CN117434262A - Novel rapid detection device for coronavirus antigen, kit and preparation method - Google Patents
Novel rapid detection device for coronavirus antigen, kit and preparation method Download PDFInfo
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Classifications
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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- 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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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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- 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
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Abstract
The invention uses the red latex microsphere as the tracer to prepare the test strip, and can qualitatively detect the novel coronavirus in the nasopharyngeal swab sample. As the surface is hydrophilic and the group content is rich, the protein binding capacity is higher, the sensitivity is higher, compared with a colloidal gold test strip, the sensitivity of the method is improved by 16 times, the detection method is simple and convenient and quick to operate, and particularly, the detection result can be obtained quickly, and the method can be used for clinical auxiliary diagnosis.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a rapid detection device, a kit and a preparation method, which are used for rapid and accurate detection of novel coronavirus antigens.
Background
The novel coronavirus pneumonia (covd-19) is an acute respiratory infectious disease caused by the novel coronavirus (SARS-CoV-2). SARS-COV-2 belongs to the genus beta coronavirus and has 5 essential genes encoding spike protein (S), matrix protein (M), viral envelope (E), nucleoprotein (N) and RNA polymerase (RdRp), respectively. Coronaviruses are a large virus family and are known to cause severe diseases such as common cold and Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a novel strain of coronavirus that has not been previously found in humans. Common signs of a person infected with coronavirus are 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. For diagnosis and treatment of patients, isolation of the intimate contact, various types of detection reagents have been developed for use in the diagnosis of covd-19, the most commonly used biomarkers being S protein and N protein. N protein is rich in coronavirus, is a high immunogenicity protein, participates in genome replication and cell signal path regulation, and has important value for diagnosis and investigation of new coronavirus.
The diagnosis method for the COVID-19 mainly comprises nucleic acid detection, antibody detection and antigen detection. At present, the nucleic acid detection is a gold standard, and the amplification detection is carried out on the virus RNA in a novel coronavirus infection sample by a full-automatic fluorescence PCR instrument, so that the detection of the novel coronavirus is realized. However, the method is complicated to operate, requires special laboratories and specialized personnel, and has long measurement period and high cost. Common methods for detection of novel coronavirus antibodies are enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay and lateral flow immunochromatography; the novel coronavirus antigen detection method is mainly a lateral flow immunochromatography method. Different detection techniques have a emphasis and cannot be replaced with each other.
Lateral Flow Immunochromatography (LFIA) is characterized by simplicity, rapidity, and intuitiveness, and currently there are more than 1000 immunochromatography test systems for the detection of various analytes. The technology has rapid development, improves the nitrocellulose membrane, develops novel markers such as carbon nano tubes, quantum dots, lanthanoid elements, superparamagnetic nano particles and the like besides colloidal gold nano particles as markers, and improves the sensitivity and stability of detection. The immunochromatography technology using colloidal gold as a marker is a traditional classical method, is still commonly adopted at present, and the domestic new crown antigen detection reagent is mostly based on the principle of colloidal gold immunochromatography. The colloidal gold immunochromatography method is simple to operate, short in reaction time and convenient to store reagents, but low in detection sensitivity, so that the accuracy of detection reagents is also affected.
Disclosure of Invention
The invention uses the red latex microsphere as the tracer to prepare the test strip, and can qualitatively detect the novel coronavirus in the nasopharyngeal swab sample. As the surface is hydrophilic and the group content is rich, the protein binding capacity is higher, the sensitivity is higher, compared with a colloidal gold test strip, the sensitivity of the method is improved by 16 times, the detection method is simple and convenient and quick to operate, and particularly, the detection result can be obtained quickly, and the method can be used for clinical auxiliary diagnosis.
The invention provides a novel rapid detection device for coronavirus antigens, which comprises a detection card (10) and a test strip (9);
the detection card comprises a detection card cover (1) and a detection card bottom (2), wherein the detection card cover (1) and the detection card bottom (2) are connected through a hinge and can be buckled together, a test strip clamping groove (8) for placing test strips is formed in the inner surface of the detection card bottom (2), the detection card cover (1) comprises a lower card cover (4), a swab placing groove (6) is formed in the inner side surface of the lower card cover (4), a swab fixing part (7) which can be buckled together with the lower card cover (4) is connected through the hinge, and a slot (7.1) corresponding to the swab placing groove (6) is formed in the swab fixing part (7);
the test strip is formed by sequentially adhering a sample pad (9.2), a microsphere pad (9.3), a cellulose acetate film (NC film) (9.4) and water absorbing paper (9.7) to a PVC bottom plate (9.1) along the chromatography direction; the NC film (9.4) is sequentially coated with a detection line (9.5) and a quality control line (9.6) along the chromatography direction; the microsphere pad (9.3) is coated with red latex microsphere of a labeled mouse anti-novel coronavirus N protein monoclonal antibody A, and the coating amount of the red latex microsphere is 5-10 mug/cm 2 The coating amount of the mouse anti-novel coronavirus N protein monoclonal antibody A is 0.3-0.6 mug/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The detection line (9.5) is coated with a mouse anti-novel coronavirus N protein monoclonal antibody B, and the coating amount is 1-1.5 mug/cm of detection line length; the quality control line (9.6) is coated with goat anti-mouse IgG, and the coating amount is 0.5-1 mug/cm; the mouse anti-novel coronavirus N protein monoclonal antibody A is different from the mouse anti-novel coronavirus N protein monoclonal antibody B.
Preferably, the detection card cover (1) further comprises an upper card cover (3), the upper card cover (3) is connected with the detection card bottom (2) through a hinge and can be buckled on the detection card bottom (2), and a detection result observation window (5) is arranged on the upper card cover (3) and is used for observing a detection line and a quality control line.
Preferably, the swab placement groove (6) is surrounded by a flange formed on the inner surface of the lower clamping cover (4).
Preferably, the swab placing groove (6) is formed by protruding from the inner surface of the lower clamping cover (4) to the rear surface.
Preferably, the slot (7.1) extends to near the upper edge of the swab fixing part (7), and a swab clamping structure (7.2) is arranged at the upper edge of the swab fixing part (7).
Preferably, a hinge structure is arranged on one side surface of the lower clamping cover (4), and a buckling structure buckled with the detection clamping bottom (2) is arranged on the other side surface.
Preferably, the test strip (9) is about 3.8mm wide; the NC film (9.4) is stuck on the PVC bottom plate (9.1) and is about 2.5cm long; the length of the microsphere pad (9.3) is about 0.7cm, and the microsphere pad (9.3) is pressed on the NC film for 1-2mm; the length of the sample pad (9.2) is about 1.7cm, and the sample pad (9.2) is pressed on the microsphere pad for 2-3mm; the length of the water absorbing paper (9.7) is about 1.7cm, and the water absorbing paper (9.7) is pressed on the NC film for 1-2mm.
Those skilled in the art will appreciate that various murine anti-novel coronavirus N protein monoclonal antibodies can be used in the present invention, and can be prepared using methods known in the art, and also using commercially available products, such as COV19-PS-MAb1, lot number, from the Phpeng biological Co., ltd: 20210302-3; COV19-PS-MAb2, lot number: 20210213-2.
It will be appreciated by those skilled in the art that a variety of sheep anti-mouse IgG can be used in the present invention, and can be prepared using methods known in the art, and that commercial products, such as sheep anti-mouse IgG from Phpeng biosystems, inc., lot number: 20201225-2.
The invention also provides a novel rapid detection kit for coronavirus antigens, which comprises the novel rapid detection device for coronavirus antigens and sample treatment fluid. Preferably, the swab is further comprised.
Preferably, the sample treatment solution is a 20-200mM phosphate buffer solution containing 0.9% -1% sodium chloride, 0.1-0.2% Tween 20, 0.1-0.5% bovine serum albumin, 0.05-0.1% proclin300, and its pH value is 8.0-8.2.
The invention also provides a preparation method of the novel rapid coronavirus antigen detection device or the kit, wherein the preparation of the test strip comprises the following steps:
(1) Coating of NC film
Cutting an NC film into a width of about 2.5cm, pasting the NC film on a PVC bottom plate, coating the NC film with a quality control wire coating liquid and a detection wire coating liquid, and drying; diluting goat anti-mouse IgG to 0.5-1mg/mL with 10mM PBS buffer solution to obtain a quality control line coating solution; diluting the novel coronavirus N protein monoclonal antibody B to 1-1.5mg/mL with 10mM PBS buffer solution to be used as a detection line coating solution;
(2) Preparation of microsphere mats
Dripping the bonding pad treatment liquid onto glass fiber to completely infiltrate the glass fiber, and drying to obtain a bonding pad; the bonding pad treatment liquid is a 20mM phosphate buffer solution containing 5-10% of sucrose, 0.1-0.5% of casein and 0.1-0.5% of polyvinylpyrrolidone 10;
dispersing the red latex microspheres into MES buffer, adding sulfo-N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, uniformly mixing and activating; centrifuging to remove supernatant, re-dissolving with MES buffer solution, adding mouse anti-novel coronavirus N protein monoclonal antibody A, mixing, and reacting; adding 10% bovine serum albumin, and sealing for reaction; centrifuging to remove supernatant, re-dissolving with re-dissolving solution, diluting, spreading on a bonding pad, and oven drying; the compound solution is 20mM phosphate buffer solution containing 50-100g/L sucrose and 3-5g/L bovine serum albumin;
(3) Preparation of sample pad
Dripping the sample pad treatment liquid onto glass fiber to completely infiltrate the glass fiber, and drying to obtain a sample pad; the sample pad treatment liquid is 100mM phosphate buffer liquid containing 9-10g/L sodium chloride, 3-5g/L bovine serum albumin, 2-5g/L tween, 2-5g/L polyvinylpyrrolidone 10 and 2-5g/L polyethylene glycol 20000;
(4) Test strip assembling and cutting
Cutting water absorbing paper into a width of about 1.7cm, and pasting the water absorbing paper on one side, close to an NC film quality control line, of the PVC bottom plate coated with the NC film, and overlapping the NC film by 1-2mm; cutting the microsphere mat to a width of about 0.7cm, and pasting the microsphere mat on one side of the PVC base plate close to the NC film detection line, and overlapping the microsphere mat with the NC film for 1-2mm; cutting the sample pad to a width of about 1.7cm, and adhering the sample pad to one side of the PVC base plate, which is close to the microsphere pad, and overlapping the sample pad with the microsphere pad for 2-3mm; the test strips were cut to a width of about 3.8 mm.
Preferably, the preparation method comprises the following steps:
(1) Coating of NC film
Cutting an NC film into about 2.5cm and about 31cm, pasting the NC film on a PVC base plate, coating the NC film with a quality control line coating liquid and a detection line coating liquid by using a metal spraying film drawing instrument, and drying; diluting goat anti-mouse IgG to 0.5mg/mL with 10mM PBS buffer solution with pH value of 7.4 as a quality control line coating solution; diluting the novel coronavirus N protein monoclonal antibody B with 10mM PBS buffer solution with pH value of 7.4 to 1mg/mL as detection line coating solution;
(2) Preparation of microsphere mats
Spreading glass fiber of about 20cm and about 30cm on a plastic plate, dripping the bonding pad treatment liquid onto the glass fiber to completely infiltrate the glass fiber, and drying to obtain a bonding pad; the binding pad treatment solution is phosphate buffer solution containing 5% of sucrose, 0.5% of casein and 0.5% of polyvinylpyrrolidone 10, wherein the pH value of 20mM is 8.2;
50 mug of red latex microspheres are dispersed into 200 mug of MES buffer with the pH value of 6.2, 7 mug of sulfo-N-hydroxysuccinimide with the concentration of 10mg/mL and 7 mug of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 1mg/mL are added, and the mixture is uniformly mixed and activated for 15 minutes at room temperature; centrifuging to remove supernatant, re-dissolving with 200 μl MES buffer with pH of 6.2, adding 5 μg mouse anti-novel coronavirus N protein monoclonal antibody A, mixing, and reacting at room temperature for about 2 hr; adding 20 mu L of 10% bovine serum albumin, and performing a blocking reaction at room temperature for about 30min; centrifuging to remove supernatant, and re-dissolving with 200 μl of a re-solution of 20mM phosphate buffer with pH of 8.2 containing 50g/L sucrose and 5g/L bovine serum albumin; further diluting with compound solution with dilution ratio of 8 times, spreading on the bonding pad, and oven drying;
(3) Preparation of sample pad
Spreading glass fiber with a thickness of about 20cm and about 30cm on a plastic plate, taking about 35mL of sample pad treatment liquid, dripping the sample pad treatment liquid on the glass fiber to completely infiltrate the glass fiber, and drying to obtain a sample pad; the sample pad treatment solution is a phosphate buffer solution with the pH value of 8.0, which contains 9g/L of sodium chloride, 5g/L of bovine serum albumin, 2g/L of tween 20, 2g/L of polyvinylpyrrolidone 10 and 2g/L of polyethylene glycol 20000 and is 100 mM;
(4) Test strip assembling and cutting
Cutting water absorbing paper into about 1.7cm and about 30cm, and pasting the water absorbing paper on one side, close to an NC film quality control line, of the PVC bottom plate coated with the NC film, and overlapping the NC film by 1-2mm; cutting the microsphere mat into about 0.7cm and about 30cm, and pasting the microsphere mat on one side of the PVC base plate, which is close to the NC film detection line, and overlapping the PVC base plate with the NC film for 1-2mm; cutting the sample pad into about 1.7cm and about 30cm, and pasting the sample pad on one side of the PVC base plate close to the microsphere pad, wherein the sample pad is overlapped with the microsphere pad by 2-3mm; the test strips were cut to a width of about 3.8 mm.
The invention also provides a method for rapidly detecting novel coronavirus antigens, wherein the rapid detection device or the kit is used. According to the invention, the detection method is not aimed at diagnosis of the disease. Specifically, a swab for collecting samples such as nasopharynx, environment, food and the like is placed in a swab placing groove (6) on the inner side surface of a lower clamping cover (4), a swab fixing component (7) is folded along a hinge, the swab fixing component (7) is buckled on the lower clamping cover (4), the swab is clamped in the swab placing groove (6), then sample treatment liquid is dripped on the swab through a slot (7.1), the swab is rotated to be uniformly mixed, the lower clamping cover (4) is folded along the hinge to buckle the lower clamping cover (4) on a detection card bottom (2), the swab passes through the slot (7.1) to be in contact with a test strip sample pad on a test strip clamping groove (8), the sample is transferred onto the test strip sample pad (9.2), and chromatography is started until a detection result is obtained. From the beginning of placing the swab in the swab placement tank (6) to the interpretation of the results, approximately 15 minutes are required.
The detection principle and advantages of the technical scheme of the invention are as follows:
the invention adopts double antibody sandwich method of immunochromatography technology to quantitatively detect novel coronavirus in nasopharyngeal swab sample, and carries out result interpretation by directly observing the outlet conditions of quality control line (C) and detection line (T). During testing, the sample treatment liquid is dripped on a sampled nasopharyngeal swab, the novel coronavirus in the sample is treated by the sample treatment liquid to release N protein, the N protein is combined with a mouse anti-novel coronavirus N protein monoclonal antibody A of a red latex microsphere which is coated on a microsphere pad in advance, the conjugate moves forward under the action of chromatography and is then combined and captured by a mouse anti-novel coronavirus N protein monoclonal antibody B which is fixed on a detection line (T), so that a red stripe appears on the detection line (T); the red latex microsphere of the non-captured conjugate and the labeled mouse anti-novel coronavirus N protein monoclonal antibody A is continuously chromatographed to a quality control line (C) to be combined with goat anti-mouse IgG fixed on the quality control line (C), and a red strip appears, and the quality control line (C) can appear regardless of whether N protein exists in a sample or not. The device omits the operation of sample independent treatment, saves time and cost, is a rapid detection device, and can obviously improve detection sensitivity by using red latex microspheres as tracers, can be used for rapid and accurate detection of novel coronavirus antigens, can be used for clinic and personal use, and can realize early discovery and early control of diseases.
Drawings
FIG. 1 is a diagram showing the structure of the outside of the test card after being snapped in
FIG. 2 is a diagram showing the structure of the inner side of the test card after the test card is unfolded
FIG. 3 is a block diagram of a test strip according to the present invention
FIG. 4 shows the results of the test in example 4 of the present invention
FIG. 5 shows the results of comparative example test of the present invention
In fig. 1-3: 1-detection card cover, 2-detection card bottom, 3-upper card cover, 4-lower card cover, 5-detection result observation window, 6-swab placing groove, 7-swab fixing component, 7.1-slot, 7.2-swab clamping structure, 8-test strip clamping groove, 9-test strip, 9.1-PVC board, 9.2-sample pad, 9.3-microsphere pad, 9.4-NC film, 9.5-detection line, 9.6-quality control line, 9.7-water absorbing paper and 10-detection card.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications of the invention will become apparent to those skilled in the art upon reading the description herein, and such equivalents are intended to fall within the scope of the invention as defined by the appended claims.
As shown in fig. 1-3, a rapid detection device for detecting novel coronavirus antigens comprises a detection card 10 and a test strip 9, wherein the detection card comprises a detection card cover 1 and a detection card bottom 2, the detection card cover 1 and the detection card bottom 2 are connected through a hinge and can be buckled together, the hinge can be integrally formed with the detection card cover 1 and has certain flexibility, the hinge can also be an independent hinge part, the inner surface of the detection card bottom 2 is provided with a test strip clamping groove 8 for placing the test strip, the detection card cover 1 comprises a lower card cover 4, the inner side surface of the lower card cover 4 is provided with a swab holding groove 6, the lower card cover 4 is connected with a swab fixing part 7 which can be buckled with the lower card cover through the hinge, the hinge can be integrally formed with the lower card cover 4 and has certain flexibility, and can also be an independent hinge part, and the swab fixing part 7 is provided with a slot 7.1 corresponding to the swab holding groove 6.
The test strip 9 is formed by sequentially adhering a sample pad 9.2, a microsphere pad 9.3, an NC film 9.4 and water absorbing paper 9.7 to a PVC bottom plate 9.1 along the chromatography direction; the NC film 9.4 is sequentially coated with a detection line 9.5 and a quality control line 9.6 along the chromatography direction; the microsphere pad 9.3 is coated with a red latex microsphere of a labeled mouse anti-novel coronavirus N protein monoclonal antibody A, the detection line 9.5 is coated with a mouse anti-novel coronavirus N protein monoclonal antibody B, the quality control line 9.6 is coated with a sheep anti-mouse IgG, and the mouse anti-novel coronavirus N protein monoclonal antibody A and the mouse anti-novel coronavirus N protein monoclonal antibody B are different.
When the device is used, a swab for collecting samples such as nasopharynx, environment, food and the like is placed in a swab placing groove 6 on the inner side surface of a lower clamping cover 4, a swab fixing part 7 is folded along a hinge, the swab fixing part 7 is buckled on the lower clamping cover 4, the swab is clamped in the swab placing groove 6, then sample treatment liquid is dripped on the swab through a slot 7.1, the swab is rotated to be uniformly mixed, the lower clamping cover 4 is folded along the hinge to buckle the lower clamping cover 4 on a detection card bottom 2, the swab passes through the slot 7.1 to be contacted with a sample pad of a test strip 9 on a test strip clamping groove 8, the sample is transferred onto the sample pad of the test strip 9.2, and the test strip 9 starts to be chromatographically processed until a detection result is obtained. From the start of placing the swab in the swab placement tank 6 to the interpretation of the results, approximately 15 minutes are required.
With further reference to fig. 1 and 2, the detection card cover 1 further includes an upper card cover 3, and the upper card cover 3 is connected with the detection card base 2 through a hinge. Preferably, the upper card cover 3 and the detection card bottom 2 are integrally formed, and the middle part is processed into a bendable folding structure. With further reference to fig. 2, the upper card cover 3 can be snapped onto the test card base 2 after being folded. The upper clamping cover 3 is provided with a detection result observation window 5 for observing a detection line 9.5 and a quality control line 9.6.
With further reference to fig. 1, the swab placement groove 6 is surrounded by a flange formed on the inner surface of the lower card cover 4 to facilitate positioning of the swab of the collected sample in the lower card cover 4.
With further reference to fig. 1, the swab placement groove 6 is formed by protruding the inner surface of the lower card cover 4 toward the rear surface, which can increase the size of the swab placement groove and reduce the thickness of the lower card cover 4 and the detection device.
With further reference to fig. 2, the slot 7.1 extends to near the upper edge of the swab fixing part 7, and a swab clamping structure 7.2 is provided at the upper edge of the swab fixing part 7, and the swab clamping structure 7.2 is adapted to the cross section of the swab, such as a semicircular structure, so as to facilitate fixing the swab in the swab holding groove 6 and prevent the relative movement of the swab.
Further referring to fig. 2, one side surface of the lower card cover 4 (the left side surface of the lower card cover 4 in fig. 2) is provided with a hinge structure, the other side surface (the right side surface of the lower card cover 4 in fig. 2) is provided with a fastening structure fastened with the detection card bottom 2, so that the lower card cover 4 is folded and fastened onto the detection card bottom 2, and is fixed through the fastening structure.
The mouse anti-novel coronavirus N protein monoclonal antibody A and the mouse anti-novel coronavirus N protein monoclonal antibody B used in the embodiment of the invention are two different antibodies aiming at different antigenic determinants of the novel coronavirus N protein, and are purchased from the Phpeng biological Co., ltd, and are respectively COV19-PS-MAb1, batch numbers: 20210302-3; COV19-PS-MAb2, lot number: 20210213-2; sheep anti-mouse IgG was purchased from fepeng biosystems, inc. As sheep anti-mouse IgG, lot number: 20201225-2; the red latex microspheres were color microspheres available from the biotechnology company, scotch: DR0400CA; absorbent paper (model S370), glass fiber (model 8975) and a metal spraying film cutter (model HGS 510) were all purchased from Hangzhou Fenghuang technology Co., ltd. Nitrocellulose membranes (NC membranes) were purchased from PALL under the model VIV12025100R.
Example 1
The novel rapid detection device for coronavirus antigen comprises a detection card 10 shown in fig. 1 and 2, wherein the detection card comprises a detection card cover 1 and a detection card bottom 2, the detection card cover 1 and the detection card bottom 2 are connected through a hinge and can be buckled together, the detection card cover 1 comprises an upper card cover 3 and a lower card cover 4, the upper card cover 3 and the detection card bottom 2 are connected through a hinge and can be buckled together, and a detection result observation window 5 is arranged on the upper card cover 3 and is used for observing a detection line 9.5 and a quality control line 9.6 of a test paper strip; the lower clamping cover 4 is connected with the detection clamping bottom 2 through a hinge and can be buckled together, a swab placing groove 6 is formed in the inner side surface of the lower clamping cover 4, a swab fixing part 7 which can be buckled together with the lower clamping cover 4 is connected with the lower clamping cover through a hinge, the swab fixing part 7 is provided with a slot 7.1 corresponding to the swab placing groove 6, the slot 7.1 extends to be close to the upper edge of the swab fixing part 7, and a swab clamping structure 7.2 is arranged at the upper edge of the swab fixing part 7; the inner surface of the detection card bottom 2 is provided with a test strip clamping groove 8 for placing a test strip.
Example 2
The test strip used in the rapid detection device of the novel coronavirus antigen is prepared according to the following method:
coating of NC film
Sticking NC film: nitrocellulose membrane (NC membrane) was cut to a width of 2.5cm and a length of 31cm, and stuck to a PVC base plate.
Preparing a coating liquid: diluting goat anti-mouse IgG to 0.5mg/mL with 10mM PBS buffer solution with pH value of 7.4 as a quality control line coating solution; the novel coronavirus N protein monoclonal antibody B is diluted to 1mg/mL with 10mM PBS buffer with pH value of 7.4 as detection line coating liquid.
Coating: and coating the NC film with the prepared quality control line coating liquid and detection line coating liquid by using a metal spraying film drawing instrument according to an operation rule, and drying in a blast drying oven at 37 ℃. The detection line is coated with novel coronavirus N protein monoclonal antibody B with the length of about 1 mug/cm; the quality control line is coated with goat anti-mouse IgG with the length of about 0.5 mug/cm.
2. Preparation of microsphere mats
(1) Preparation of the bond pad: phosphate buffer with pH 8.2 at 20mM containing 5% sucrose, 0.5% casein, 0.5% polyvinylpyrrolidone 10 (PVP 10) was prepared as a conjugate pad treatment. Spreading glass fiber (20 cm. Times.30 cm) on a plastic plate, collecting 35mL of the bonding pad treatment liquid, dripping onto the glass fiber to completely infiltrate, and oven drying in a blast drying oven at 37deg.C.
(2) Preparation of red latex microspheres for labeling antibodies:
activation of microspheres: 50. Mu.g of microspheres were dispersed in 200. Mu.L of MES buffer pH 6.2, and 10mg/mL of Sulfo-NHS (Sulfo-N-hydroxysuccinimide) 7. Mu.L and 1mg/mL of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) 7. Mu.L were added, mixed and activated at room temperature for 15 minutes.
Antibody coupling: taking activated microspheres, centrifuging to remove supernatant, re-dissolving the supernatant with 200 mu L of MES buffer solution with the pH value of 6.2, adding 5 mu g of mouse anti-novel coronavirus N protein monoclonal antibody A, uniformly mixing, and reacting for 2 hours at room temperature.
Closing: 10% bovine serum albumin (20. Mu.L) was added thereto, and the reaction was blocked at room temperature for 30 minutes.
And (3) centrifuging and redissolving: taking the closed microsphere, centrifuging to remove supernatant, and redissolving with 200 mu L of redissolution. The reconstituted solution was a 20mM phosphate buffer pH 8.2 containing 50g/L sucrose, 5g/L bovine serum albumin.
(3) Ball laying: diluting the red latex microsphere marked with the mouse anti-novel coronavirus N protein monoclonal antibody A by using a compound solution, wherein the dilution ratio is 8 times, spreading the diluted microsphere on the bonding pad, and drying in a blast drying oven at 37 ℃. Micro-scaleThe coating amount of the red latex microspheres on the ball pad is about 7 mug/cm 2 The coating amount of the mouse anti-novel coronavirus N protein monoclonal antibody A is about 0.35 mug/cm 2 。
3. Preparation of sample pad
A phosphate buffer solution of pH 8.0, 100mM, containing 9g/L sodium chloride, 5g/L bovine serum albumin, 2g/L Tween 20, 2g/L polyvinylpyrrolidone 10 (PVP 10), and 2g/L polyethylene glycol 20000, was prepared as a sample pad treatment solution. Spreading glass fiber (20 cm. Times.30 cm) on a plastic plate, collecting 35mL sample pad treatment solution, dripping onto glass fiber to completely infiltrate, and oven drying in a blast drying oven at 37deg.C.
4. Test strip assembling and cutting
Assembling a test strip: as shown in fig. 3, the absorbent paper is cut into 1.7cm x 30cm pieces, and the absorbent paper is adhered to one side, close to the NC film quality control line, of the NC film-coated PVC base plate, and is lapped 1-2mm over the NC film. Cutting the microsphere mat into 0.7cm by 30cm, and pasting the microsphere mat on one side of the PVC base plate close to the NC film detection line, and overlapping the microsphere mat with the NC film by 1-2mm. The sample pad was cut to 1.7cm by 30cm and adhered to the PVC base plate on the side near the microsphere pad, overlapping the microsphere pad by 2-3mm.
Cutting the assembled large plate into test strips according to the width of 3.8 mm.
Example 3
The novel rapid detection kit for coronavirus antigens comprises the detection card of the embodiment 1, the test strip prepared in the embodiment 2 and a sample treatment liquid, wherein the sample treatment liquid is a 20mM phosphate buffer solution containing 9g/L sodium chloride, 2g/L Tween 20, 5g/L bovine serum albumin and 0.5g/L proclin300, and the pH value is 8.2.
Example 4
The using method of the novel rapid detection kit for coronavirus antigen comprises the following steps:
prior to testing, the kit was returned to room temperature (15-30 ℃);
taking out the detection device, opening the lower clamping cover 4 and the swab fixing part 7, and horizontally placing;
opening the package from the swab handle end, removing the swab, sampling from the bilateral nostrils or sampling from the pharynx;
fixing the sampled swab to the swab placing groove 6, and fastening the swab fixing part 7;
sample processing was taken by dropping 8 drops (about 200 μl) into the slot 7.1 and spinning the swab to ensure that the entire swab was wetted by the sample processing fluid.
After the upper and lower clamping covers are buckled for 15min, the result is read and is effective within 5min.
Interpretation of the results:
positive (+): C. red outgoing lines were observed on the T line, and the result was positive. Negative (-). The C line observed a red outgoing line, the T line had no red outgoing line, and the result was negative. Invalidation: the C line had no red outgoing line at all.
Using the above method, the lowest limit of detection reference in the national reference (purchased from China food and drug assay institute) was tested for novel coronavirus antigen, and the results S1-S9 were judged positive and S10 negative, wherein the dilutions of S1-S10 were 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600, respectively. The chromatographic results are shown in the following table:
TABLE 1 detection results of minimum limit of detection reference
Comparative example 1
The colloidal gold test strip was prepared as follows:
coating of NC film
Sticking NC film: nitrocellulose membrane (NC membrane) was cut to a width of 2.5cm and a length of 31cm, and stuck to a PVC base plate.
Preparing a coating liquid: diluting goat anti-mouse IgG to 0.5mg/mL with 10mM PBS buffer solution with pH value of 7.4 as a quality control line coating solution; the novel coronavirus N protein mab B was diluted to 1.5mg/mL as detection line coating with 10mM PBS buffer at pH 7.4.
Coating: and coating the NC film with the prepared quality control line coating liquid and detection line coating liquid by using a metal spraying film drawing instrument according to an operation rule, and drying in a blast drying oven at 37 ℃.
2. Preparation of gold mark pad
(1) Preparation of the bond pad: an aqueous solution containing 4% sucrose, 1% trehalose, and 0.3% surfactant S9 (TETRONIC 1307) was prepared as a conjugate pad treatment solution for use. Spreading glass fiber (20 cm. Times.30 cm) on a plastic plate, collecting 35mL of the bonding pad treatment liquid, dripping onto the glass fiber to completely infiltrate, and oven drying in a blast drying oven at 37deg.C.
(2) Preparation of colloidal gold-labeled antibody:
taking 10mL of four parts per million colloidal gold solution, adding 100 μL of 0.2M K 2 CO 3 Adding 200 mug of novel coronavirus N protein monoclonal antibody A after stirring for 5min, adding 1mL of 10% bovine serum albumin after stirring for 5min, and stirring for 5min; centrifuging at 10000rpm for 8-10min, collecting precipitate, and redissolving to 1mL with the redissolved solution. The reconstituted solution was a 20mM phosphate buffer pH 8.2 containing 50g/L sucrose, 5g/L bovine serum albumin.
(3) Gold paving: diluting the colloidal gold solution marked with the mouse anti-N protein monoclonal antibody A by using a compound solution, wherein the dilution ratio is 8 times, spreading the diluted colloidal gold solution on the bonding pad, and drying in a blast drying oven at 37 ℃.
3. Preparation of sample pad
A phosphate buffer solution of pH 8.0, 100mM, containing 9g/L sodium chloride, 5g/L bovine serum albumin, 2g/L Tween 20, 2g/L polyvinylpyrrolidone 10, 2g/L polyethylene glycol 20000 was prepared as a sample pad treatment solution. Spreading glass fiber (20 cm. Times.30 cm) on a plastic plate, collecting 35mL sample pad treatment solution, dripping onto glass fiber to completely infiltrate, and oven drying in a blast drying oven at 37deg.C.
4. Test strip assembling and cutting
Assembling a test strip: cutting the water absorbing paper into 1.7cm which is 30cm, and pasting the water absorbing paper on one side of the PVC bottom plate coated with the NC film, which is close to the NC film quality control line, and overlapping the NC film by 1-2mm. Cutting the gold mark pad into 0.7cm or 30cm, and pasting the gold mark pad on one side of a PVC base plate close to an NC film detection line, and overlapping the gold mark pad with the NC film by 1-2mm. The sample pad is cut into 1.7cm by 30cm, and is stuck on one side of the PVC bottom plate close to the gold mark pad, and is lapped with the gold mark pad for 2-3mm.
Cutting the assembled large plate into test strips according to the width of 3.8 mm.
Using the kit of example 1, the colloidal gold test strip described above, and the sample processing solution of example 3, the procedure was followed in accordance with example 4 to test the lowest detection limit reference among the national references for novel coronavirus antigen detection reagents, and the results S1-S5 were positive, S6-S10 negative, and the chromatographic results were as shown in the following table:
TABLE 2 detection results of minimum limit of detection reference
Example 4 compared to comparative example 1, the lowest limit of detection of example 4 is 16 times that of comparative example 1.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A novel rapid detection device for coronavirus antigen comprises a detection card (10) and a test strip (9);
the detection card (10) comprises a detection card cover (1) and a detection card bottom (2), wherein the detection card cover (1) and the detection card bottom (2) are connected through a hinge and can be buckled together, a test strip clamping groove (8) for placing a test strip is formed in the inner surface of the detection card bottom (2), the detection card cover (1) comprises a lower card cover (4), a swab placing groove (6) is formed in the inner side surface of the lower card cover (4), a swab fixing part (7) which can be buckled together with the lower card cover (4) is connected through the hinge, and a slot (7.1) corresponding to the swab placing groove (6) is formed in the swab fixing part (7);
the test strip (9) is formed by sequentially adhering a sample pad (9.2), a microsphere pad (9.3), an NC film (9.4) and water absorbing paper (9.7) to a PVC bottom plate (9.1) along the chromatography direction; NC film (9.4) is coated in sequence along the chromatographic directionA detection line (9.5) and a quality control line (9.6) are arranged; the microsphere pad (9.3) is coated with red latex microsphere of a labeled mouse anti-novel coronavirus N protein monoclonal antibody A, and the coating amount of the red latex microsphere is 5-10 mug/cm 2 The coating amount of the mouse anti-novel coronavirus N protein monoclonal antibody A is 0.3-0.6 mug/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The detection line (9.5) is coated with a mouse anti-novel coronavirus N protein monoclonal antibody B, and the coating amount is 1-1.5 mug/cm of detection line length; the quality control line (9.6) is coated with goat anti-mouse IgG, and the coating amount is 0.5-1 mug/cm; the mouse anti-novel coronavirus N protein monoclonal antibody A is different from the mouse anti-novel coronavirus N protein monoclonal antibody B.
2. The rapid detection device according to claim 1, wherein the detection card cover (1) further comprises an upper card cover (3), the upper card cover (3) is connected with the detection card bottom (2) through a hinge and can be buckled on the detection card bottom (2), and a detection result observation window (5) is arranged on the upper card cover (3) and is used for observing the detection line (9.5) and the quality control line (9.6).
3. The rapid test device according to claim 1, wherein the swab placement groove (6) is surrounded by a flange formed on the inner surface of the lower snap cap (4).
4. The rapid test device according to claim 1, wherein the swab placement groove (6) is formed by a protrusion of the inner surface of the lower card cover (4) toward the rear surface.
5. A rapid detection device according to claim 1, the slot (7.1) extending to near the upper edge of the swab fixing part (7), a swab clamping structure (7.2) being provided at the upper edge of the swab fixing part (7).
6. The rapid detection device according to claim 1, wherein one side surface of the lower clamping cover (4) is provided with a hinge structure, and the other side surface is provided with a buckling structure buckled with the detection clamping bottom (2).
7. The rapid test device of claim 1, wherein the test strip is about 3.8mm wide; the NC film is stuck on the PVC bottom plate and is about 2.5cm long; the length of the microsphere pad is about 0.7cm, and the microsphere pad is pressed on the NC film for 1-2mm; the length of the sample pad is about 1.7cm, and the sample pad is pressed on the microsphere pad for 2-3mm; the length of the absorbent paper is about 1.7cm, and the absorbent paper is pressed on the NC film for 1-2mm.
8. A novel rapid assay kit for coronavirus antigens comprising the rapid assay device of claim 1 and a sample processing fluid.
Preferably, the swab is further comprised.
Preferably, the sample treatment solution is a 20-200mM phosphate buffer solution containing 0.9% -1% sodium chloride, 0.1-0.2% Tween 20, 0.1-0.5% bovine serum albumin, 0.05-0.1% proclin300, and its pH value is 8.0-8.2.
9. The rapid assay device according to claim 1 or the rapid assay kit according to claim 8, wherein the preparation of the test strip comprises the steps of:
(1) Coating of NC film
Cutting an NC film into a width of about 2.5cm, pasting the NC film on a PVC bottom plate, coating the NC film with a quality control wire coating liquid and a detection wire coating liquid, and drying; diluting goat anti-mouse IgG to 0.5-1mg/mL with 10mM PBS buffer solution to obtain a quality control line coating solution; diluting the novel coronavirus N protein monoclonal antibody B to 1-1.5mg/mL with 10mM PBS buffer solution to be used as a detection line coating solution;
(2) Preparation of microsphere mats
Dripping the bonding pad treatment liquid onto glass fiber to completely infiltrate the glass fiber, and drying to obtain a bonding pad; the bonding pad treatment liquid is a 20mM phosphate buffer solution containing 5-10% of sucrose, 0.1-0.5% of casein and 0.1-0.5% of polyvinylpyrrolidone 10;
dispersing the red latex microspheres into MES buffer, adding sulfo-N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, uniformly mixing and activating; centrifuging to remove supernatant, re-dissolving with MES buffer solution, adding mouse anti-novel coronavirus N protein monoclonal antibody A, mixing, and reacting; adding 10% bovine serum albumin, and sealing for reaction; centrifuging to remove supernatant, re-dissolving with re-dissolving solution, diluting, spreading on a bonding pad, and oven drying; the compound solution is 20mM phosphate buffer solution containing 50-100g/L sucrose and 3-5g/L bovine serum albumin;
(3) Preparation of sample pad
Dripping the sample pad treatment liquid onto glass fiber to completely infiltrate the glass fiber, and drying to obtain a sample pad; the sample pad treatment liquid is 100mM phosphate buffer liquid containing 9-10g/L sodium chloride, 3-5g/L bovine serum albumin, 2-5g/L tween, 2-5g/L polyvinylpyrrolidone 10 and 2-5g/L polyethylene glycol 20000;
(4) Test strip assembling and cutting
Cutting water absorbing paper into a width of about 1.7cm, and pasting the water absorbing paper on one side, close to an NC film quality control line, of the PVC bottom plate coated with the NC film, and overlapping the NC film by 1-2mm; cutting the microsphere mat to a width of about 0.7cm, and pasting the microsphere mat on one side of the PVC base plate close to the NC film detection line, and overlapping the microsphere mat with the NC film for 1-2mm; cutting the sample pad to a width of about 1.7cm, and adhering the sample pad to one side of the PVC base plate, which is close to the microsphere pad, and overlapping the sample pad with the microsphere pad for 2-3mm; the test strips were cut to a width of about 3.8 mm.
10. A method of detecting a novel coronavirus antigen, wherein the rapid detection device of claim 1 or the rapid detection kit of claim 8 is used.
Preferably, the swab for collecting the sample is placed in a swab placing groove (6) on the inner side surface of the lower clamping cover (4), the swab fixing component (7) is folded along the hinge, the swab fixing component (7) is buckled on the lower clamping cover (4), the swab is clamped in the swab placing groove (6), then sample treatment liquid is dripped on the swab through the slot (7.1), the swab is rotated to be uniformly mixed, the lower clamping cover (4) is folded along the hinge to buckle the lower clamping cover (4) on the detection card bottom (2), the swab passes through the slot (7.1) to be in contact with a test strip sample pad on the test strip clamping groove (8), the sample is transferred onto the test strip sample pad (9.2), and the test strip starts to be chromatographed until a detection result is obtained.
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