CN113466452A - Novel coronavirus IgM/IgG antibody detection kit - Google Patents
Novel coronavirus IgM/IgG antibody detection kit Download PDFInfo
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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Abstract
The invention relates to a 2019 novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit, which comprises a chromatography test paper, wherein the chromatography test paper comprises: a sample pad, a colloidal gold pad, a chromatographic membrane, a water absorbing material and a bottom plate; wherein, the colloidal gold pad is coated with a novel coronavirus protein colloidal gold conjugate; the chromatographic membrane is coated with a mouse anti-human IgG monoclonal antibody and a mouse anti-human IgM antibody. The detection kit provided by the invention has the advantages of high sensitivity, low false positive rate and the like, and is suitable for rapid diagnosis of novel coronavirus infection.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a 2019 novel coronavirus (SARS-CoV-2) IgM/IgG antibody detection kit.
Background
The coronavirus is a large virus family widely existing in nature, is named as crown like observed under an electron microscope, and mainly causes respiratory system diseases. Coronaviruses belong to the single-stranded positive-strand RNA viruses, the genetic material of which is the largest of all RNA viruses, and are classified into four genera, α, β, γ, and δ.
On day 11/2/2020, the world health organization named "COVID-19" for pneumonia infected with the novel coronavirus. Meanwhile, the International Committee for Classification of viruses has named the novel coronavirus "SARS-CoV-2". It belongs to the genus beta, is classified as a seventh group of coronaviruses, and can cause viral pneumonia, which is clinically manifested as respiratory symptoms, fever, cough, shortness of breath, dyspnea, and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. After a human is infected with a virus, the body can generate an immune response against the virus and produce antibodies. IgM antibodies are produced quickly (5-7 days), and gradually disappear after weeks or months; IgG antibody production is late (10-14 days), has higher affinity, and can persist for many years or even lifetime.
According to the results of the present study, the genome of the novel coronavirus (SARS-CoV-2) is arranged in the order of 5 '-replicase poly-glycoprotein (orf1/ab) -structural protein [ spike glycoprotein (spike, S) -small envelope glycoprotein (envelope, E) -membrane glycoprotein (membrane, M) -nucleocapsid protein (N) ] -3'. There is a key S protein in coronaviruses (spike protein) comprising 2 subunits: s1 and S2. S1 promotes binding of the virus to host cell receptors and contains an important C-terminal RBD domain responsible for receptor binding.
It is found that although SARS-CoV-2 belongs to the same beta-CoV as SARS-CoV and MERS-CoV, its genome shows higher sequence homology with SARS-CoV genome. However, the SARS-CoV-2 genome has a high degree of variability at two core positions. One is a silent variation in the ORF1ab gene and the other is a polymorphism in amino acid ORF 8. It is predicted that mutations in ORF8 result in two variants thereof (i.e., ORF8-L and ORF8-S), resulting in structural abnormalities of the protein. When SARS-CoV-2 is subjected to structural analysis, mutation occurs in its spike glycoprotein and nucleocapsid protein. This suggests that SARS-CoV-2 has unique genomic and structural features, which require further investigation.
Due to the short time of discovery of the novel coronavirus (SARS-CoV-2), its genomic and structural characteristics are under constant investigation. The existing novel coronavirus antibody detection kit has short development period, and the detection sensitivity needs to be further improved.
Disclosure of Invention
In order to solve the above problems, the present invention provides, in a first aspect, a novel coronavirus IgM/IgG antibody detection kit, which can achieve simultaneous detection of an IgM antibody and an IgG antibody of a novel coronavirus, and improve detection sensitivity. The kit comprises chromatographic test paper, wherein the chromatographic test paper comprises: a sample pad, a colloidal gold pad, a chromatographic membrane, a water absorbing material and a bottom plate. Wherein, the colloidal gold pad is coated with a novel coronavirus protein colloidal gold conjugate; the chromatographic membrane is coated with a mouse anti-human IgG monoclonal antibody and a mouse anti-human IgM monoclonal antibody. Wherein the novel coronavirus protein colloidal gold conjugate on the colloidal gold pad is selected from 2 or more than 2 of novel coronavirus S1 protein colloidal gold conjugate, novel coronavirus S2 protein colloidal gold conjugate, novel coronavirus S protein colloidal gold conjugate, novel coronavirus RBD protein colloidal gold conjugate and novel coronavirus N protein colloidal gold conjugate.
Preferably, the novel coronavirus IgM/IgG antibody detection kit of the present invention further comprises a streptavidin colloidal gold conjugate coated on the colloidal gold pad, and biotinylated BSA coated on the chromatographic membrane.
Preferably, in the detection kit of the present invention, the chromatographic membrane is further coated with a mouse anti-human IgA antibody to detect IgA/IgM/IgG antibodies of the novel coronavirus.
According to the detection kit, the colloidal gold pad is of a single-layer structure, a double-layer structure or a three-layer structure. If the structure is a single-layer structure, more than two colloidal gold combination materials are mixed and coated on the colloidal gold pad; if the structure is a double-layer structure or a three-layer structure, 1 or more than 1 colloidal gold conjugate is coated on each colloidal gold pad.
Preferably, the novel coronavirus IgM/IgG antibody detection kit disclosed by the invention is coated with a mouse anti-human erythrocyte monoclonal antibody on the sample pad, is suitable for detecting serum and plasma samples and whole blood samples, and widens the application range of the kit.
Preferably, the novel coronavirus IgM/IgG antibody detection kit of the present invention further comprises a sample diluent. The sample diluent comprises: sodium chloride: 8.5-9.0 g/L, NP-40: 20-40 g/L. In the research and development process, the applicant unexpectedly finds that the detection sensitivity is obviously improved by adopting the detection result obtained by diluting the sample to be detected by the normal saline added with the NP-40 compared with the normal saline not added with the NP-40. In addition, the applicants have also found that the amount of NP-40 added has an influence on the results of the assay, and that, for example, when the concentration of NP-40 is 50g/L, the false positive rate obtained by the assay is high; when the concentration of NP-40 is 20, 23, 25, 28, 30, 32, 34, 35, 37, 39 or 40g/L, the detection effect is good.
The invention provides another novel coronavirus IgM/IgG antibody detection kit, which comprises chromatography test paper, wherein the chromatography test paper comprises: a sample pad, a colloidal gold pad, a chromatographic membrane, a water absorbing material and a bottom plate. Wherein the colloidal gold pad is coated with a novel coronavirus S protein colloidal gold conjugate and a novel coronavirus N protein colloidal gold conjugate; the chromatographic membrane is coated with a mouse anti-human IgG monoclonal antibody and a mouse anti-human IgM monoclonal antibody.
In the present invention, the "novel coronavirus S protein colloidal gold conjugate", i.e., the colloidal gold-labeled novel coronavirus S protein, refers to a product obtained by mixing and reacting the novel coronavirus S protein with colloidal gold. The colloidal gold conjugate of the novel coronavirus N protein, namely the colloidal gold labeled novel coronavirus N protein, is a product obtained by mixing and reacting the novel coronavirus N protein with colloidal gold.
The novel coronavirus S protein, the novel coronavirus N protein, the novel coronavirus S1 protein, the novel coronavirus S2 protein and the novel coronavirus RBD protein can be natural proteins or recombinant proteins obtained by expression through a conventional genetic engineering technology. The amino acid sequence of a novel coronavirus (SARS-CoV-2) and its encoding nucleic acid sequence have been disclosed at NCBI, and the genome arrangement order thereof is 5 '-replicase poly-glycoprotein (orf1/ab) -structural protein [ spike glycoprotein (spike, S) -small envelope glycoprotein (envelope, E) -membrane glycoprotein (membrane, M) -nucleocapsid protein (nucleocapsid, N) ] -3'. There is a key S protein in coronaviruses (spike protein) comprising 2 subunits: s1 and S2. S1 promotes binding of the virus to host cell receptors and contains an important C-terminal RBD domain responsible for receptor binding.
There are many commercially available products of recombinant proteins of novel coronavirus, such as recombinant novel coronavirus S protein (product number is XCL1830) produced by Sichuan Mike BioNew Material technology, Inc., recombinant novel coronavirus N protein (product number is XCL1829) produced by Sichuan Mike BioNew Material technology, Inc., and recombinant novel coronavirus S protein (product number is FB2019HIS) produced by Beijing Fubo Biotechnology, Inc.
Preferably, the novel coronavirus IgM/IgG antibody detection kit of the present invention further comprises a streptavidin colloidal gold conjugate coated on the colloidal gold pad, and biotinylated BSA coated on the chromatographic membrane. "biotinylated BSA" refers to biotin-modified bovine serum albumin or a derivative thereof.
Preferably, the novel coronavirus IgM/IgG antibody detection kit of the present invention has a double-layer structure of a colloidal gold pad, wherein a first layer of the colloidal gold pad is coated with a streptavidin colloidal gold conjugate and a novel coronavirus N protein colloidal gold conjugate; and a second layer of colloidal gold pad is coated with streptavidin colloidal gold conjugate and novel coronavirus S protein colloidal gold conjugate. The applicant unexpectedly finds that the colloidal gold pad is set to be of a double-layer structure, so that a better color development effect can be realized, and the judgment on the detection result is facilitated.
Preferably, the novel coronavirus IgM/IgG antibody detection kit disclosed by the invention is coated with a mouse anti-human erythrocyte monoclonal antibody on the sample pad, is suitable for detecting serum and plasma samples and whole blood samples, and widens the application range of the kit.
Preferably, the novel coronavirus IgM/IgG antibody detection kit of the present invention further comprises a sample diluent. The sample diluent comprises: sodium chloride: 8.5-9.0 g/L, NP-40: 20-40 g/L. In the research and development process, the applicant unexpectedly finds that the detection sensitivity is obviously improved by adopting the detection result obtained by diluting the sample to be detected by the normal saline added with the NP-40 compared with the normal saline not added with the NP-40. In addition, the applicants have also found that the amount of NP-40 added has an influence on the results of the assay, and that, for example, when the concentration of NP-40 is 50g/L, the false positive rate obtained by the assay is high; when the concentration of NP-40 is 20, 23, 25, 28, 30, 32, 34, 35, 37, 39 or 40g/L, the detection effect is good.
In another aspect of the invention, a preparation method of the novel coronavirus IgM/IgG antibody detection kit is provided, which comprises the following steps:
(1) preparation of sample pad
Coating the mouse anti-human erythrocyte monoclonal antibody on a glass fiber membrane to obtain the mouse anti-human erythrocyte monoclonal antibody;
(2) preparation of colloidal gold pad
Mixing streptavidin and a colloidal gold solution for reaction to obtain a streptavidin colloidal gold conjugate;
mixing the novel coronavirus N protein with a colloidal gold solution for reaction to obtain a novel coronavirus N protein colloidal gold conjugate;
mixing the novel coronavirus S protein with a colloidal gold solution for reaction to obtain a novel coronavirus S protein colloidal gold conjugate;
mixing the streptavidin colloidal gold conjugate with the novel coronavirus N protein colloidal gold conjugate, and then paving the mixture on a first glass fiber membrane to obtain a first layer of colloidal gold pad;
mixing the streptavidin colloidal gold conjugate with the novel coronavirus S protein colloidal gold conjugate, and then paving the mixture on a second glass fiber membrane to obtain a second layer of colloidal gold pad;
(3) preparation of chromatographic membranes
Respectively marking a mouse anti-human IgG monoclonal antibody as a detection line G, a mouse anti-human IgM monoclonal antibody as a detection line M and biotinylation BSA as a quality control line C on a nitrocellulose membrane to obtain a chromatographic membrane;
(4) assembly of chromatography test paper
And assembling the sample pad, the colloidal gold pad, the chromatographic membrane, the water absorbing material and the bottom plate to obtain the chromatographic test paper.
Preferably, in the first layer of colloidal gold pad, the streptavidin colloidal gold conjugate and the novel coronavirus N protein colloidal gold conjugate are mixed according to a concentration ratio of 1: 1-1: 5, and more preferably, the mixing ratio is 1: 2.5; and in the second layer of colloidal gold pad, mixing the streptavidin colloidal gold conjugate and the novel coronavirus S protein colloidal gold conjugate according to a concentration ratio of 1: 1-1: 5, and more preferably, the mixing ratio is 1: 2.5.
In the present invention, the expressions "first", "second", etc. are used for descriptive purposes only to distinguish defined objects, and not to define an order or primary or secondary in any way.
As used herein, the terms "novel coronavirus", "2019 novel coronavirus", "SARS-CoV-2", "2019-nCoV" and "novel coronavirus" are interchangeable and are all novel coronaviruses designated by the International Committee for viral Classification as "SARS-CoV-2".
Advantageous effects
Compared with the prior art, the applicant unexpectedly finds that the SARS-CoV-2 coronavirus N protein and the SARS-CoV-2 coronavirus S protein are used as diagnostic antigens to simultaneously detect the SARS-CoV-2 coronavirus IgM antibody and the IgG antibody in a sample, and can improve the sensitivity of a detection kit and reduce false positive. In addition, the applicant also unexpectedly finds that the detection sensitivity can be further improved and the false positive can be reduced by adopting a special sample diluent in the detection kit.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly demonstrated. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
The reagent components mentioned in the following examples are commercially available products, for example, the novel coronavirus RBD protein is obtained from Sichuan Michael BioNew Material technology Co., Ltd. (cargo number: XCL1831), the novel coronavirus N protein is obtained from Sichuan Michael BioNew Material technology Co., Ltd. (cargo number: XCL1829), the novel coronavirus S protein is obtained from Sichuan Michael BioNew Material technology Co., Ltd. (cargo number: XCL1830), the mouse anti-human IgG monoclonal antibody is obtained from Sichuan Michael BioNew Material technology Co., Ltd. (cargo number: XCL1833), and the mouse anti-human IgM monoclonal antibody is obtained from Sichuan Michael BioNew Material technology Co., Ltd. (cargo number: XCL 1832).
EXAMPLE 1 novel coronavirus IgM/IgG antibody detection kit of the present invention
Novel coronavirus IgM/IgG antibody detection kit, including the chromatography test paper: a sample pad, a colloidal gold pad, a chromatographic membrane, a water absorbing material and a bottom plate. Wherein, the sample pad is coated with a mouse anti-human erythrocyte monoclonal antibody; the colloidal gold pad has two layers, wherein one layer of the colloidal gold pad is coated with a streptavidin colloidal gold conjugate and a novel coronavirus N protein colloidal gold conjugate, and the other layer of the colloidal gold pad is coated with the streptavidin colloidal gold conjugate and a novel coronavirus S protein colloidal gold conjugate; the chromatographic membrane is provided with a detection line G, a detection line M and a quality control line C which are respectively coated with a mouse anti-human IgG monoclonal antibody, a mouse anti-human IgM monoclonal antibody and biotinylated BSA.
The detection kit further comprises a sample diluent, wherein the sample diluent comprises: sodium chloride: 8.5-9.0 g/L, NP-40: 20-40 g/L.
The use method of the detection kit comprises the following steps:
(1) taking 10 mu L of serum/plasma sample or 20uL of whole blood sample into the sample diluent, and fully and uniformly mixing for 5-10 s;
(2) dripping 2-3 drops vertically onto the sample pad, and standing at room temperature;
(3) the results were observed within 10 to 15 minutes and after 15 minutes, the results were judged to be invalid:
if the sample contains IgG antibody or IgM antibody resisting the new coronavirus antigen, firstly reacting with coronavirus protein (antigen) marked by colloidal gold on a test paper colloidal gold pad, moving on a chromatographic membrane along with a lateral flow chromatography effect, and reacting a reaction complex formed by the IgG antibody resisting the coronavirus antigen and the protein (antigen) marked by the colloidal gold on a detection line G with a mouse anti-human IgG monoclonal antibody to form an antigen-antibody colloidal gold complex which is in a regular red strip G and has no judgment result according to the red strip; a reaction complex formed by the IgM antibody of the anti-new coronavirus antigen in the sample and the protein marked by the colloidal gold is sent to a detection line M to react with the mouse anti-human IgM monoclonal antibody to form an antigen-antibody colloidal gold complex which is a regular red strip M and has no judgment result according to the red strip; the colloidal gold labeled avidin continues to move forward until the quality control line C reacts with the biotin, and a neat red strip C is formed, which indicates that the detection reaction system is effective.
Example 2 preparation method of the novel coronavirus IgM/IgG antibody detection kit of the present invention
1. Sample pad preparation:
taking a mouse anti-human red blood cell monoclonal antibody solution with the final concentration of 0.1mg/mL, and mixing according to the ratio of 40 mu l/cm2Uniformly pouring the sample on a glass fiber membrane, balancing for about 30min at room temperature, and drying at 30-42 ℃ for 6-24 h to obtain the sample pad.
2. Preparing a colloidal gold pad:
(1) preparing a colloidal gold solution with the diameter of about 40nm by a chloroauric acid-trisodium citrate reduction method, putting 100ml of the colloidal gold solution in a beaker after the preparation is finished, adjusting the pH to about 10.0 by using 0.1M potassium carbonate solution, adding 2.5mg of streptavidin into the 100ml of the colloidal gold solution, uniformly mixing, reacting for 30min, adding 5g/L of casein solution (the adding amount is 10 percent of the total volume), uniformly mixing, and reacting for 30 min. After the marking is finished, centrifuging for 30 minutes at 10000r/m, removing supernatant, redissolving to 5ml, taking 50 mu l of colloidal gold protein compound, diluting the colloidal gold protein compound by ultrapure water until OD is about 1, scanning at the wavelength of 450 nm-700 nm, recording the maximum absorption wavelength and the OD value thereof, and calculating the original OD value of the colloidal gold protein compound;
(2) preparing a colloidal gold solution with the diameter of about 40nm by a chloroauric acid-trisodium citrate reduction method, putting 100ml of the colloidal gold solution in a beaker after the preparation is finished, adjusting the pH to about 10.0 by using 0.1M sodium hydroxide solution, adding 2.5mg of SARS-CoV-2 coronavirus N protein into 100ml of the colloidal gold solution, mixing uniformly, reacting for 30min, adding 5g/L of casein solution (the adding amount is 10 percent of the total volume), mixing uniformly, and reacting for 30 min. After the marking is finished, centrifuging for 30 minutes at 10000r/m, removing supernatant, redissolving to 5ml, taking 50 mu l of colloidal gold protein compound, diluting the colloidal gold protein compound by ultrapure water until OD is about 1, scanning at the wavelength of 450 nm-700 nm, recording the maximum absorption wavelength and the OD value thereof, and calculating the original OD value of the colloidal gold protein compound;
the concentration of the new coronavirus N protein colloidal gold protein complex in the gold pad coating solution to be prepared is about OD 10, the concentration of the streptavidin colloidal gold protein complex is about OD 4, the new coronavirus N protein colloidal gold protein complex, the dosage of the streptavidin colloidal gold protein complex and the compound solution are calculated, the new coronavirus N protein colloidal gold protein complex, the dosage of the streptavidin colloidal gold protein complex and the compound solution are uniformly mixed, and the mixture is paved into a solution of 22cm according to the volume of 1ml2Uniformly spreading the glass fiber membrane in the proportion, placing the glass fiber membrane in a drying room, drying for 6-24 hours at the temperature of 30-42 ℃ and the humidity of less than 30%;
(3) preparing a colloidal gold solution with the diameter of 40nm by a chloroauric acid-trisodium citrate reduction method, putting 100ml of the colloidal gold solution in a beaker after the preparation is finished, adjusting the pH to be about 10.0 by using 0.1M sodium hydroxide solution, adding 1.5mg of SARS-CoV-2 coronavirus S protein into 100ml of the colloidal gold solution, mixing uniformly, reacting for 30min, adding 5g/L of casein solution (the adding amount is 10 percent of the total volume), mixing uniformly, and reacting for 30 min. After the marking is finished, centrifuging for 30 minutes at 10000r/m, removing supernatant, redissolving to 5ml, taking 50 mu l of colloidal gold protein compound, diluting the colloidal gold protein compound by ultrapure water until OD is about 1, scanning at the wavelength of 450 nm-700 nm, recording the maximum absorption wavelength and the OD value thereof, and calculating the original OD value of the colloidal gold protein compound;
the concentration of the new coronavirus S protein colloidal gold protein complex in the gold pad coating solution to be prepared is about OD 10, and the concentration of the streptavidin colloidal gold protein complex isOD is about 4, then the dosage of the new coronavirus S protein colloidal gold protein complex, the dosage of the streptavidin colloidal gold protein complex and the redissolution are calculated, the new coronavirus S protein colloidal gold protein complex, the streptavidin colloidal gold protein complex and the redissolution are mixed evenly, and the mixture is paved into 22cm per 1ml of solution2The glass fiber membrane is uniformly paved on a glass fiber membrane in proportion, and then the glass fiber membrane is placed in a drying room, the temperature is 30-42 ℃, the humidity is less than 30%, and the drying is carried out for 6-24 hours.
3. Preparing a chromatographic membrane:
diluting biotinylated BSA with 30mM Tris buffer solution with pH10.0 to final concentration of 2mg/mL as quality control line coating solution, diluting mouse anti-human IgG monoclonal antibody and mouse anti-human IgM monoclonal antibody with 10mM phosphate buffer solution with pH 7.4 to final concentrations of 2mg/mL and 2mg/mL respectively as mouse anti-human IgM monoclonal antibody detection line coating solution and mouse anti-human IgG monoclonal antibody detection line coating solution, adding the coating solutions into pumps of a striping machine respectively, taking a PVC bottom plate with the specification of 300mM multiplied by 60mM/15mM +25mM +20mM, tearing off a non-drying adhesive with the width of 25mM in the middle, pasting a nitrocellulose membrane with the width of 25mM along the lower edge of the non-drying adhesive at the lower end of the middle, fixing the nitrocellulose membrane on the PVC bottom plate as a chromatographic membrane, striping a mouse anti-human IgG monoclonal antibody detection line G, a mouse anti-human IgM monoclonal antibody detection line M and a quality control line C on the chromatographic membrane respectively, after scribing, placing the chromatographic membrane in a drying room, drying for 5 hours at the temperature of 20 ℃ and the humidity of less than 30%;
4. assembling chromatography test paper:
the chromatographic carrier with detection line G, detection line M and quality control line C is close to C line one side and pastes 18 mm' S absorbent paper, absorbent paper upper edge aligns with PVC bottom plate upper edge, cut into 5mm rectangular with the guillootine with the colloidal gold pad of cladding reorganization N albumen, press lower limb 1mm to paste, cut into 5mm rectangular with the colloidal gold pad of cladding new coronavirus S albumen again, press 3-4mm of cladding new coronavirus N albumen gold pad to paste, the sample pad cuts into 17mm rectangular, the lower extreme aligns PVC bottom plate lower limb and pastes, cut into 4mm wide test paper strip with the slitter with the big board of dress, obtain.
Example 3 laboratory Performance testing of the kits
The performance of the test kit prepared in example 2 was examined.
1. Minimum detection limit
Detecting with 3 parts of SARS-CoV-2-IgG enterprise minimum detection limit reference, wherein L1 should be positive, L2 can be positive or negative, and L3 should be negative;
when the reagent is detected by using 3 parts of SARS-CoV-2-IgM enterprise minimum detection limit reference substances, L1 should be positive, L2 can be positive or negative, and L3 should be negative.
2. Positive reference compliance rate
When 5 parts of SARS-CoV-2-IgM/IgG enterprise positive reference substance is used for detection, IgG antibodies of P1, P2 and P3 are positive, IgM antibodies of P4 and P5 are positive, and the positive coincidence rate is not lower than 5/5.
3. Negative reference product compliance rate
When 20 parts of SARS-CoV-2-IgM/IgG enterprise negative reference is used for detection, the IgG antibody coincidence rate should not be lower than 20/20, and the IgM antibody negative reference coincidence rate should not be lower than 18/20.
4. Repeatability of
1 part of SARS-CoV-2-IgM/IgG enterprise repetitive reference substance is used for parallel detection for 10 times, and the IgM antibody/IgG antibody should be positive and have consistent color development.
5. The anti-interference capability: the drugs commonly used for the treatment of pathogen infection, such as alpha-interferon, zanamivir, ribavirin, oseltamivir, peramivir, lopinavir, ritonavir, abidol, levofloxacin, azithromycin, ceftriaxone, meropenem, tobramycin and the like, have no obvious influence on the determination of the kit.
6. HOOK (HOOK) effect: the kit has no obvious hook effect on high-concentration specific IgM antibodies and IgG antibodies.
7. Sensitivity and specificity of the kit
64 positive serum samples with confirmed diagnosis were detected by using the detection kit prepared in example 2, and the detection results are shown in Table 1.
TABLE 1
60 normal human serum samples were tested using the test kit prepared in example 2, and the test results are shown in Table 2.
TABLE 2
As can be seen from tables 1 and 2, the detection kit of the present invention has high sensitivity and specificity.
Comparative example
Comparative example 1
The comparative example differs from example 1 only in that: the colloidal gold pad is of a single-layer structure, is coated with a streptavidin colloidal gold conjugate and a coronavirus N protein colloidal gold conjugate, and the rest is the same.
Comparative example 2
The comparative example differs from example 1 only in that: the colloidal gold pad is of a single-layer structure, is coated with a streptavidin colloidal gold conjugate and a coronavirus S protein colloidal gold conjugate, and the rest is the same.
Comparative example 3
The comparative example differs from example 1 only in that: the colloidal gold pad is still in a double-layer structure, and coronavirus S protein is replaced by coronavirus RBD protein, and the rest is the same.
The kits of comparative examples 1-3 were used to test 64 positive serum samples with confirmed diagnosis, and the test results are shown in Table 3.
TABLE 3
As can be seen from Table 3, the positive detection rates of the test kits of comparative examples 1 to 3 were to be further improved.
Claims (10)
1. The utility model provides a novel coronavirus IgM/IgG antibody detection kit, includes the chromatography test paper, its characterized in that, the chromatography test paper includes: a sample pad, a colloidal gold pad, a chromatographic membrane, a water absorbing material and a bottom plate; the colloidal gold pad is coated with a novel coronavirus protein colloidal gold conjugate; the chromatographic membrane is coated with a mouse anti-human IgG monoclonal antibody and a mouse anti-human IgM monoclonal antibody.
2. The detection kit according to claim 1, wherein the novel coronavirus protein colloidal gold conjugate is selected from the group consisting of a novel coronavirus S1 protein colloidal gold conjugate, a novel coronavirus S2 protein colloidal gold conjugate, a novel coronavirus S protein colloidal gold conjugate, a novel coronavirus RBD protein colloidal gold conjugate, and a novel coronavirus N protein colloidal gold conjugate, and any combination of two or more thereof.
3. The utility model provides a novel coronavirus IgM/IgG antibody detection kit, includes the chromatography test paper, its characterized in that, the chromatography test paper includes: a sample pad, a colloidal gold pad, a chromatographic membrane, a water absorbing material and a bottom plate; the colloidal gold pad is coated with a novel coronavirus S protein colloidal gold conjugate and a novel coronavirus N protein colloidal gold conjugate; the chromatographic membrane is coated with a mouse anti-human IgG monoclonal antibody and a mouse anti-human IgM monoclonal antibody.
4. The detection kit of claim 1, wherein the colloidal gold pad is further coated with streptavidin colloidal gold conjugate, and the chromatographic membrane is further coated with biotinylated BSA.
5. The detection kit according to claim 2, wherein the colloidal gold pad has a double-layer structure, and the first layer of colloidal gold pad is coated with a streptavidin colloidal gold conjugate and a novel coronavirus N-protein colloidal gold conjugate; the second layer of colloidal gold pad is coated with streptavidin colloidal gold conjugate and novel coronavirus S protein colloidal gold conjugate.
6. The test kit of claim 1, wherein the sample pad is coated with a murine anti-human red blood cell monoclonal antibody.
7. The test kit of claim 1, further comprising a sample diluent comprising: sodium chloride: 8.5-9.0 g/L, NP-40: 20-40 g/L; preferably, the sample diluent comprises: sodium chloride: 8.5g/L, NP-40: 25 g/L.
8. The method for preparing the novel coronavirus IgM/IgG antibody detection kit according to any one of claims 1 to 7, which comprises the steps of:
(1) preparation of sample pad
Coating the mouse anti-human erythrocyte monoclonal antibody on a glass fiber membrane to obtain the mouse anti-human erythrocyte monoclonal antibody;
(2) preparation of colloidal gold pad
Mixing streptavidin and a colloidal gold solution for reaction to obtain a streptavidin colloidal gold conjugate;
mixing the novel coronavirus N protein with a colloidal gold solution for reaction to obtain a novel coronavirus N protein colloidal gold conjugate;
mixing the novel coronavirus S protein with a colloidal gold solution for reaction to obtain a novel coronavirus S protein colloidal gold conjugate;
mixing the streptavidin colloidal gold conjugate with the novel coronavirus N protein colloidal gold conjugate, and then paving the mixture on a first glass fiber membrane to obtain a first layer of colloidal gold pad;
mixing the streptavidin colloidal gold conjugate with the novel coronavirus S protein colloidal gold conjugate, and then paving the mixture on a second glass fiber membrane to obtain a second layer of colloidal gold pad;
(3) preparation of chromatographic membranes
Respectively marking a mouse anti-human IgG monoclonal antibody as a detection line G, a mouse anti-human IgM monoclonal antibody as a detection line M and biotinylation BSA as a quality control line C on a nitrocellulose membrane to obtain a chromatographic membrane;
(4) assembly of chromatography test paper
And assembling the sample pad, the colloidal gold pad, the chromatographic membrane, the water absorbing material and the bottom plate to obtain the chromatographic test paper.
9. The method for preparing the novel coronavirus IgM/IgG antibody detection kit according to claim 8, wherein in the first layer of colloidal gold pad, streptavidin colloidal gold conjugate and novel coronavirus N protein colloidal gold conjugate are mixed in a concentration ratio of 1: 1-1: 5; and in the second layer of colloidal gold pad, mixing the streptavidin colloidal gold conjugate and the novel coronavirus S protein colloidal gold conjugate according to the concentration ratio of 1: 1-1: 5.
10. The method for preparing the novel coronavirus IgM/IgG antibody detection kit according to claim 9, wherein in the first layer of colloidal gold pad, streptavidin colloidal gold conjugate and novel coronavirus N-protein colloidal gold conjugate are mixed in a concentration ratio of 1: 2.5; in the second layer of colloidal gold pad, the streptavidin colloidal gold conjugate and the novel coronavirus S protein colloidal gold conjugate are mixed according to the concentration ratio of 1: 2.5.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265800A1 (en) * | 2003-06-30 | 2004-12-30 | Sysmex Corporation | Sample pretreatment solution for immunological test and method for using the same |
| CN103059132A (en) * | 2012-12-17 | 2013-04-24 | 华中农业大学 | Monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein and application thereof |
| CN104764884A (en) * | 2014-08-20 | 2015-07-08 | 苏州和锐医药科技有限公司 | Preparation method and application of anti-alpha-enolase antibody chromatography test strip |
| CN105859843A (en) * | 2016-04-29 | 2016-08-17 | 兰州雅华生物技术有限公司 | Preparation method of respiratory syncytial virus antigen and rapid respiratory syncytial virus antibody detection kit prepared from antigen |
| CN205910196U (en) * | 2016-05-26 | 2017-01-25 | 江苏雷森生物科技有限公司 | Colloidal gold test paper card of short -term test dog coronavirus antibody |
| CN106771123A (en) * | 2016-12-14 | 2017-05-31 | 苏州万木春生物技术有限公司 | A kind of preparation method of dengue virus IgM/IgG antibody Test paper |
| CN106841601A (en) * | 2016-12-30 | 2017-06-13 | 广州市达瑞生物技术股份有限公司 | The preparation of zika virus multiple clips fusion protein and IgG/IgM antibody assay kits |
| CN107966561A (en) * | 2017-11-24 | 2018-04-27 | 德诺杰亿（北京）生物科技有限公司 | A kind of test strips and kit for detecting HIV antibody |
| CN108181473A (en) * | 2017-12-25 | 2018-06-19 | 迈克生物股份有限公司 | Schistosome antibody detection kit |
| CN209656716U (en) * | 2019-04-09 | 2019-11-19 | 潍坊市康华生物技术有限公司 | A kind of 6 type of Coxsackie virus A and A10 type IgM antibody combined detection kit |
-
2020
- 2020-03-31 CN CN202010240846.8A patent/CN113466452A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040265800A1 (en) * | 2003-06-30 | 2004-12-30 | Sysmex Corporation | Sample pretreatment solution for immunological test and method for using the same |
| CN103059132A (en) * | 2012-12-17 | 2013-04-24 | 华中农业大学 | Monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein and application thereof |
| CN104764884A (en) * | 2014-08-20 | 2015-07-08 | 苏州和锐医药科技有限公司 | Preparation method and application of anti-alpha-enolase antibody chromatography test strip |
| CN105859843A (en) * | 2016-04-29 | 2016-08-17 | 兰州雅华生物技术有限公司 | Preparation method of respiratory syncytial virus antigen and rapid respiratory syncytial virus antibody detection kit prepared from antigen |
| CN205910196U (en) * | 2016-05-26 | 2017-01-25 | 江苏雷森生物科技有限公司 | Colloidal gold test paper card of short -term test dog coronavirus antibody |
| CN106771123A (en) * | 2016-12-14 | 2017-05-31 | 苏州万木春生物技术有限公司 | A kind of preparation method of dengue virus IgM/IgG antibody Test paper |
| CN106841601A (en) * | 2016-12-30 | 2017-06-13 | 广州市达瑞生物技术股份有限公司 | The preparation of zika virus multiple clips fusion protein and IgG/IgM antibody assay kits |
| CN107966561A (en) * | 2017-11-24 | 2018-04-27 | 德诺杰亿（北京）生物科技有限公司 | A kind of test strips and kit for detecting HIV antibody |
| CN108181473A (en) * | 2017-12-25 | 2018-06-19 | 迈克生物股份有限公司 | Schistosome antibody detection kit |
| CN209656716U (en) * | 2019-04-09 | 2019-11-19 | 潍坊市康华生物技术有限公司 | A kind of 6 type of Coxsackie virus A and A10 type IgM antibody combined detection kit |
Non-Patent Citations (3)
| Title |
|---|
| RONGQING ZHAO 等: "Serological diagnostic kit of SARS-CoV-2 antibodies using CHO-expressed full-length SARS-CoV-2 S1 proteins", MEDRXIV, pages 3 - 4 * |
| 四川省人民医院: "我院杨正林团队成功研制出新冠病毒的早期快速准确检测诊断试剂盒", pages 2, Retrieved from the Internet <URL:四川省人民医院官网> * |
| 晏彩霞;李佳;沈鑫;罗丽;李燕;李明远;: "用于新型冠状病毒肺炎防治的生物制品研究策略", 四川大学学报(医学版), no. 02, pages 14 - 20 * |
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