CN115420895A - Test paper strip and kit for detecting influenza A/B virus and respiratory syncytial virus antigens - Google Patents
Test paper strip and kit for detecting influenza A/B virus and respiratory syncytial virus antigens Download PDFInfo
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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
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- 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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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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
- G01N33/558—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
- 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
- G01N33/577—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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- 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
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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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
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/135—Respiratory syncytial virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention provides an influenza A/B virus and respiratory syncytial virus antigen detection test strip and a kit, wherein the test strip comprises: the bottom plate is bonded on the bottom plate, and the sample pad, the combination pad, the chromatography matrix and the water absorption pad are sequentially overlapped; wherein, a biological blocking agent is added in the sample pad, and the biological blocking agent comprises the following components in final concentration: 10-20mg/mL bovine serum albumin, 1-5mg/mL sheep IgG and 0.1-3mg/mL chicken IgY; the combination pad is added with a chemical blocking agent, and the chemical blocking agent comprises the following components in final concentration: 0.1 to 1 percent of polyvinylpyrrolidone, 0.1 to 5 percent of PEG4000, 0.1 to 2 percent of Triton X-100 and 0.1 to 2 percent of Tween-20. Meanwhile, the kit can detect influenza A virus, influenza B virus and respiratory syncytial virus, and has strong specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of medical diagnosis, and relates to an antigen detection test strip and a kit for influenza A/B virus and respiratory syncytial virus.
Background
Respiratory viruses are a group of viruses that can cause respiratory and other systemic diseases through respiratory infections. Common respiratory viruses include influenza viruses of the orthomyxoviridae family, parainfluenza viruses, measles viruses, mumps viruses, respiratory syncytial viruses of the paramyxoviridae family, coronaviruses of the coronaviridae family, rubella viruses of the togaviridae family, adenoviruses of the adenoviridae family, and the like. In the case of respiratory viruses, the same virus can cause multiple clinical symptoms, and different viruses can cause the same clinical symptoms, which causes great difficulty in clinical diagnosis and treatment, so that the detection and diagnosis of the viruses are very important for taking effective preventive and therapeutic measures.
Influenza viruses are classified into influenza A, influenza B and influenza C viruses according to the antigenic characteristics of virus Nucleoprotein (NP), and the influenza A, influenza B and influenza C viruses belong to the orthomyxoviridae, and have the characteristics that the influenza A viruses are most easily epidemic, the influenza B viruses are second-grade influenza, and the influenza C viruses rarely cause epidemic. Influenza a viruses are divided into subtypes according to differences in the antigenicity of Hemagglutinin (HA) and Neuraminidase (NA) proteins. Influenza virus is mainly transmitted by air droplets, often causing fever, weakness, muscle soreness and mild to moderate respiratory symptoms, and severe cases can cause pneumonia, myocarditis and heart failure.
Respiratory Syncytial Virus (RSV) is an enveloped, single-stranded RNA virus belonging to the Paramyxoviridae family, the pneumovirus genus. RSV nucleotides encode at least 10 specific proteins, and RSV can be divided into two subtypes, a (RSVA) and B (RSVB), with up to 20% nucleotide variation within the two subtypes. Respiratory syncytial virus is widely prevalent in the global range, and is mainly fulminated in winter and spring in temperate regions; however, in cold regions, it is detectable almost all the year round. A. The two subtypes B are often co-prevalent, differing in relative proportions per year, with the subtype a predominating in the more prevalent season overall. RSV is not only the most common viral pathogen in lower respiratory tract infections in infants and young children, but also has a high incidence in the elderly and at high risk of immunocompromised individuals and can cause serious complications leading to prolonged hospital stays and high mortality rates.
In recent years, the incidence rate of respiratory virus infection is remarkably increased, and the method poses a huge threat to global public health and human health, so that the method is crucial to timely and accurately identifying the respiratory virus infection type and the epidemic characteristics thereof and is important for clinical treatment. The clinical detection of main viruses of respiratory tract infection mostly adopts a virus isolation culture method, immunofluorescence, a molecular biological method such as a PCR (polymerase chain reaction) technology and the like at present, and the methods can be executed only by specific equipment and professional operators and need to be operated in a laboratory, so the method does not have the characteristics of simplicity and convenience in operation, rapidness and the like. The pathogen culture method is used as a gold standard for detection, but the culture time is generally 7 days, the time consumption is long, and the method is not beneficial to guiding clinical treatment in time.
Therefore, establishing a rapid and simple method for detecting respiratory viruses is particularly important for diagnosing and treating viral infections in time.
Disclosure of Invention
In order to solve the technical problem, the invention provides an influenza A/B virus and respiratory syncytial virus antigen detection test strip and a kit, which have strong specificity, no non-specificity of the same test strip detection and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided an influenza a/b virus, respiratory syncytial virus antigen detection test strip, comprising:
the sample pad, the combination pad, the chromatography matrix and the water absorption pad are adhered to the bottom plate and are sequentially overlapped; wherein, the first and the second end of the pipe are connected with each other,
the sample pad is added with a biological blocker, and the biological blocker comprises the following components in final concentration: 10-20mg/mL bovine serum albumin, 1-5mg/mL sheep IgG and 0.1-3mg/mL chicken IgY;
the combination pad is coated with a colloidal gold compound coated by an influenza A virus monoclonal antibody A2, a colloidal gold compound coated by an influenza B virus monoclonal antibody B2 and a colloidal gold compound coated by a respiratory syncytial virus monoclonal antibody R2; and a chemical blocking agent is added into the bonding pad, and comprises the following components in final concentration: 0.1 to 1 percent of polyvinylpyrrolidone, 0.1 to 5 percent of PEG4000, 0.1 to 2 percent of Triton X-100 and 0.1 to 2 percent of Tween-20;
a quality control line C is arranged on one side, close to the water absorption pad, of the chromatography matrix, and is coated with anti-mouse IgG secondary antibody;
a detection line T is arranged on one side, close to the combination pad, of the chromatography matrix; the detection line T includes: the detection line A coated with the influenza A virus monoclonal antibody A1, the detection line B coated with the influenza B virus monoclonal antibody B1 and the detection line R coated with the respiratory syncytial virus monoclonal antibody R1 (murine source).
Further, the pH value of the colloidal gold compound coated by the influenza A virus monoclonal antibody A2 is 7.0-7.5, and the labeling amount is 15 mu g/mL colloidal gold solution.
Further, the pH value of the colloidal gold compound coated by the influenza B virus monoclonal antibody B2 is 7.0-7.5, and the labeling amount is 15 mug/mL colloidal gold solution.
Further, the pH value of the colloidal gold compound coated by the respiratory syncytial virus monoclonal antibody R2 is 7.0-7.5, and the labeling amount is 18 mu g/mL colloidal gold solution.
Furthermore, the concentration of the influenza A virus monoclonal antibody A1 is 1mg/mL, and the coating amount is 0.7-1.5 muL/cm.
Further, the concentration of the influenza B virus monoclonal antibody B1 is 1mg/mL, and the coating amount is 0.7-1.5 muL/cm.
Furthermore, the concentration of the respiratory syncytial virus monoclonal antibody R1 (murine source) is 1mg/mL, and the coating amount is 0.7-1.5 mu L/cm.
Furthermore, the concentration of the anti-mouse IgG secondary antibody is 1mg/mL, and the coating amount is 0.7-1.5 mu L/cm.
In a second aspect of the present invention, the present invention provides a method for preparing the influenza a/b virus and respiratory syncytial virus antigen detection test strip, wherein the method comprises:
coating the biological blocking agent on the sample pad to obtain a treated sample pad;
treating a colloidal gold compound coated by an influenza A virus monoclonal antibody A2, a colloidal gold compound coated by an influenza B virus monoclonal antibody B2 and a colloidal gold compound coated by a respiratory syncytial virus monoclonal antibody R2 by a stabilizing agent, spraying the mixture on a bonding pad, and spraying a chemical blocking agent on the bonding pad to obtain a treated bonding pad;
spraying an anti-mouse IgG secondary antibody on a quality control line C on a chromatography matrix, spraying an influenza A virus monoclonal antibody A1 on a detection line A on the chromatography matrix, spraying an influenza B virus monoclonal antibody B1 on a detection line B on the chromatography matrix, and spraying a respiratory syncytial virus monoclonal antibody R1 (mouse source) on a detection line R on the chromatography matrix to obtain the chromatography matrix containing the detection line T and the quality control line C;
and the processed sample pad, the processed combination pad, the chromatography matrix containing the detection line T and the quality control line C and the water absorption pad are sequentially and mutually overlapped and stuck on the bottom plate to obtain the test paper strip for detecting the antigens of the influenza A/B virus and the respiratory syncytial virus.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
1. the test paper strip for detecting the influenza A/B virus and respiratory syncytial virus antigens provided by the invention has no cross reaction with other pathogenic microorganisms, and has strong specificity. And the sensitivity is high: h 1 Minimum detection limit of N: 1.58X 10 3 TCID 50 /mL；H 3 N 2 Minimum detection limit of (c): 4.2X 10 2 TCID 50 Per mL; minimum detection limit of FluB: 6.3X 10 2 TCID 50 Per mL; RSVA minimum detection limit: 7.2X 10 2 TCID 50 Per mL; RSVB minimum detection limit: 7.9X 10 2 TCID 50 /mL。
2. The test strip for detecting the influenza A/B virus and respiratory syncytial virus antigens is simple and convenient to operate, can detect the influenza A/B virus and respiratory syncytial virus after the virus infection has symptoms, can obtain results in 15min, and can quickly diagnose diseases.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic view of the appearance of the influenza A/B virus and respiratory syncytial virus antigen detection reagent card shell of the present invention.
FIG. 2 is a schematic structural diagram of the test strip for detecting influenza A/B virus and respiratory syncytial virus antigens; the reference signs are: 1: a water absorbent pad; 2: a chromatography matrix; 3: a bonding pad; 4: a sample pad; 5: a base plate; the transverse arrows indicate the direction of chromatography;
FIG. 3 is a diagram showing the results of the detection according to the present invention.
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 apparent therefrom. 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.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically indicated, various raw materials, reagents, instruments, equipment and the like used in the present invention may be commercially available or may be prepared by existing methods.
The influenza A virus monoclonal antibody, the influenza B virus monoclonal antibody, the respiratory syncytial virus monoclonal antibody, the anti-mouse IgG secondary antibody, the sheep IgG and the chicken IgY used in the embodiment of the invention are all purchased commercial products.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
according to an exemplary embodiment of the present invention, there is provided a test strip for detecting antigens of influenza a/b virus and respiratory syncytial virus, comprising:
the sample pad, the combination pad, the chromatography matrix and the water absorption pad are adhered to the bottom plate and sequentially overlapped; wherein the content of the first and second substances,
the sample pad is added with a biological blocking agent, and the biological blocking agent comprises the following components in final concentration: 10-20mg/mL bovine serum albumin, 1-5mg/mL sheep IgG and 0.1-3mg/mL chicken IgY;
the combination pad is coated with a colloidal gold compound coated by an influenza A virus monoclonal antibody A2, a colloidal gold compound coated by an influenza B virus monoclonal antibody B2 and a colloidal gold compound coated by a respiratory syncytial virus monoclonal antibody R2; and a chemical blocking agent is added into the bonding pad, and comprises the following components in final concentration: 0.1 to 1 percent of polyvinylpyrrolidone, 0.1 to 5 percent of PEG4000, 0.1 to 2 percent of Triton X-100 and 0.1 to 2 percent of Tween-20;
a quality control line C is arranged on one side, close to the water absorption pad, of the chromatography matrix, and anti-mouse IgG secondary antibodies are coated on the quality control line C;
a detection line T is arranged on one side, close to the combination pad, of the chromatography matrix; the detection line T comprises: the detection line A coated with the influenza A virus monoclonal antibody A1, the detection line B coated with the influenza B virus monoclonal antibody B1 and the detection line R coated with the respiratory syncytial virus monoclonal antibody R1 (murine source).
The test strip provided by the invention has the following principle: respectively coating the influenza A virus monoclonal antibody A1, the influenza B virus monoclonal antibody B1 and the respiratory syncytial virus monoclonal antibody R1 (mouse source) at a detection line (A/B/R) of a nitrocellulose membrane, and respectively marking the influenza A virus monoclonal antibody A2, the influenza B virus monoclonal antibody B2 and the respiratory syncytial virus monoclonal antibody R2 as tracers to be fixed on a glass fiber membrane at a sample hole. During detection, a sample to be detected is added into a sample adding hole, a substance to be detected in the sample can firstly react with a colloidal gold labeled antibody pre-coated on a glass fiber membrane to form an antigen-antibody complex, then the complex is subjected to chromatography under capillary effect and finally combined with an antibody pre-coated on a nitrocellulose membrane detection line (A/B/R) to form a mauve strip, and the sample can be judged to be influenza A virus positive, influenza B virus positive, respiratory syncytial virus positive or composite positive according to the strip color development condition; if the sample does not contain the substance to be detected, a mauve strip does not appear at the detection line, and the sample is negative. No matter whether the sample contains the substance to be detected or not, a mauve strip should appear at the C line of the quality control line, and the mauve strip is used as an internal control standard for judging whether the chromatography process is normal or not and whether the reagent is invalid or not.
The chromatography matrix can be a nitrocellulose membrane, the combination pad is a glass cellulose membrane containing gold-labeled antibodies, and the bottom plate can be a PVC bottom plate.
The invention only needs one test strip, reduces the consumption of consumables to the maximum extent, and can achieve the purpose of simultaneously detecting the influenza A virus, the influenza B virus and the respiratory syncytial virus.
In the process of experimental exploration, the inventor of the application finds that: when the sample pad and the combination pad are only treated by phosphate and the like, false positive results can be generated when the three index antibodies are mixed and detected on the same test strip, and in order to eliminate the non-specificity of detection of the same test strip, the combination pad and the sample pad are added with blocking agents. Wherein, the blocker added in the combination pad is a chemical blocker and comprises 0.1 to 1 percent of polyvinylpyrrolidone, 0.1 to 5 percent of PEG4000, 0.1 to 2 percent of Triton X-100 and 0.1 to 2 percent of Tween-20; the sample pad is added with biological blocker, including bovine serum albumin (10-20 mg/mL), sheep IgG (1-5 mg/mL), and chicken IgY (0.1-3 mg/mL). Therefore, the antigen detection kit has great advantages compared with other kits in the whole view.
The test strip for detecting antigens of influenza a/b virus and respiratory syncytial virus of the present application will be described in detail with reference to examples and experimental data.
Example 1 preparation of antigen detection kit for influenza A/B virus and respiratory syncytial virus
1. Detection kit for influenza A/B virus and respiratory syncytial virus antigens
1. Sample pad treatment
Preparing a sample pad treatment solution: 0.296g of sodium dihydrogen phosphate, 2.901g of disodium hydrogen phosphate, 10g of bovine serum albumin and 2.14g of Triton X-100 were weighed into 1L of ultrapure water solution, and goat IgG and chicken IgY were simultaneously added to make the final concentrations 2mg/mL and 0.5mg/mL, respectively. Putting a 200mm 300mm sample pad into a stainless steel disc, uniformly soaking the sample pad treatment solution on the sample pad, turning over every 10min, taking out after 20min, putting the sample pad into a 37 ℃ drying oven for drying treatment (about 12-16 h), cutting into 15mm 300mm, and storing.
2. Bond pad treatment
Preparing a bonding pad treatment solution: 0.296g of sodium dihydrogen phosphate, 2.901g of disodium hydrogen phosphate, 10g of bovine serum albumin and 60g of sucrose are weighed into 1L of ultrapure water solution, and Tween-20, PEG4000, polyvinylpyrrolidone and Triton X-100 are added simultaneously to make the final concentrations reach 0.5%, 1%, 0.2% and 0.2% respectively. Placing 240mm 300mm of bonding pad in stainless steel plate, soaking the bonding pad with the treating solution uniformly until it is completely soaked, taking out after 10min, drying in a 37 deg.C drying oven (about 12-16 hr), cutting into 6mm 300mm, and storing.
3. Preparation of colloidal gold solution
Adding 10mL of chloroauric acid into 990mL of ultrapure water solution by a trisodium citrate chemical reduction method, heating to boil, adjusting to 100-150 ℃, rapidly adding 15mL of trisodium citrate into the chloroauric acid water solution, continuously boiling for 10min until the chloroauric acid solution turns to purple red, cooling to room temperature, and recovering the volume to 1L.
4. Preparation of gold-labeled antibody of influenza A virus, influenza B virus and respiratory syncytial virus
(1) The colloidal gold solution was adjusted to the appropriate pH using 0.1M potassium carbonate.
(2) Stock solutions of influenza A virus monoclonal antibody, influenza B virus monoclonal antibody and respiratory syncytial virus monoclonal antibody were diluted with 10mMPB so that the respective antibody concentrations were 0.1mg/mL.
(3) And adding the diluted antibody with the corresponding volume according to the volume during labeling, and placing the mixture on a shaking table at room temperature for shaking reaction for 30min.
(4) Adding a volume of 2% PEG20000/20% BSA for blocking, and further placing in a shaker for shaking reaction for 30min.
(5) 14000rpm, centrifuging for 25min, discarding the supernatant, adding a certain volume of gold-labeled antibody re-suspension, adjusting the antibody to 4OD, and standing at 4 deg.C.
5. Preparation of gold label pad
And (3) uniformly mixing the adjusted 4OD gold-labeled antibodies according to a certain proportion, taking out the treated combination pad, uniformly dripping 1.2mL of gold-labeled antibody mixed solution on the combination pad by using a pipette, and placing the combination pad in a drying oven at 37 ℃ for treatment (2-3 hours) and storing.
6. NC film coating
Diluting a C line coating antibody, an influenza A virus coating antibody, an influenza B virus coating antibody and a respiratory syncytial virus coating antibody to 0.8mg/mL by using a coating solution to obtain a C line coating solution, an influenza A virus coating solution, an influenza B virus coating solution and a respiratory syncytial virus coating solution, uniformly scratching each coating solution on a nitrocellulose membrane by using a scratching instrument at 1 mu L/cm to form a quality control line C, an influenza A virus detection line A1, an influenza B virus detection line B1 and a respiratory syncytial virus detection line R1, wherein the distance from the C line to an upper end water absorption pad is 5 +/-1mm, the distance from the R line to a lower end NC membrane lower edge is 6 +/-1 mm, and the distance between the A line, the B line and the R line is 4mm.
7. Test strip assembly
(1) Cutting the water absorption pad into the size of 20 +/-1 mm multiplied by 300 +/-1 mm by using a cutting machine; and sequentially building the treated sample pad, the gold label pad, the NC membrane and the water absorption pad on a PVC (polyvinyl chloride) base plate to form the colloidal gold chromatography test strip, wherein the water absorption pad and the NC membrane are overlapped for 2mm, the NC membrane and the gold label pad are overlapped for 2mm, the gold label pad and the sample pad are overlapped for 2mm, and the residual visible length of the NC membrane is 21mm.
(2) Cutting the large card into test strips with the thickness of 3mm +/-0.1 mm.
(3) Fixing the prepared test paper strip in the membrane strip area of the shell-bottom of the detection card, covering the surface card shell, pressing, putting the installed detection card into a shell pressing machine for pressing, and covering the card shell without a gap.
8. Sample extraction liquid
0.296g of sodium dihydrogen phosphate, 2.901g of disodium hydrogen phosphate, 5g of casein sodium salt, 0.6g of NP-4010.6g and 150uL of preservative were weighed and dissolved in 1L of ultrapure aqueous solution.
9. Sampling a sample tube: comprises a tube and a dropper, wherein 500 mu L (about 20 drops) of sample extract is added into the sample tube, and a sampling swab is placed.
10. Packaging: the final product kit comprises a detection card, a sample extraction liquid, a sampling sample tube and an instruction.
2. Detection method
(1) Collecting samples: the head of the person to be collected is held by one hand of the sampling person, the person holds the swab with the other hand and then sticks into the swab, and the swab slowly and deeply penetrates backwards along the bottom of the inferior nasal passage, and the nasal passage is arc-shaped and cannot be forcefully too violent, so that traumatic bleeding is avoided. When the top end of the swab reaches the back wall of the nasopharyngeal cavity, the swab is gently rotated for a circle (if reflex cough is encountered, the swab should stay for a moment), then the swab is slowly taken out, and the sample extraction liquid treatment should be immediately carried out after the sample is collected.
(2) Sample treatment: the sample tube was removed and 500. Mu.L (about 20 drops) of the sample extract was added to the sample tube (the lowermost scale of the sample tube was about 500. Mu.L). Placing the collected swab in a sample tube, rotating the swab about 10 times close to the inner wall of the sample tube to completely soak the swab with the sample extraction liquid, squeezing the swab several times with a finger from the outside of the sample tube to leave as much of the solution in the tube as possible, removing the swab and capping the dropper.
(3) Sample detection: the sample tube is placed upside down on the sample hole of the detection card, the sample tube is slightly pressed, 3 drops (about 70 mu L) are dripped into the sample hole of the detection card, timing is started, the sample tube is placed at room temperature for 15 minutes, and then the result is observed and judged.
The judgment method comprises the following steps:
a. negative: only the quality control line region (C) has a stripe, and the detection line region (A/B/R) has no stripe.
b. Positive: the quality control line area (C) and the detection line area (A) both have strips, which indicate that the influenza A virus is infected;
the quality control line area (C) and the detection line area (B) both have strips, which indicate that the influenza B virus is infected;
the quality control line area (C) and the detection line area (R) are provided with strips, which indicate that the respiratory syncytial virus is infected;
the quality control line area (C) and the detection line area (A/B) both have a strip, which indicates that the influenza A/B virus is infected;
the quality control line area (C) and the detection line area (A/R) both have a strip, which indicates that the influenza A virus and the respiratory syncytial virus are infected;
the quality control line area (C) and the detection line area (B/R) are provided with strips, which indicate that the influenza B virus and the respiratory syncytial virus are infected;
the quality control line area (C) and the detection line area (A/B/R) are provided with strips, which indicates that the three areas are infected;
c. and (4) invalidation: the red band does not appear in the quality control line region (C). As shown in particular in figure 3.
Comparative example 1
In this comparative example, a chemical blocking agent-free conjugate pad + a biological blocking agent-free sample pad were used, all other things being equal to example 1.
Comparative example 2
In this comparative example, a chemical blocking agent-containing conjugate pad + a sample pad without a biological blocking agent was used, all other things being equal to example 1.
Comparative example 3
In this comparative example, a conjugate pad without a chemical blocking agent + a sample pad with a biological blocking agent was used, and the rest was the same as in example 1.
Experimental example 1 specificity test
And (3) verifying the detection effect of the chemical blocking agent added into the binding pad and the biological blocking agent added into the sample pad on the test strip, and testing the kits of the example 1 and the comparative examples 1-3 respectively.
Virus stocks of ATCC-derived H1N1 (ATCC VR-1469), H3N2 (ATCC VR-1680), fluB (ATCCVR-1735), RSVA (ATCCVR-26) and RSVB (ATCC VR-1580) were diluted to 7.9X 10 4 TCID 50 /mL、2.1×10 4 TCID 50 /mL、3.15×10 4 TCID 50 /mL、3.6×10 4 TCID 50 /mL、3.95×10 4 TCID 50 The specificity test is carried out on the/mL and the sample extract, and the detection result is as follows:
TABLE 1
As can be seen from table 1, compared with comparative examples 1 to 3, in example 1 of the present invention, the test strip has higher specificity due to the combined use of the binding pad containing the chemical blocking agent and the sample pad containing the biological blocking agent.
Experimental example 2 sensitivity test
The virus stock solution of ATCC (American Petroleum institute) H1N1 (ATCC VR-1469), H3N2 (ATCC VR-1680), fluB (ATCCVR-1735), RSVA (ATCCVR-26) and RSVB (ATCC VR-1580) is subjected to gradient dilution and then is subjected to determination of the lowest detection limit, the virus dilution solution of each gradient is repeated for 3 to 5 times, repeated detection is carried out for 20 times, and the virus level with 90 to 95 percent of positive detection rate is taken as the lowest detection limit. The kit of the embodiment 1 of the invention is adopted for detection, and the detection results are as follows:
1. H1N1 minimum detection limit detection
TABLE 2 actual data for different titres of H1N1 assay
TABLE 3-actual data of minimum detection limit of H1N1
2. H3N2 minimum detection limit detection
TABLE 4 actual data for different titres of H3N2 detection
TABLE 5 actual data of lowest detection limit for H3N2
3. FluB minimum detection Limit detection
TABLE 6 actual data for different titer FluB assays
TABLE 7 FluB minimum detection Limit actual data
4. RSVA minimum detection limit detection
TABLE 8 actual data for different titres of RSVA tested
TABLE 9 RSVA minimum detection limit actual data
5. RSVB minimum detection limit detection
TABLE 10 actual data for different titres of RSVB test
TABLE 11 RSVB lowest detection Limit actual data
3. Conclusion
From the above, the detection sensitivity of the kit according to the embodiment of the present invention is finally determined as follows: as shown in table 12.
TABLE 12
| Detecting the index | Viral strains | Minimum limit of detection |
| H1N1 | ATCC VR-1469 | 1.58×10 3 TCID 50 /mL |
| H3N2 | ATCC VR-1680 | 4.2×10 2 TCID 50 /mL |
| FluB | ATCCVR-1735 | 6.3×10 2 TCID 50 /mL |
| RSVA | ATCCVR-26 | 7.2×10 2 TCID 50 /mL |
| RSVB | ATCC VR-1580 | 7.9×10 2 TCID 50 /mL |
Experimental example 3, cross validation
1. Test strains
The different microorganisms in the table below were diluted to the corresponding concentrations/titers using the sample extracts, verifying the specificity of the kit of the invention, the relevant pathogens and titers are as in table 13 below.
TABLE 13 Cross-validation Strain test information
2. Test results
TABLE 14-Cross-compatibility verification of strains test results
3. Conclusion
As can be seen from the data, the results of the kit for detecting other pathogenic microorganisms are negative, and the kit for detecting other pathogenic microorganisms is proved to have no cross reaction with other pathogenic microorganisms and have strong specificity.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. An influenza A/B virus and respiratory syncytial virus antigen detection test strip is characterized by comprising:
a bottom plate, a plurality of first connecting plates,
the sample pad, the combination pad, the chromatography matrix and the water absorption pad are adhered to the bottom plate and are sequentially overlapped; wherein the content of the first and second substances,
the sample pad is added with a biological blocker, and the biological blocker comprises the following components in final concentration: 10-20mg/mL bovine serum albumin, 1-5mg/mL sheep IgG and 0.1-3mg/mL chicken IgY;
the combination pad is coated with a colloidal gold compound coated by an influenza A virus monoclonal antibody A2, a colloidal gold compound coated by an influenza B virus monoclonal antibody B2 and a colloidal gold compound coated by a respiratory syncytial virus monoclonal antibody R2; and a chemical blocking agent is added into the bonding pad, and comprises the following components in final concentration: 0.1-1% of polyvinylpyrrolidone, 0.1-5% of PEG4000, 0.1-2% of Triton X-100 and 0.1-2% of Tween-20;
a quality control line C is arranged on one side, close to the water absorption pad, of the chromatography matrix, and is coated with anti-mouse IgG secondary antibodies;
a detection line T is arranged on one side, close to the combination pad, of the chromatography matrix; the detection line T comprises: the detection line A coated with the influenza A virus monoclonal antibody A1, the detection line B coated with the influenza B virus monoclonal antibody B1 and the detection line R coated with the respiratory syncytial virus monoclonal antibody R1 (murine source).
2. The test strip for detecting the influenza A/B virus and respiratory syncytial virus antigens according to claim 1, wherein the pH of the colloidal gold compound coated by the influenza A virus monoclonal antibody A2 is 7.0 to 7.5, and the labeling amount is 15 mug/mL of colloidal gold solution.
3. The test strip for detecting the influenza A/B virus and respiratory syncytial virus antigens according to claim 1, wherein the pH of the colloidal gold compound coated by the influenza B virus monoclonal antibody B2 is 7.0 to 7.5, and the labeling amount is 15 mug/mL of colloidal gold solution.
4. The test strip for detecting the influenza A/B virus and the respiratory syncytial virus antigens according to claim 1, wherein the pH of the colloidal gold compound coated by the respiratory syncytial virus monoclonal antibody R2 is 7.0 to 7.5, and the labeling amount is 18 mug/mL of colloidal gold solution.
5. The test strip for detecting the influenza A/B virus and respiratory syncytial virus antigens according to claim 1, wherein the concentration of the influenza A virus monoclonal antibody A1 is 1mg/mL, and the coating amount is 0.7 to 1.5 muL/cm.
6. The test strip for detecting the influenza A/B virus and the respiratory syncytial virus antigens according to claim 1, wherein the concentration of the influenza B virus monoclonal antibody B1 is 1mg/mL, and the coating amount is 0.7 to 1.5 muL/cm.
7. The test strip for detecting the influenza A/B virus and the respiratory syncytial virus antigens according to claim 1, wherein the concentration of the respiratory syncytial virus monoclonal antibody R1 (murine source) is 1mg/mL, and the coating amount is 0.7 to 1.5 muL/cm.
8. A method for preparing an antigen test strip for detecting influenza a/b virus and respiratory syncytial virus according to any one of claims 1 to 7, wherein the method comprises:
coating the biological blocking agent on the sample pad to obtain a treated sample pad;
treating a colloidal gold compound coated by an influenza A virus monoclonal antibody A2, a colloidal gold compound coated by an influenza B virus monoclonal antibody B2 and a colloidal gold compound coated by a respiratory syncytial virus monoclonal antibody R2 by a stabilizing agent, spraying the mixture on a bonding pad, and spraying a chemical blocking agent on the bonding pad to obtain a treated bonding pad;
spraying an anti-mouse IgG secondary antibody on a quality control line C on a chromatography matrix, spraying an influenza A virus monoclonal antibody A1 on a detection line A on the chromatography matrix, spraying an influenza B virus monoclonal antibody B1 on a detection line B on the chromatography matrix, and spraying a respiratory syncytial virus monoclonal antibody R1 (mouse source) on a detection line R on the chromatography matrix to obtain the chromatography matrix containing the detection line T and the quality control line C;
and sequentially and mutually overlapping and sticking the processed sample pad, the processed combination pad, the chromatography matrix containing the detection line T and the control line quality control C and the water absorption pad on the bottom plate to obtain the test paper strip for detecting the antigens of the influenza A/B virus and the respiratory syncytial virus.
9. An antigen detection kit for influenza A/B virus and respiratory syncytial virus, which is characterized by comprising 1-8 antigen detection test strips for influenza A/B virus and respiratory syncytial virus
10. The kit for detecting the antigens of the influenza A/B virus and the respiratory syncytial virus according to claim 9, which further comprises a detection card or/and a sample extraction solution or/and a sample sampling tube.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116660520A (en) * | 2023-03-07 | 2023-08-29 | 江苏一诺生物科技有限公司 | Four-in-one antigen detection card and kit |
| CN116718771A (en) * | 2023-08-08 | 2023-09-08 | 北京库尔科技有限公司 | Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116660520A (en) * | 2023-03-07 | 2023-08-29 | 江苏一诺生物科技有限公司 | Four-in-one antigen detection card and kit |
| CN116718771A (en) * | 2023-08-08 | 2023-09-08 | 北京库尔科技有限公司 | Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit |
| CN116718771B (en) * | 2023-08-08 | 2023-11-03 | 北京库尔科技有限公司 | Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit |
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