibody bind to different nCoV-N protein epitopes. The second nCoV-N protein monoclonal antibody marked by gold nanoparticles is coated on the bonding pad, so that the nCoV-N protein can be specifically identified, virus infection can be directly indicated, a measurement result can be obtained within 5 minutes, any sample polluted by 2019-nCoV can be detected, and the method has the characteristics of convenience, rapidness, sensitivity and low cost.
Description
Technical Field
The invention relates to the technical field of immunological detection, in particular to a nCoV-N protein detection reagent kit and an nCoV-N protein detection method.
Background
2019 novel coronaviruses (2019-nCoV) are easily transmitted from person to person, not only causing pulmonary inflammation, but also damaging the immune system and other organs. Nowadays, 2019-nCoV has become a sudden public health event of international concern, and the rapid and accurate detection of coronavirus is important in epidemic prevention. In the propagation of virus, Nucleocapsid Protein (N) is responsible for virus genome replication and regulation of cell signal path, and has high conservation, so nCoV-N Protein is often used as 2019-nCoV diagnosis and detection tool and is the core raw material of immunological rapid diagnosis reagent.
For two detection reagents currently used in China, the Joint defense Joint Committee of the State Council, 20 days 3 and 20 months 2020 is introduced at the News-issuing society: the new coronary pneumonia detection reagents approved to be on the market at present mainly comprise two types, one type is a nucleic acid detection reagent, and the other type is an antibody detection reagent. The nucleic acid detection process comprises a plurality of steps of sample treatment, nucleic acid extraction, PCR detection and the like, and the average detection time is 2-3 hours. The method has high sensitivity, strong specificity and relatively long detection time. Antibody detection comprises a colloidal gold method and a magnetic microparticle chemiluminescence method, wherein the colloidal gold method is used for detecting the antibody for about 15 minutes on average, a blood drop can be observed by naked eyes within 15 minutes, and the magnetic microparticle chemiluminescence method generally needs 30-60 minutes. However, the antibody detection is to detect the antibody level in human blood, if antibodies are not generated in human bodies in the early stage of virus infection, the window period of detection exists in the antibody detection, and the antibody detection can only be used as an auxiliary means of nucleic acid detection, and the development of an accurate and rapid nCoV-N protein detection kit can be used for examining and screening cases.
Disclosure of Invention
In view of the above, the invention provides an nCoV-N protein detection kit and an nCoV-N protein detection method, wherein the nCoV-N protein detection kit can specifically identify nCoV-N protein, directly indicate virus infection, obtain a measurement result within 5 minutes, and realize quick and sensitive detection.
The specific technical scheme is as follows:
the invention provides an nCoV-N protein detection kit, which comprises: bonding a board;
the bonding plate is sequentially provided with a sample pad, a combination pad, a reaction film and an absorption pad, and the reaction film is provided with a detection line and a control line;
the combination pad is coated with a first nCoV-N protein monoclonal antibody solution marked by gold nanoparticles, the control line is coated with a second nCoV-N protein antibody solution, and the control line is coated with a goat anti-mouse IgG solution;
the epitopes of the nCoV-N protein to which the first nCoV-N protein monoclonal antibody and the second nCoV-N protein monoclonal antibody bind are different.
In the invention, the amino acid sequence of the epitope of the nCoV-N protein is shown as SEQ ID NO.1, and the amino acid sequence of the epitope of the nCoV-N protein bound by the second nCoV-N protein monoclonal antibody is shown as SEQ ID NO. 2.
In the present invention, the reaction membrane is preferably a cellulose acetate membrane.
In the present invention, the sample pad is coated with a 50mM pH8.0 boric acid-borax buffer containing 0.5% Triton, 1% BSA, 2% sucrose;
the solvent of the gold nanoparticle-labeled first nCoV-N protein monoclonal antibody solution is 20mM Na containing 5% BSA, 0.25% Tween-20 and 10% sucrose3PO4；
The concentration of the gold nanoparticle-labeled first nCoV-N protein monoclonal antibody solution is 0.25-0.5mg/mL, preferably 0.5 mg/mL;
the concentration of the second nCoV-N protein antibody solution is 0.5-2.5mg/mL, preferably 1 mg/mL;
the concentration of the goat anti-mouse IgG solution is 0.5-2.5mg/mL, preferably 1 mg/mL;
the dosage of the first nCoV-N protein monoclonal antibody marked by the gold nanoparticles is 1-5 mu g/cm, preferably 2 mu g/cm;
the dosage of the second nCoV-N protein antibody is 1-5 mug/cm, preferably 2 mug/cm;
the dosage of the goat anti-mouse IgG is 1-5 mug/cm, preferably 2 mug/cm.
In the invention, the preparation of the first nCoV-N protein monoclonal antibody solution marked by gold nanoparticles specifically comprises the following steps:
a) 1ml of 0.01 percent HAuCl is taken4The solution was heated and stirred rapidly, 4mL of 1% trisodium citrate was added after boiling, the solution was further heated and stirred until the solution turned red, the solution was boiled for another 10min and cooled to room temperature with slow stirring. By centrifugation 4 times (12X 10)3rpm, 20min), concentrating and collecting gold nanoparticles;
b) 70 mu L0.1M K was added in advance to 10mL of gold nanoparticle aqueous solution2CO3Then, 1000. mu.L (50. mu.g) of nCoV-N protein antibody was added thereto, gently shaken at room temperature for 30min, then, 1mL of 1% bovine serum albumin was added, the uncoated site of colloidal gold was blocked at room temperature for 30min, and the particles were centrifuged (12X 10)3rpm, 20min) and rinsed three times with buffer B to remove unbound antibody, resulting in a platinum nanoflower-labeled dehydroepiandrosterone polyclonal antibody.
In the invention, the preparation method of the first nCoV-N protein monoclonal antibody comprises the following steps:
1) adopting the protein coded by SEQ ID NO.1 as immunogen to immunize mice, taking spleen cells of the mice with high serum titer to perform cell fusion with myeloma cells SP2/0, and then adopting a limiting dilution method to screen hybridoma cells to obtain completely homogeneous monoclonal antibodies and stable monoclonal hybridoma cell strains;
2) injecting the monoclonal hybridoma cell suspension into the abdominal cavity of a mouse, collecting ascites, and purifying the ascites by an octanoic acid-ammonium sulfate precipitation method to obtain a purified first nCoV-N protein monoclonal antibody.
In the step 1) of the invention, the immunization of mice by using the protein coded by SEQ ID NO.1 as immunogen is specifically as follows: emulsifying a complete Freund's adjuvant and a protein solution coded by SEQ ID NO.1 in an equal volume for the first immunization, emulsifying the incomplete Freund's adjuvant and the protein coded by SEQ ID NO.1 in an equal volume for each subsequent boosting immunization, wherein the dosage is the same as that of the first immunization, and the boosting immunization is performed once every 14 days after the first immunization, and the boosting is performed for 3 times;
the dose of the primary immunization is 100 mu g/mouse;
the concentration of the protein solution coded by the SEQ ID NO.1 is 100 mug-500 mug/mL, preferably 200 mug/mL;
the ratio of the mouse spleen cells to the myeloma cells SP2/0 is 10:1, cell fusion is carried out.
The invention uses the immunologic adjuvant to change the physical state of the antigen and prolong the retention time of the antigen in vivo, so that the antigen is slowly released, and meanwhile, the nonspecific promotion of local phagocyte reaction is carried out to enhance the immune effect of animals, thereby obtaining more stable monoclonal antibodies.
The step 2) of the invention is specifically as follows: mice are taken and sensitized by adopting intraperitoneal injection of incomplete adjuvant one week before, and the dosage is 0.5 mL/mouse. The number of cells was 106Individual monoclonal hybridoma cell suspensions were injected into the abdominal cavity of mice. Inoculating hybridoma for 7-10 days, collecting ascites, collecting repeatedly for several times, and storing in refrigerator at 4 deg.C. And carrying out ascites purification by an octanoic acid-ammonium sulfate precipitation method to obtain a purified nCoV-N protein monoclonal antibody, thereby improving the sensitivity and accuracy of the kit.
In the invention, the protein coded by SEQ ID NO.1 is synthesized by the following method:
designing a primer by using a sequence of SEQ ID NO.1, amplifying by using PCR, purifying to obtain a polypeptide, connecting the polypeptide to BamH-I and Xho-sites of a plasmid pET-28a, and constructing a recombinant plasmid pET-28 a-nCoV;
the recombinant plasmid pET-28a-nCoV is transformed into Escherichia coli, and the protein coded by SEQ ID NO.1 is expressed.
According to the invention, the polypeptide expressed by escherichia coli is modified, so that the structure and chemical stability of the polypeptide are stabilized, and a more stable antigen is obtained.
It should be noted that, in the present invention, the first nCoV-N protein monoclonal antibody is self-made by the present invention, the second nCoV-N protein monoclonal antibody is provided by the second hospital of people in Guangdong province, and the preparation method is the same as the present application, and the preparation of the second nCoV-N protein monoclonal antibody in the present invention is not described again.
In the invention, the preparation method of the nCoV-N protein detection kit comprises the following steps:
1) preparation of sample pad: immersing the glass fiber in 50mM boric acid-borax buffer solution with pH8.0 containing 0.5% Triton, 1% BSA and 2% sucrose, drying, and storing at low humidity at room temperature;
2) preparation of the bonding pad: adding a gold nanoparticle-labeled first nCoV-N protein monoclonal antibody solution onto glass fiber to serve as a binding pad;
3) preparation of a reaction film: respectively scratching a solution containing a second nCoV-N protein antibody and a solution containing goat anti-mouse IgG on a reaction membrane to serve as a detection line and a control line, drying at room temperature for 12 hours, and storing in a moisture-proof cabinet in a sealed and vacuum manner for later use;
4) assembling a kit: the sample conjugate pad, reaction membrane and absorbent pad head-tail portions were connected in an overlapping direction of 1-2mm along the long axis of the adhesive plate, and cut into 4mm wide strips with a paper cutter to obtain nCoV-N protein kits.
The sample pad, the conjugate pad, the reaction film and the absorbent pad were attached in an overlapping direction of 1-2mm along the long axis of the adhesive sheet, which had a width of 4 mm.
The invention also provides a nCoV-N protein detection method, which comprises the following steps:
and (3) dripping a sample to be detected into the sample pad in the nCoV-N protein detection kit, wherein the sample to be detected contains nCoV-N protein if the detection line and the control line are colored, and the sample to be detected does not contain the nCoV-N protein if the detection line is not colored and the quality control line is colored.
The nCoV-N protein detection kit provided by the invention has the following principle:
combining the nCoV-N protein monoclonal antibody with the gold nanoparticles to form a gold-labeled antibody which is fixed on the combination pad, and when a sample to be detected is dripped on the sample pad, the sample flows through the combination pad along the nitrocellulose membrane due to the siphonage effect caused by the nitrocellulose membrane and the absorption pad. If 2019-nCoV exists in the sample, the virus can be specifically recognized with the first 2019-nCoV-N protein monoclonal antibody and is connected together; then the liquid flows through the detection line, a second nCoV-N protein antibody on the detection line is combined with the 2019-nCoV-N protein again, so that the second nCoV-N protein antibody is fixed at the detection line, and the detection line generates signals and positive characters; if 2019-nCoV is not contained in the sample, the protein connected with the colloidal gold nanoparticles cannot be finally fixed at the detection line, so that no signal exists at the detection line, and the detection line is negative.
The control line plays a role in product self-checking. No matter whether 2019-nCoV exists in the sample or not, the liquid can flow through the control line with the nCoV-N protein monoclonal antibody marked by the gold nanoparticles on the sample pad, and can be combined with the goat anti-mouse IgG fixed on the control line, so that the nCoV-N protein monoclonal antibody is fixed on the control line. If the control line is positive in each detection, the detection is effective.
According to the technical scheme, the invention has the following advantages:
the invention provides an nCoV-N protein detection kit, which comprises: bonding a board; the bonding plate is sequentially provided with a sample pad, a combination pad, a reaction film and an absorption pad, and the reaction film is provided with a detection line and a control line; the combination pad is coated with a first nCoV-N protein monoclonal antibody solution marked by gold nanoparticles, the control line is coated with a second nCoV-N protein antibody solution, and the control line is coated with a goat anti-mouse IgG solution.
The first nCoV-N protein monoclonal antibody marked by gold nanoparticles is coated on the bonding pad, so that the nCoV-N protein can be specifically identified, virus infection can be directly indicated, a measurement result can be obtained within 5 minutes, any sample polluted by 2019-nCoV can be detected, and the method has the characteristics of convenience, rapidness, sensitivity and low cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a gel electrophoresis diagram of nCoV-N protein of example 1 of the present invention;
FIG. 2 is a graph showing the sensitivity and accuracy of the nCoV-N protein detection kit prepared in example 1 of the present invention.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the examples of the present invention, 5-week-old female Balb/c mice (20 g/mouse, obtained from Shanghai SLAC laboratory animals, Inc.. Escherichia coli BL21(DE3) and Escherichia coli DH 5. alpha. were stored in the laboratory of the subject group of the present inventors.
Example 1
The nCoV-N protein detection kit provided by the embodiment specifically comprises the following steps:
1) solution preparation
Preparing buffer A: 50mM boric acid-borax buffer (pH8.0), 0.5% Triton, 1% BSA, 2% sucrose.
Preparing buffer B: 20mM Na3PO45% BSA, 0.25% Tween-20 and 10% sucrose, distilled water.
Preparing buffer C: 0.1% Triton-100 in PBS.
Preparing buffer D: 20mM Tris-HCl, pH8.0, 500mM NaCl and 10% glycerol, distilled water.
Preparing buffer E: 20mM Tris-HCl, pH8.0, 20mM imidazole, 500mM NaCl and 10% glycerol, distilled water.
Preparing buffer F: 20mM Tris-HCl, pH8.0, 200mM imidazole, 500mM NaCl and 10% glycerol, distilled water.
Preparing buffer G: 20mM Tris-HCl, pH8.0, 10mM EDTA，5mM MgCl2And 10% of glycerol and distilled water.
2) Construction of recombinant plasmid for preparing protein gene encoded by SEQ ID NO.1
The RNA for nCoV was from the second national hospital, Guangdong province. Firstly, primers are designed according to the nucleotide sequence SEQ ID NO.3 of the protein gene coded by SEQ ID NO.1, and a forward primer (5'-CGCGGATCCATGAGGAAGAAGCGGAGACAGCGACGAATTAGGCCTG AGTTGAGTCAG-3') with a BamH-I site and a reverse primer (5'-CCGCTCGAGCACGGCGAATGTCTGATAATGGACCCC-3') with an Xho-I site are added at both ends of the sequence.
Reverse transcription-polymerase chain reaction (RT-PCR reaction) was performed in the Superscript One-step system (Invitrogen, USA) according to the following cycle parameters: reverse transcription at 50 ℃ for 30min, denaturation at 94 ℃ for 3min, and circulation for 35 times according to the duration of 15s per cycle, wherein the denaturation at 98 ℃ is 15s, the renaturation at 58 ℃ is 15s, the elongation at 68 ℃ is 30s, the elongation at 68 ℃ is 10min after the last circulation, and the product is stored in a refrigerator at 4 ℃. The PCR product was digested with BamH I and Xho I, purified by 1% agarose gel electrophoresis, and ligated into BamH-I and Xho-I digested pET-28a vector (containing a histidine tag) to generate recombinant plasmid pET-28 a-nCoV.
The recombinant plasmid was transformed into competent E.coli DH5 alpha (laboratory preservation of this group of subjects) and sequenced to confirm nucleotide identity. Then, Escherichia coli was spread on an agar plate containing kanamycin (50. mu.g/mL), and colonies in which the plasmid was successfully integrated were selected; extracting plasmid DNA by using a high-purity plasmid small-quantity extraction kit (Tiangen-Biotech, Beijing, China), and digesting and identifying the plasmid by using restriction endonuclease; the digestion products were separated on an agarose gel containing 1% ethidium bromide. Nucleotide sequencing was performed in Sangon Biotech (shanghai, china).
3) Expressing the protein encoded by SEQ ID NO.1
The pET-28a-nCoV-N plasmid vector was transferred into E.coli BL21(DE3) cells, cultured for one day in LB agar plates containing kanamycin (50. mu.g/mL), and after picking one colony, it was inoculated in 5mL of LB broth containing 50. mu.g/mL of kanamycin, and incubated overnight at 37 ℃ in a shaking incubator with continuous shaking at 210 rpm. Then 5mL of LB meatThe broth was inoculated into 500mL LB broth containing 50. mu.g/mL kanamycin and incubated on a shaker at 37 ℃ until OD600To about 0.5-0.6.
The LB broth was cooled to 22 ℃ and IPTG was added to a final concentration of 0.5 mM; then incubated at 22 ℃ for 16 h. The bacteria were harvested by centrifugation (4 ℃, 5000rpm, 10min) and the bacterial pellet was resuspended in 20mL buffer D. The bacteria were sonicated and the supernatant was collected by centrifugation (4 ℃, 10000rpm, 20min) to obtain the expressed protein. The results of gel chromatography electrophoresis analysis of protein distribution of the supernatant and the precipitate are shown in figure 1, and compared with the supernatant after bacterial disruption induced by not adding IPTG, the protein expression level at 80KD is obviously increased, and the protein is nCOV-N protein required by the invention.
4) Purification of the protein encoded by SEQ ID NO.1
The protein to be purified was stored at 4 ℃. The Ni-NTA affinity column was equilibrated with 10 column volumes of buffer D, and then the supernatant after disruption of the bacteria was injected into the Ni-NTA affinity chromatography column, and the protein was naturally bound to the column under gravity, and repeated 1-2 times. Then, the column was washed with 10 volumes of buffer D, and the filtrate was collected. The eluate was washed with 20 volumes of buffer E and collected. The target protein was then eluted with 5 volumes of buffer F and the eluate was collected and stored at 4 ℃. The serum donated by the new coronary pneumonia convalescent is used for identifying the eluent through an ELISA experiment.
The purified protein was packed into a 10kDa dialysis bag (Thermo Fisher Scientific, America) and dialyzed overnight at 4 ℃ in bufffer G. The protein concentration after dialysis was determined at 4 ℃ using the bicinchoninic acid protein assay (Tiangen-Biotech).
5) Preparation and purification of monoclonal antibodies
The synthesized protein coded by SEQ ID NO.1 is used as immunogen to immunize female Balb/c mice (20 g/mouse) with age of 5 weeks. The prepared protein coded by SEQ ID NO.1 is diluted in PBS to be used as antigen solution (the concentration is 200 mug/mL), the first immunization is carried out by emulsifying the antigen solution with the equal volume of complete Freund's adjuvant, and the dosage of the antigen solution is 100 mug/mouse; each subsequent booster immunization was performed using an equal volume of incomplete Freund's adjuvant emulsified with the antigen solution at the same dose as the initial immunization. After the primary immunization, the booster was given every 14 days for a total of 3 boosts. After immunization, tail blood was taken to test serum titer. Taking a mouse with the serum titer of more than 256000, and taking spleen cells of the mouse to perform cell fusion with a osteomyelitis cell SP2/0 according to a ratio of 10:1 under a sterile condition. And (3) screening the hybridoma cells by adopting a limiting dilution method to obtain a completely homogeneous monoclonal antibody and a stable monoclonal hybridoma cell strain.
Preparation and purification of monoclonal antibody: Balb/C mice were sensitized by intraperitoneal injection with an incomplete adjuvant at a dose of 0.5 mL/mouse one week before administration. The number of cells was 106Individual hybridoma cell suspensions were injected into the abdominal cavity of mice. After inoculating hybridoma cells for 7-10 days, ascites is collected, repeatedly collected for several times, and stored in a refrigerator at 4 ℃. Purifying ascites by caprylic acid-ammonium sulfate precipitation. The specific method comprises the following steps: adding 3 parts of sodium acetate buffer solution (with concentration of 0.05mol/L and pH of 4.0) into 1 part of ascites, adjusting pH to 4.5 with 0.1mmol/L NaOH, stirring at 4 deg.C for 30min, slowly adding octanoic acid during the period, and calculating by ascites volume before dilution to 40 μ L/mL; standing at 4 deg.C for 3 hr, centrifuging (11000r/min, 30min), collecting supernatant, maintaining at 4 deg.C, and adding (NH) within 30min4)2SO4The final concentration was made 0.277g/mL, and the mixture was allowed to stand for 1h, centrifuged at 4 deg.C (11000r/min, 30min), and the supernatant was discarded to obtain a monoclonal antibody precipitate, which was diluted with a concentrated sample diluent to a concentration of 2. mu.g/mL.
6) Preparation and purification of gold nanoparticle (AuNPs) -antibody conjugates
1ml of 0.01 percent HAuCl is taken4The solution was added to a 10mL beaker, heated and stirred rapidly, after boiling 4mL of 1% trisodium citrate was added, heating and stirring continued until the solution turned red, the solution was boiled for a further 10min and cooled to room temperature with slow stirring. By centrifugation 4 times (12X 10)3rpm, 20min), concentrate and collect colloidal gold. The resulting 0.01 wt% colloidal gold solution was stored at 4 ℃ until use.
To 10mL of the colloidal gold solution, 70. mu.L of 0.1M K was previously added2CO3Then, 1000. mu.L (50. mu.g) of nCoV-N protein antibody was added thereto, and the mixture was gently shaken at room temperature for 30 min. Then, 1mL of 1% Bovine Serum Albumin (BSA) was added and the uncoated sites of colloidal gold were blocked for 30min at room temperature. The particles were then centrifuged (12X 10)3rpm, 20min) and rinsed three times with buffer B to remove unbound antibody. The red precipitate was resuspended in 150. mu.L buffer B and then stored in a refrigerator at 4 ℃ until use.
7) Bond pad preparation
Gold nanoparticle (AuNPs) -antibody conjugate solution was dispensed onto glass fibers as a conjugate pad.
8) Preparation of nitrocellulose membranes
The nitrocellulose membrane is from a commercial company, after a laminator is assembled, a detection line and a control line are respectively scribed, wherein 30 mu L of another nCoV-N protein antibody with the concentration of 1mg/mL is scribed on the detection line (T line), 30 mu L of goat anti-mouse IgG with the concentration of 1mg/mL is scribed on the control line (C line), the coated nitrocellulose membrane is dried for 12h at room temperature, and sealed and stored in a vacuum manner in a moisture-proof cabinet for later use.
9) Preparation of sample pad
After the glass fiber was immersed in the sample pad buffer a, it was used as a sample pad, which was dried and stored at room temperature under low humidity.
10) Sample diluent: buffer C as sample diluent
11) Test strip assembly
The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad head-tail portion were connected in an overlapping direction of 1 to 2mm along the long axis of the adhesive sheet, and cut into 4mm wide strips with a paper cutter. Obtaining a nCoV-N protein detection kit.
In use, sputum, tears, blood or other samples are diluted in a sample diluent. They were mixed well and then placed in the sample pad of the biosensor and the results were observed within 5 minutes.
Example 2
This example is the determination of the sensitivity and specificity of the nCoV-N protein detection kit prepared in example 1
Diluting the serum of a patient into different concentrations by using a sample diluent: 100 times, 50 times and 10 times of samples are put into a sample pad of the biosensor. The results were observed within 5 minutes.
The results are shown in FIG. 2, with the biosensor from left to right containing serum from a healthy person and 100-fold, 50-fold, 10-fold diluted serum from a patient. The result shows that the serum detection result of the healthy person is that only the control line (C line) is developed; the serum of the patient diluted by 100 times is positive at low concentration, the C line develops color, and the control line (T line) develops light color; the serum of the patient diluted by 50 times is positive, the C line is developed, and the T line is developed obviously; the serum of the patient diluted by 10 times is positive, the C line is developed, and the T line is obviously developed.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
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Claims (10)
1. An nCoV-N protein detection kit, which is characterized by comprising: bonding a board;
the bonding plate is sequentially provided with a sample pad, a combination pad, a reaction film and an absorption pad, and the reaction film is provided with a detection line and a control line;
the combination pad is coated with a first nCoV-N protein monoclonal antibody solution marked by gold nanoparticles, the control line is coated with a second nCoV-N protein antibody solution, and the control line is coated with a goat anti-mouse IgG solution;
the epitopes of the nCoV-N protein to which the first nCoV-N protein monoclonal antibody and the second nCoV-N protein monoclonal antibody bind are different.
2. The nCoV-N protein detection kit according to claim 1, wherein the amino acid sequence of the epitope of the nCoV-N protein is shown as SEQ ID No.1, and the amino acid sequence of the epitope of the nCoV-N protein bound by the second nCoV-N protein monoclonal antibody is shown as SEQ ID No. 2.
3. The nCoV-N protein detection kit according to claim 1, wherein the amount of the gold nanoparticle-labeled first nCoV-N protein monoclonal antibody is 1-5 μ g/cm;
the dosage of the second nCoV-N protein antibody is 1-5 mug/cm;
the dosage of the goat anti-mouse IgG is 1-5 mu g/cm.
4. The nCoV-N protein detection kit according to claim 1, wherein the concentration of the gold nanoparticle-labeled first nCoV-N protein monoclonal antibody solution is 0.25-0.5 mg/mL;
the concentration of the second nCoV-N protein antibody solution is 0.5-2.5 mg/mL;
the concentration of the goat anti-mouse IgG solution is 0.5-2.5 mg/mL.
5. The nCoV-N protein detection kit according to claim 1, wherein the concentration of the gold nanoparticle-labeled first nCoV-N protein monoclonal antibody solution is 0.5 mg/mL;
the concentration of the second nCoV-N protein antibody solution is 1 mg/mL;
the concentration of the goat anti-mouse IgG solution is 1 mg/mL.
6. The nCoV-N protein assay kit of claim 1, wherein said sample pad is coated with a 50mM ph8.0 borate-borax buffer containing 0.5% Triton, 1% BSA, 2% sucrose.
7. The nCoV-N protein detection kit according to claim 1, wherein the solvent of the gold nanoparticle-labeled first nCoV-N protein monoclonal antibody solution is 20mM Na containing 5% BSA, 0.25% Tween-20 and 10% sucrose3PO4。
8. The nCoV-N protein detection kit according to claim 1, wherein the preparation method of the first nCoV-N protein monoclonal antibody comprises the following steps:
1) adopting protein coded by SEQ ID NO.1 as immunogen to immunize mice, taking spleen cells of the mice with high serum titer to perform cell fusion with myeloma cells SP2/0, and then adopting a limiting dilution method to screen hybridoma cells to obtain a completely homogeneous monoclonal antibody and a stable monoclonal hybridoma cell strain;
2) injecting the monoclonal hybridoma cell suspension into the abdominal cavity of a mouse, collecting ascites, and purifying the ascites by an octanoic acid-ammonium sulfate precipitation method to obtain a purified first nCoV-N protein monoclonal antibody.
9. The nCoV-N protein detection kit according to claim 8, wherein the immunization of a mouse with the protein encoded by SEQ ID No.1 as an immunogen comprises: emulsifying a complete Freund's adjuvant and a protein solution coded by SEQ ID NO.1 in an equal volume for the first immunization, emulsifying the incomplete Freund's adjuvant and the protein coded by SEQ ID NO.1 in an equal volume for each subsequent boosting immunization, wherein the dose is the same as that of the first immunization, and the boosting immunization is performed once every 14 days after the first immunization and 3 times in total;
the dose of the primary immunization is 100 mu g/mouse;
the concentration of the first nCoV-N protein antigen solution or the second nCoV-N protein antigen solution is 100 mu g-500 mu g/mL.
10. A nCoV-N protein detection method is characterized by comprising the following steps:
dropping a sample to be tested into the nCoV-N protein detection kit of any one of claims 1 to 9, wherein if the detection line and the control line are colored, the sample to be tested contains nCoV-N protein, and if the detection line is not colored and the quality control line is colored, the sample to be tested does not contain nCoV-N protein.
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