WO2023040026A1 - Bandelette réactive de détection immunochromatographique et trousse la comprenant, et leurs applications respectives - Google Patents

Bandelette réactive de détection immunochromatographique et trousse la comprenant, et leurs applications respectives Download PDF

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WO2023040026A1
WO2023040026A1 PCT/CN2021/130321 CN2021130321W WO2023040026A1 WO 2023040026 A1 WO2023040026 A1 WO 2023040026A1 CN 2021130321 W CN2021130321 W CN 2021130321W WO 2023040026 A1 WO2023040026 A1 WO 2023040026A1
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influenza
virus
protein
detection
reagent strip
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PCT/CN2021/130321
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Chinese (zh)
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陈科达
陈喆
王燕青
陈秋强
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杭州宝临生物科技有限公司
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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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/58—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
    • 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
    • G01N33/58—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
    • G01N33/587—Nanoparticles
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure relates to the technical field of biological detection, in particular to an immunochromatographic detection reagent strip, a kit containing the same and the application of the two.
  • the novel coronavirus Delta (Delta) mutant strain (B.1.617.2) has become the main pathogenic strain worldwide.
  • the viral load of the delta mutant strain is 1260 times higher than that of the original strain, and it is highly contagious, fast in transmission and strong in vaccine resistance. People infected with the Delta mutant strain had a higher risk of hospitalization, severe pneumonia, and death than those infected with the non-mutated strain.
  • the delta variant strain has caused a new wave of outbreaks around the world. Rapid screening and effective detection of the new coronavirus delta and lambda variant strains have become the key to the current epidemic prevention battle.
  • influenza is another infectious disease that endangers human life and health.
  • the mixed infection of new coronavirus/influenza virus has caused great troubles in clinical diagnosis and treatment. People with influenza infection have increased susceptibility to the new crown, and the aggravation of the mixed infection of new coronavirus influenza can easily develop into severe disease. pneumonia.
  • the disclosure provides an immunochromatographic detection reagent strip, the reagent strip includes a bottom plate, and the bottom plate is provided with a sample pad, a marker binding pad, a chromatographic reaction membrane and absorbent paper;
  • the marker binding pad is adsorbed with specific protein complexes, and the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins;
  • the specific proteins include Novel coronavirus N protein primary antibody, influenza A virus NP protein primary antibody and influenza B virus NP protein primary antibody, used to bind novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigen;
  • the chromatographic reaction membrane is provided with a detection line, and the detection line contains the second antibody of the novel coronavirus N protein, the second antibody of the NP protein of the influenza A virus, and the second antibody of the NP protein of the influenza B virus, in order to bind the novel coronavirus.
  • the detection lines of influenza A and influenza B positive samples are blue, and the detection lines of novel coronavirus positive samples are red, which is convenient Visual recognition.
  • the specific protein complex is obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon to specific proteins.
  • amino acid sequence of the novel coronavirus N protein is shown in SEQ ID NO:01;
  • the amino acid sequence of the influenza A virus NP protein is shown in SEQ ID NO:02;
  • the amino acid sequence of the influenza B virus NP protein is shown in SEQ ID NO:03.
  • the concentration of the second antibody to the novel coronavirus N protein in the detection line is 0.1-1 mg/mL
  • the concentration of influenza A virus NP second protein antibody in the detection line is 0.1 ⁇ 1mg/mL;
  • the concentration of the second protein antibody of influenza B virus NP in the detection line is 0.1-1 mg/ml.
  • the concentration of the specific protein complex in the marker-binding pad is 0.001-0.01 mg/mL.
  • the chromatographic reaction membrane is arranged in the middle of the bottom plate, the marker binding pad and the sample pad are sequentially lapped on one end of the chromatographic reaction membrane, and the absorbent paper is lapped on the chromatographic reaction membrane on the other end.
  • a quality control line is also provided on the chromatographic reaction membrane.
  • the LOD value for the new coronavirus delta strain is 20TCID 50 /mL, corresponding to a nucleic acid Ct value of 34.97;
  • the LOD value of the original strain of the novel coronavirus was 20TCID 50 /mL, corresponding to a nucleic acid Ct value of 34.64.
  • the LOD value for the new coronavirus delta strain is 40TCID 50 /mL, corresponding to the nucleic acid Ct value of 33.60; for the new coronavirus The LOD value of the original virus strain was 40TCID 50 /mL, corresponding to the nucleic acid Ct value of 33.72.
  • the LOD value for the new coronavirus delta strain is 80 TCID 50 /mL, corresponding to the nucleic acid Ct value of 32.35;
  • the LOD value of the original coronavirus strain was 80TCID 50 /mL, corresponding to a nucleic acid Ct value of 32.61.
  • the detected LOD value for influenza A virus is 1.5 ⁇ 10 3 TCID 50 /mL
  • the detected LOD value for influenza B virus It is 2 ⁇ 10 4 TCID 50 /mL.
  • the present disclosure provides an immunoassay kit, which includes the above-mentioned immunochromatography detection kit.
  • the present disclosure provides an immunochromatographic detection reagent strip according to any one of the above or the immunochromatographic detection kit described above in joint detection of novel coronavirus, influenza A virus and influenza B virus use.
  • the present disclosure provides an immunochromatographic detection reagent strip according to any one of the above or the immunochromatographic detection kit described above, which is used for joint detection of novel coronavirus, influenza A virus and influenza B virus use in .
  • the present disclosure provides a method for joint detection of novel coronavirus, influenza A virus and influenza B virus, including:
  • the present disclosure provides a method for diagnosing subjects infected with diseases related to novel coronavirus, influenza A virus and influenza B virus, comprising:
  • the novel coronavirus-related diseases include: fever, dry cough, severe acute respiratory syndrome, shortness of breath, diarrhea, pneumonia;
  • influenza A H1N1 is an acute respiratory infectious disease
  • influenza A H2N2 is an acute respiratory infectious disease
  • influenza A H3N2 is an acute respiratory infectious disease
  • Figure 1 is a schematic structural diagram of the immunochromatographic detection reagent strip provided in Example 1 of the present disclosure, wherein the arrow indicates the direction of chromatography;
  • FIG. 2 is a schematic diagram of the structure of the immunoassay kit provided in Example 1 of the present disclosure, wherein, A represents: influenza A virus detection line, B represents: influenza B virus detection line, C represents: quality control line, SARS CoV-2 represents: COVID-19 testing line.
  • quality control line refers to: the "C (Control) line” on the reagent strip, which is used to indicate whether the quality of the reagent strip is passed and whether the test result is valid. If there is no quality control line in the test result, it means that the test result is invalid.
  • the term "subject” refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • the term “antibody” refers to a protective protein produced by the body due to stimulation by an antigen. It is understood that, herein, the term “specific protein” may refer to an antibody and/or include a combination of two or more antibodies.
  • sensitivity refers to the positive coincidence rate between the detection results of the detection method of the present disclosure and the detection results of the fluorescent PCR method in a statistical sense.
  • the present disclosure provides an immunochromatographic detection reagent strip, the reagent strip includes a bottom plate 110, on which a sample pad 120, a marker binding pad 140, a chromatographic reaction membrane 160 and an absorbent paper 180 are arranged;
  • the marker binding pad is adsorbed with specific protein complexes, and the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins;
  • the specific proteins include Novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein;
  • a detection line is provided on the chromatographic reaction membrane, and the detection line contains antibodies to novel coronavirus N protein, influenza A virus NP protein, and influenza B virus NP protein.
  • the present disclosure provides an immunochromatographic detection reagent strip, the reagent strip includes a bottom plate 110, on which a sample pad 120, a marker binding pad 140, a chromatographic reaction membrane 160 and an absorbent paper 180 are arranged;
  • the marker binding pad 140 is adsorbed with a specific protein complex, and the specific protein complex is mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific protein Including the first antibody of novel coronavirus N protein, the first antibody of influenza A virus NP protein and the first antibody of influenza B virus NP protein, which are used to bind the new coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein antigen;
  • Chromatographic reaction membrane 160 is provided with detection line 162, and described detection line 162 contains novel coronavirus N protein second antibody, influenza A virus NP protein second antibody, influenza B virus NP protein second antibody, in order to combine Antigens of novel coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein.
  • the sample to be tested is firstly contacted with the marker binding pad 140, and the viral antigen first interacts with the first antibody of the novel coronavirus N protein, the first antibody of the NP protein of the influenza A virus, and the first antibody of the influenza B virus.
  • the first antibody of the virus NP protein specifically binds, and then the new coronavirus antigen, influenza A virus antigen, and influenza B virus antigen in the sample to be tested bind to the second antibody of the new coronavirus N protein of the detection line, and the Influenza virus NP protein secondary antibody, influenza B virus NP protein secondary antibody.
  • the double-antibody sandwich method is adopted, the first antibody and the second antibody respectively act on different epitopes of the antigen, and the first antibody, the second antibody and the antigen form a sandwich structure.
  • the detection lines of influenza A and influenza B positive samples show blue, and the positive samples of novel coronavirus The detection line is displayed in red, which is easy to identify with the naked eye.
  • the reagent strip includes a bottom plate 110, on which a sample pad 120, a marker binding pad 140, a chromatographic reaction membrane 160 and an absorbent paper 180 are arranged; wherein the marker The binding pad 140 is adsorbed with specific protein complexes, which are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include novel coronavirus N protein
  • the chromatographic reaction membrane 160 is provided with a detection line 162, and the detection line 162 contains the first antibody of novel coronavirus N protein Secondary antibody, Influenza A virus NP protein secondary antibody, Influenza B virus NP protein secondary antibody.
  • the above-mentioned detection reagent strip adopts immunochromatography technology, uses the new coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein as detection antigens, and uses fluorescent microspheres, latex, colloidal gold or colloidal carbon and specific proteins Conjugation yields specific protein complexes, which are subsequently tested on patient nasopharyngeal swabs, oropharyngeal swabs, or saliva.
  • the above-mentioned reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent.
  • the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.
  • the above-mentioned reagent strips of the present disclosure adopt immunochromatography technology, and according to the actual application situation, latex method, fluorescence method, colloidal gold method or colloidal carbon method can be used to convert the N protein of the new coronavirus, type A and type B influenza virus
  • the NP protein can be used to detect biological samples of suspected novel coronavirus or influenza A and B virus infections.
  • amino acid sequence of the novel coronavirus N protein is shown in SEQ ID NO: 01, as follows:
  • the amino acid sequence of the influenza A virus NP protein is shown in SEQ ID NO:02, as follows:
  • the amino acid sequence of the influenza B virus NP protein is shown in SEQ ID NO:03, as follows:
  • amino acid sequences of SEQ ID NO:01, SEQ ID NO:02 and SEQ ID NO:03 in this disclosure may have mutations at individual points, and mutations at high-frequency mutation sites of amino acid sequences also occur in this disclosure. within the scope of the claims. Because there are various mutants in the amino acid sequence, but its conserved region is the main site for binding to our antibody. Amino acid sequences with mutations at the above-mentioned high-frequency mutation sites should also be within the protection scope of the present disclosure. For example, mutations in the N protein of the new coronavirus: R203K, G204R.
  • the concentration of the second antibody to the novel coronavirus N protein in the detection line 162 is 0.1-1 mg/mL;
  • the concentration of the second antibody to the influenza A virus NP protein in the detection line 162 is 0.1-1 mg/mL;
  • the concentration of the second antibody to the NP protein of influenza B virus in the detection line 162 is 0.1-1 mg/ml.
  • the concentration of the specific protein complex in the marker-binding pad 140 is 0.001-0.01 mg/mL.
  • the novel coronavirus N protein first antibody and the second antibody are each independently selected from: novel coronavirus N protein polyclonal antibody (mouse source), novel coronavirus N protein monoclonal antibody (mouse source), Any one of the polyclonal antibody to the new coronavirus N protein (rabbit source) and the monoclonal antibody to the new coronavirus N protein (rabbit source).
  • the first antibody to the NP protein of influenza A virus and the second antibody are each independently selected from: polyclonal antibody to NP protein of influenza A virus (mouse origin), monoclonal antibody to NP protein of influenza A virus (mouse) source), influenza A virus NP protein polyclonal antibody (rabbit source), any one of influenza A virus NP protein monoclonal antibody (rabbit source).
  • the first antibody to the NP protein of influenza B virus and the second antibody are each independently selected from: polyclonal antibody to NP protein of influenza B virus (mouse origin), monoclonal antibody to NP protein of influenza B virus (mouse origin) source), influenza B virus NP protein polyclonal antibody (rabbit source), any one of influenza B virus NP protein monoclonal antibody (rabbit source).
  • antibodies are obtained by:
  • the chromatographic reaction membrane 160 is disposed in the middle of the bottom plate 110 .
  • the chromatographic reaction membrane 160 includes a proximal end (ie, the upper end in the chromatographic direction) and a distal end (ie, the lower end in the chromatographic direction).
  • the marker binding pad 140 and the sample pad 120 are lapped on one end (the proximal end) of the chromatography reaction membrane 160 in turn, and the absorbent paper 180 is lapped on the other end (the far side end) of the chromatography reaction membrane 160 superior.
  • a quality control line 164 is also provided on the chromatographic reaction membrane 160 .
  • the LOD value for the new coronavirus delta strain is 20TCID 50 /mL, corresponding to The nucleic acid Ct value is 34.97; the LOD value for the original strain of the new coronavirus is 20TCID 50 /mL, corresponding to the nucleic acid Ct value of 34.64;
  • the LOD value for the new coronavirus delta strain is 40TCID 50 /mL, corresponding to the nucleic acid Ct value of 33.60; for the new coronavirus original The LOD value of the strain was 40TCID 50 /mL, corresponding to the nucleic acid Ct value of 33.72;
  • the LOD value for the new coronavirus delta strain is 80TCID 50 /mL, corresponding to the nucleic acid Ct value of 32.35; for the new coronavirus The LOD value of the original strain was 80TCID 50 /mL, corresponding to the nucleic acid Ct value of 32.61.
  • the new coronavirus clinical sample testing status of the new coronavirus/influenza A virus/influenza B virus one-card three-test antigen detection kit (fluorescence method, latex method) provided by the present disclosure is as follows: :
  • the detected LOD value for influenza A virus is 1.5 ⁇ 10 3 TCID 50 /mL, and for influenza B The detected LOD value of influenza virus was 2 ⁇ 10 4 TCID 50 /mL.
  • the LOD value of the above-mentioned latex method reagent strip for detecting influenza A virus is 1.5 ⁇ 10 3 TCID 50 /mL, the detection sensitivity of clinical samples is 90.00%, the specificity is >99%, and the accuracy is 98.57%;
  • the LOD value of the latex method reagent strip for detecting influenza B virus was 2 ⁇ 10 4 TCID 50 /mL, the sensitivity was 92.86%, the specificity was >99%, and the accuracy was 98.44%.
  • an immunoassay kit includes the above-mentioned immunochromatographic detection reagent strip.
  • the immunoassay kit provided by the present disclosure, the kit includes the above-mentioned immunochromatography detection reagent strip. Determined by the characteristics of the above-mentioned reagent strips, the disclosed kit has the advantages of high sensitivity, high specificity, and quantitative detection, and can quickly detect novel coronavirus, influenza A virus, and influenza B virus antigens by adding a sample. At the same time, the missed detection of weak positive samples and the false detection of false positive samples can be avoided to the greatest extent.
  • the latex method novel coronavirus/influenza A virus/influenza B virus antigen one-card-three test reagent Influenza A and influenza B positive sample T line shows blue, and the new coronavirus positive sample T line shows Red, easy to identify with the naked eye.
  • an application of the above-mentioned immunochromatographic detection reagent strip or the above-mentioned immunochromatographic detection kit in the preparation of a joint detection product for novel coronavirus, influenza A virus and influenza B virus.
  • immunochromatographic detection reagent strips or the above-mentioned immunochromatographic detection kits provided in the present disclosure can be widely used in the preparation process of joint detection products for novel coronavirus, influenza A virus and influenza B virus.
  • the sample is dropped onto an immunochromatographic detection reagent strip to detect viruses.
  • the antigen in the sample runs toward the absorbent paper 180 through the suction of the absorbent paper 180 , and binds to the primary antibody on the marker-binding pad 140 .
  • the combination of the antigen and the first antibody continues to run on the plate, moves to the detection line 162, combines with the second antibody coated with anti-"antigen" on the detection line 162 and develops color, wherein the first antibody and the second antibody Acting on different epitopes of the antigen respectively, the first antibody, the second antibody and the antigen form a sandwich structure.
  • One embodiment of the present disclosure discloses the use of an immunochromatographic detection reagent strip or an immunochromatographic detection kit in joint detection of novel coronavirus, influenza A virus and influenza B virus.
  • One embodiment of the present disclosure discloses a method for joint detection of novel coronavirus, influenza A virus and influenza B virus, including:
  • One embodiment of the present disclosure discloses a method for diagnosing subjects infected with diseases related to novel coronavirus, influenza A virus and influenza B virus, including:
  • diseases related to the novel coronavirus include: fever, dry cough, severe acute respiratory syndrome, shortness of breath, diarrhea, pneumonia;
  • influenza A H1N1 is an acute respiratory infectious disease
  • influenza A H2N2 is an acute respiratory infectious disease
  • influenza A H3N2 is an acute respiratory infectious disease
  • the reagent strip provided by the present disclosure has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent.
  • the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.
  • the reagent strip includes a bottom plate on which a sample pad, a marker binding pad, a chromatographic reaction membrane, and an absorbent paper are arranged; wherein, the marker binding pad is adsorbed with a specific Protein complexes, the specific protein complexes are mainly obtained by coupling fluorescent microspheres, latex, colloidal gold or colloidal carbon with specific proteins; the specific proteins include the first antibody of the new coronavirus N protein, influenza A Viral NP protein first antibody and influenza B virus NP protein first antibody; a detection line is provided on the chromatographic reaction membrane, and the detection line contains the new coronavirus N protein second antibody, influenza A virus NP protein first antibody Secondary antibody, influenza B virus NP protein secondary antibody.
  • the above-mentioned detection reagent strip adopts immunochromatography technology, uses the new coronavirus N protein, influenza A virus NP protein and influenza B virus NP protein as detection antigens, and uses fluorescent microspheres, latex, colloidal gold or colloidal carbon and specific proteins Conjugation yields specific protein complexes, which are then tested on patient nasopharyngeal swabs, oropharyngeal swabs, or saliva.
  • the above-mentioned reagent strip has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent.
  • the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.
  • the immunoassay kit provided by the present disclosure, the kit includes the above-mentioned immunochromatography detection reagent strip. Determined by the characteristics of the above-mentioned reagent strips, the disclosed kit has the advantages of high sensitivity, high specificity, and quantitative detection, and can quickly detect novel coronavirus, influenza A virus, and influenza B virus antigens by adding a sample. At the same time, the missed detection of weak positive samples and the false detection of false positive samples can be avoided to the greatest extent.
  • immunochromatographic detection reagent strips or the above-mentioned immunochromatographic detection kits provided in the present disclosure can be widely used in the preparation process of joint detection products for novel coronavirus, influenza A virus and influenza B virus.
  • A, B, C, and D represent the test results.
  • the novel coronavirus N protein monoclonal antibody can be prepared by the following:
  • the mouse myeloma cell line SP2/0 is preserved by our company;
  • PEG1500, hypoxanthine, thymine, aminopterin, and DMSO were purchased from Sigma;
  • RPMI1640 basal medium was purchased from Gibco;
  • Fetal bovine serum was purchased from LONSASCIENCESRL;
  • Embodiment 1 prepares immunochromatography detection reagent strip:
  • a kind of immunochromatographic detection reagent strip the preparation method of described reagent strip comprises the following steps:
  • the detection line 162 will contain the second antibody of novel coronavirus N protein (material code: N34), the second antibody of influenza A virus NP protein (material code: FA12), the second antibody of type B influenza virus NP protein (material code: The chromatographic reaction membrane 160 of FB11);
  • the marker binding pad 140 prepared above, and the sample pad 120 and the absorbent paper 180 are lapped and adhered to the PVC backboard (ie the bottom plate 110);
  • the chromatographic reaction membrane 160 includes a proximal end (ie, the upper end in the chromatographic direction) and a distal end (ie, the lower end in the chromatographic direction).
  • the sample pad 120 and the quantum dot marker binding pad 140 are lapped successively on one end (the proximal end) of the chromatography reaction membrane 160, and the absorbent paper 180 is lapped on the other end (the far side end) of the chromatography reaction membrane 160, and then adjusted
  • the parameters of the strip cutting machine are to cut into strips and cut into test strips with a width of 3-4mm.
  • an immunoassay kit the immunoassay kit includes the above-mentioned immunochromatographic detection reagent strip.
  • Example 2 Sensitivity test of novel coronavirus/influenza A virus/influenza B virus antigen one card three test kit to novel coronavirus antigen:
  • kits prepared in Example 1 were used to test the novel coronavirus/influenza A virus/influenza B virus antigen in one card and three tests by fluorescence method, latex method and colloidal gold method respectively, and the reagents were selected from fluorescence method, latex method and The colloidal gold method 2019-nCoV antigen one card single test reagent was used as the control reagent.
  • the latex method, the fluorescence method and the colloidal gold method have one card with three tests (that is, one reagent strip detects three viruses at the same time) and one card with a single test for the new crown antigen reagent.
  • the sensitivity is similar, and the corresponding nucleic acid detection Ct value is 33.72. , 34.64, and 32.61;
  • the latex method, fluorescence method, and colloidal gold method have similar sensitivities to one-card three-test and one-card single-test new crown antigen reagents, corresponding to nucleic acid detection Ct values of 33.60, 34.97 and 32.35, respectively.
  • the test results are shown in Table 1 and Table 2:
  • the fluorescence method, latex method and colloidal gold method of the present disclosure have the same sensitivity for one card three tests and one card single test for the new crown antigen reagent.
  • the sensitivity of the new crown antigen latex method reagent is higher than that of the colloidal gold method
  • the sensitivity of the fluorescence method is higher than that of the latex method.
  • the detection buffer (buffer) (composed of PBS and TWEEN20 volume ratio: 200:1) to dilute the 4 strains of inactivated influenza virus to their respective LOD, each diluted 3ml; take out the example 1 Novel coronavirus/influenza A virus/influenza B virus antigen combined detection kit, respectively detect 4 strains of inactivated influenza virus solutions diluted to their respective LODs, each solution is repeated 20 times, and interpreted separately.
  • the test results are shown in Table 3.
  • Table 3 Fluorescence method and latex method Novel coronavirus/influenza A virus/influenza B virus antigen one card three test reagents Influenza A virus and influenza B virus LOD test:
  • the LOD concentration of inactivated virus samples of influenza A virus strain Flu A/GUANGDONG/2019 and influenza B virus strain Flu B/Washington/02/2019 was taken to make mixed standard 1, and influenza A virus strain Flu A/
  • the LOD concentration of NYMC X-223A and influenza B virus strain Flu B/phuket/3073/2013 inactivated virus samples was prepared as a mixed standard 2, and this product was compared with Japan's competitor products (QuickNavi-Flu A&B, latex method) experiment. According to the sample volume and running time of the respective product manuals, test and interpret respectively, and judge the brightness of the ribbon according to the color chart, and the results are shown in Table 4.
  • the experimental data of this example proves that the latex method new coronavirus/influenza A virus/influenza B virus antigen combined detection reagent product provided by the present disclosure has both the detection sensitivity of influenza A virus and the detection sensitivity of influenza B virus. It is about 1 times higher than that of Shengyan's competing products (QuickNavi-Flu A&B, latex method). The test results are shown in Table 4.
  • Table 4 New coronavirus/influenza A virus/influenza B virus antigen one card three test reagent (latex method) and Sanken influenza A virus/influenza B virus antigen one card dual test reagent (QuickNavi-Flu A&B, latex method) method) sensitivity comparison:
  • the limit of detection (LOD) of influenza A virus and influenza B virus in the 2019-nCoV/Influenza A virus/Influenza B virus antigen one-card three test reagent is a gradient using virus samples Measured in diluent.
  • inactivated sample stock solutions of 10 types of influenza A virus and 7 types of influenza B virus were selected for this test.
  • the initial material concentration was 3.0 ⁇ 10 5 TCID 50 /mL.
  • the initial detection range of the samples was determined using three PBS buffer gradient dilution series, and then the LOD was obtained by further refining the test through a 2-fold dilution series.
  • the detection limit of the detection reagent provided by the disclosure is about 1.5 ⁇ 10 3 for influenza A virus TCID 50 /mL, the detection limit of influenza B virus is about 2.0 ⁇ 10 4 TCID 50 /mL.
  • Embodiment 5 multiple novel coronavirus/influenza A virus/influenza B virus antigen cross-reaction test:
  • this embodiment designs experiments for multiple subtypes of A Cross-reactivity was assessed between Type 2 and Type B influenza virus strains.
  • This example designs an experiment to study the cross-interference and non-specific interference of the latex method, fluorescence method, and colloidal gold method for novel coronavirus/influenza A/influenza B virus antigen detection reagents provided by this disclosure.
  • Cross-interference experiment design for common respiratory pathogens use inactivated positive samples of novel coronavirus, influenza A virus and influenza B virus for cross-interference test, and confirm the following 12 virus samples (1.0 ⁇ 10 5 Pfu/mL), 8 There is no cross-interference between bacteria (1.0 ⁇ 10 6 Pfu/mL), Chlamydia pneumoniae (1.0 ⁇ 10 6 Pfu/mL) and Mycoplasma pneumoniae (1.0 ⁇ 10 6 Pfu/mL) and this reagent.
  • Operation steps Take 500 ⁇ L of the sample solution of each sample and 500 ⁇ L of the buffer solution of the sample detection kit and mix it. After standing for 2 minutes, take 90 ⁇ L of the above buffer solution-treated sample and add it to the reagent injection hole. Repeat for 1 person for each diluted sample and each reagent. Take the reading after 15 minutes of dropping the sample, use the instrument to read the fluorescence method, and use the color card to read the latex method and colloidal gold method.
  • Table 7 shows the cross-interference and interference test results of common respiratory pathogens in the novel coronavirus/influenza A/influenza B virus antigen detection kit.
  • Table 7 Influenza A/Influenza B virus multi-subtype antigen cross-interference test results (latex method, fluorescence method, colloidal gold method)
  • Cross-interference experimental design of common non-specific substances in the respiratory tract use inactivated positive samples of novel coronavirus, influenza A virus, and influenza B virus for cross-interference test, and confirm that the following 21 sample substances have no cross-interference with this reagent. Take 500 ⁇ L of the sample solution of each sample and 500 ⁇ L of the buffer solution of the sample detection kit and mix it. After standing for 2 minutes, take 90 ⁇ L of the above-mentioned buffer-treated sample and add it to the reagent injection hole. Repeat for 1 person for each diluted sample and each reagent. Take the reading after 15 minutes of dropping the sample, use the instrument to read the fluorescence method, and use the color card to read the latex method and colloidal gold method. According to the test results, at the indicated concentrations, no interference from non-specific substances as shown in Table 8 was observed.
  • Novel coronavirus/influenza A/influenza B virus antigen detection kit fluorescence method, latex method
  • new coronavirus clinical positive sample test fluorescence method, latex method
  • a clinical sample test experiment is designed to verify the performance of the detection reagent provided by the present disclosure.
  • Novel coronavirus clinical sample testing a total of 579 samples. 358 clinical samples of patients with new crown virus, including 23 nasopharyngeal swab samples, 93 sputum samples, 236 saliva samples and 6 alveolar lavage fluid samples; 96 new coronavirus negative volunteer saliva samples, negative volunteer throat swab The sub-sample is 125 cases.
  • Sputum or alveolar lavage fluid sample processing method After the sputum or alveolar lavage fluid sample is centrifuged at high speed to remove coarse particles, the centrifuged supernatant is mixed with buffer solution 1:1 (v/v); the nasopharyngeal swab is processed Method: take the virus preservation solution containing nasopharyngeal swab, and mix it with the detection buffer 1:1 (v/v); saliva sample processing method: take 100 ⁇ L of the saliva sample, and mix it with the detection buffer 1:3 (v/v) Mix well. All samples were checked with the fluorescent PCR detection kit of Daan Gene Company, and the operation was carried out according to the instructions to determine the corresponding Ct value of each sample.
  • Example 9 Novel coronavirus/influenza A virus/influenza B virus antigen detection kit (latex method) influenza A virus/influenza B clinical positive sample test:
  • a clinical sample test experiment is designed to verify the performance of the detection reagent provided by the present disclosure.
  • Influenza A virus/influenza B virus clinical sample test a total of 20 cases of influenza A virus positive clinical samples (18 cases of nasopharyngeal swabs, 2 cases of saliva), 28 cases of influenza B virus positive clinical samples (nasopharyngeal swabs 19 cases, 11 cases of saliva).
  • Nasopharyngeal swab sample processing method take the virus preservation solution containing the nasopharyngeal swab sample, and mix it with the detection buffer 1:1; saliva sample processing method: after the saliva sample is centrifuged at high speed to remove coarse particles, take the centrifuged supernatant and Buffer 1:3 was mixed well.
  • Sensitivity 90.00% (93.37-96.63%)*; specificity: >99% (98.37-101.63%)*; precision: 98.57% (97.54-100.79%)*; *95% confidence interval.
  • Sensitivity 92.86% (90.71-95.01%)*; specificity: >99% (97.85-102.15%)*; precision: 98.44% (96.29-100.59%)*; *95% confidence interval.
  • the present disclosure provides an immunochromatographic detection reagent strip and a kit containing the same and applications thereof.
  • the reagent strip provided by the present disclosure has the advantages of high sensitivity, high specificity and quantitative detection, and avoids missed detection of weak positive samples and false positive samples to the greatest extent.
  • the immunochromatographic method used in the present disclosure is rapid in detection, and the result can be obtained within 15 minutes, and can be operated on site with simple training.
  • the immunoassay kit provided by the present disclosure has the advantages of high sensitivity, high specificity, and quantitative detection.
  • One-time sample addition can quickly detect novel coronavirus, influenza A virus, and influenza B virus antigens, and at the same time avoid The missed detection of weak positive samples and the false detection of false positive samples were eliminated.
  • the above-mentioned immunochromatographic detection reagent strip or the above-mentioned immunochromatographic detection kit provided in the present disclosure can be widely used in the preparation process of joint detection products for novel coronavirus, influenza A virus and influenza B virus, and has wide application value.

Abstract

L'invention concerne une bandelette réactive de détection immunochromatographique et une trousse la comprenant, ainsi que leurs applications, se rapportant au domaine technique de la détection biologique. Selon la bandelette réactive de détection immunochromatographique, des microsphères fluorescentes, du latex, de l'or colloïdal ou du carbone colloïdal sont associés à une protéine spécifique en utilisant l'immunochromatographie pour obtenir un complexe protéique spécifique. La protéine spécifique comprend un premier anticorps de la nouvelle protéine N du coronavirus, un premier anticorps de la protéine NP du virus de l'influenza A et un premier anticorps de la protéine NP du virus de l'influenza B, qui sont utilisés pour lier les antigènes de la nouvelle protéine N du coronavirus, de la protéine NP du virus de l'influenza A et de la protéine NP du virus de l'influenza B. Des échantillons prélevés dans le nez ou dans la gorge d'un patient, ou de la salive sont ensuite analysés. La bandelette réactive de détection immunochromatographique présente les avantages d'une sensibilité élevée, d'une spécificité élevée et d'une détection quantitative, et évite dans une large mesure la détection manquée d'échantillons faiblement positifs et la fausse détection d'échantillons faussement positifs.
PCT/CN2021/130321 2021-09-15 2021-11-12 Bandelette réactive de détection immunochromatographique et trousse la comprenant, et leurs applications respectives WO2023040026A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116715760A (zh) * 2023-07-31 2023-09-08 南京佰抗生物科技有限公司 抗乙型流感病毒核衣壳蛋白的单克隆抗体及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115947835B (zh) * 2022-11-15 2023-07-28 杭州华葵金配生物科技有限公司 靶向乙型流感病毒核蛋白的抗体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111398593A (zh) * 2020-04-03 2020-07-10 天津华科泰生物技术有限公司 一种快速联合检测卡及其制备方法和应用
CN111458499A (zh) * 2020-02-17 2020-07-28 江苏美克医学技术有限公司 新型冠状病毒和流感病毒检测试剂盒及检测方法
CN111948389A (zh) * 2020-08-30 2020-11-17 深圳市信一生物技术有限公司 一种检测甲、乙型流感病毒和新型冠状病毒抗原的时间分辨荧光免疫层析试纸条及制备方法
CN112198312A (zh) * 2020-10-12 2021-01-08 南京佰抗生物科技有限公司 一种新型冠状病毒抗原与流感病毒抗原联合检测试剂条及其制备方法
CN112334478A (zh) * 2018-06-11 2021-02-05 葛兰素史克消费保健（美国）控股有限责任公司 用于快速甲型流感诊断测试的抗体对
CN113189333A (zh) * 2021-04-28 2021-07-30 杭州宝临生物科技有限公司 量子点免疫荧光检测试剂条包含其的试剂盒及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101336375A (zh) * 2005-12-26 2008-12-31 株式会社比尔生命 用于检测流感a型病毒的强毒株的方法
US8163474B2 (en) * 2008-03-15 2012-04-24 Arbor Vita Corporation NS1-NP diagnostics of influenza virus infection

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112334478A (zh) * 2018-06-11 2021-02-05 葛兰素史克消费保健（美国）控股有限责任公司 用于快速甲型流感诊断测试的抗体对
CN111458499A (zh) * 2020-02-17 2020-07-28 江苏美克医学技术有限公司 新型冠状病毒和流感病毒检测试剂盒及检测方法
CN111398593A (zh) * 2020-04-03 2020-07-10 天津华科泰生物技术有限公司 一种快速联合检测卡及其制备方法和应用
CN111948389A (zh) * 2020-08-30 2020-11-17 深圳市信一生物技术有限公司 一种检测甲、乙型流感病毒和新型冠状病毒抗原的时间分辨荧光免疫层析试纸条及制备方法
CN112198312A (zh) * 2020-10-12 2021-01-08 南京佰抗生物科技有限公司 一种新型冠状病毒抗原与流感病毒抗原联合检测试剂条及其制备方法
CN113189333A (zh) * 2021-04-28 2021-07-30 杭州宝临生物科技有限公司 量子点免疫荧光检测试剂条包含其的试剂盒及其应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116715760A (zh) * 2023-07-31 2023-09-08 南京佰抗生物科技有限公司 抗乙型流感病毒核衣壳蛋白的单克隆抗体及应用
CN116715760B (zh) * 2023-07-31 2023-10-03 南京佰抗生物科技有限公司 抗乙型流感病毒核衣壳蛋白的单克隆抗体组合物及应用

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