WO2023202271A1 - 一种多项病原体联合检测装置及其制备方法 - Google Patents

一种多项病原体联合检测装置及其制备方法 Download PDF

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WO2023202271A1
WO2023202271A1 PCT/CN2023/081483 CN2023081483W WO2023202271A1 WO 2023202271 A1 WO2023202271 A1 WO 2023202271A1 CN 2023081483 W CN2023081483 W CN 2023081483W WO 2023202271 A1 WO2023202271 A1 WO 2023202271A1
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colloidal gold
antigen
chitosan
solution
detection line
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PCT/CN2023/081483
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English (en)
French (fr)
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王洪波
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吉林迅准生物技术有限公司
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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
    • G01N33/569—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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • G01N33/56911—Bacteria
    • G01N33/56927—Chlamydia
    • 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
    • G01N33/56911—Bacteria
    • G01N33/56933—Mycoplasma
    • 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
    • G01N2333/01—DNA viruses
    • G01N2333/075—Adenoviridae
    • 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
    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • G01N2333/095—Rhinovirus
    • 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
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/295—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Chlamydiales (o)
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • 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
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • 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 invention belongs to the field of medical detection devices, and particularly relates to a multiple pathogen joint detection device and a preparation method thereof.
  • Influenza is an acute respiratory infectious disease caused by influenza virus infection. It spreads rapidly through the respiratory tract, has a short incubation period and obvious seasonality. It can easily cause large-scale outbreaks and is difficult to control. Influenza viruses are divided into 3 types: A (A), B (B), and C (C). Among them, influenza A virus and influenza B virus infections are more common, highly contagious, spread rapidly, and pose a serious threat to human health. Because influenza viruses spread quickly and mutate easily, epidemics of varying scales occur every year, and even a global pandemic occurs every 10 to 15 years.
  • influenza The main clinical manifestations of influenza are fever, headache, myalgia and general malaise, which may be accompanied by respiratory symptoms such as cough and sputum, as well as digestive system symptoms such as vomiting and diarrhea.
  • respiratory symptoms such as cough and sputum
  • digestive system symptoms such as vomiting and diarrhea.
  • Children, pregnant women, the elderly, and people with various chronic diseases or weak constitutions are prone to serious complications or aggravation of existing chronic diseases, or even life-threatening organ failure. Therefore, understanding the characteristics of influenza is helpful for early identification and timely treatment.
  • influenza viruses The mutation of influenza viruses is the most important, and it is often closely related to worldwide pandemics.
  • antigenic variation of influenza viruses refers to changes in the H and N antigen structures. Small mutations (quantitative changes) often occur within subtypes, which is called antigenic drift. Antigenic variation occurs only in type A viruses. It may be caused by genetic reassortment between the viruses when the same cell is infected with two viruses, both human and animal. The resulting viral hemagglutinin and neuraminidase combine completely new, leaving the population without immunity. Antigenic shift is responsible for global influenza pandemics. Generally, there is an obvious alternation between the old and new subtypes.
  • Influenza B viruses also have large and small variations, but they are not divided into subtype changes. No antigenic variation has been found in influenza C viruses.
  • the clinical treatment plans for influenza virus types A and B, HRV, HPIV, RSV, HAdV, MP, and CP are different, so it is necessary to make a differential diagnosis to determine which causative factor is the cause, and then to treat it accordingly; therefore, influenza virus type A
  • the joint qualitative detection project with IgM antibodies of type B, HRV, HPIV, RSV, HAdV, MP, and CP can detect which virus is infected in a timely, convenient, accurate and comprehensive manner, bringing great benefits to clinical diagnosis and treatment, and also to various fields.
  • test samples are whole blood, serum, or plasma, which is convenient and safe to draw; IgM antibodies of influenza virus types A and B, HRV, HPIV, RSV, HAdV, MP, and CP are indicators of current infection, and there is no false positive problem. Therefore, it is more accurate and has great value for clinical diagnosis.
  • the virus is generally confirmed by isolation and culture of chicken embryos or MDCK cells and hemagglutination inhibition method.
  • the operation is cumbersome, time-consuming and labor-intensive, and it is difficult to meet the needs of rapid diagnosis.
  • nucleic acid diagnostics PCR method
  • ELISA method enzyme-linked immunosorbent assay
  • colloidal gold immune layer analysis method colloidal gold immune layer analysis method.
  • the PCR method has the highest accuracy and is the preferred method for diagnosis, but the detection cost is high and the detection time is long; the ELISA method is a classic method in hospital laboratory departments and disease control system laboratories; the colloidal gold method is the fastest, mature and stable technology, It is simple and easy to use and suitable for universal screening and diagnosis in medical institutions at all levels, CDCs and other places.
  • Colloidal gold immunochromatography assay is a solid-phase membrane immunoassay technology that combines colloidal gold labeling technology and protein chromatography technology and uses a microporous filter membrane as a carrier.
  • Colloidal gold immunochromatography is a commonly used immunochromatographic detection method. Due to its simple operation, time saving, low manufacturing cost, and easy interpretation of results, it is very suitable for on-site detection and is widely used in biology, medicine, and food. and other fields. Since colloidal gold immunochromatography technology completes the detection in one step, there are many interference factors in the detection process. Its low sensitivity is the main factor limiting the application range of colloidal gold immunochromatography. The detection limit of traditional colloidal gold immunochromatography technology is higher than ELISA and other methods.
  • NC membrane nitrocellulose membrane
  • the detection results completely depend on the good adsorption effect of the capture reagent on the membrane, uniform and good adsorption of proteins on the membrane is very important for the colloidal gold detection results. If the amount of protein bound to the NC membrane is insufficient or the protein binding force is not strong enough, there will be quite a few problems, which will be very obvious on the test line of the test results. If the amount of protein bound to the membrane is too low, the line detected in the results The color development is weak and the detection sensitivity is reduced.
  • the protein cannot be firmly adsorbed on the NC membrane, diffusion will occur before the protein is adsorbed on the NC membrane, resulting in a wider detection line and weaker color development instead of bright and clear, making the detection results difficult to interpret.
  • the flowing protein detector and surfactant solution may wash the fixed protein from the NC membrane, resulting in a wider or unclear detection. lines, making it difficult to interpret test results.
  • the invention provides a multi-pathogen joint detection device and a preparation method thereof to solve the problems of insufficient adsorbed protein and weak binding force of NC membranes existing in the prior art.
  • Influenza virus types A and B, HRV prepared by the invention HPIV, RSV, HAdV IgM antibody six-in-one, or influenza virus type A and B, HRV, HPIV, RSV, HAdV, MP, CP IgM antibody eight-in-one definite combined detection device, improves the detection of patients with respiratory diseases Reasonable comprehensive and comprehensive judgment can quickly and accurately judge the risk of respiratory tract infections.
  • the invention provides a multi-pathogen joint detection device.
  • a sample pad, an immune colloidal gold antibody glass fiber membrane, an immune nitrocellulose membrane, and an absorption pad are respectively pasted on a plastic plate.
  • the two ends of the nitrocellulose membrane are respectively connected to
  • the absorption pad and the immune colloidal gold antibody glass fiber membrane are overlapped, and the other end of the immune colloidal gold antibody glass fiber membrane is overlapped with the sample pad;
  • the immune nitrocellulose membrane is provided with a first detection line T1 and a second detection line T2, the third detection line T3, the fourth detection line T4, the fifth detection line T5, the sixth detection line T6 and the quality control line C.
  • the first detection line T1 has influenza virus A antigen on the solid phase
  • influenza virus B antigen on the solid phase on line T2 influenza virus B antigen on the solid phase
  • rhinovirus antigen on the solid phase on the third detection line T3 rhinovirus antigen on the solid phase on the third detection line T3
  • parainfluenza virus antigen on the solid phase on the fourth detection line T4 influenza virus B antigen on the solid phase on the third detection line T3
  • parainfluenza virus antigen on the solid phase on the fourth detection line T4 and respiratory syncytium on the solid phase on the fifth detection line T5.
  • the sixth detection line T6 has adenovirus antigen on the solid phase
  • the quality control line C is sprayed with sheep anti-mouse IgG polyclonal antibody
  • the first detection line T1 and the second detection line set on the immune nitrocellulose membrane
  • the line T2, the third detection line T3, the fourth detection line T4, the fifth detection line T5 and the sixth detection line T6 are respectively set on six lines, and a quality control line C is set on each line.
  • the immune nitrocellulose membrane of the present invention is also provided with a seventh detection line T7 and an eighth detection line T8.
  • the solid phase on the seventh detection line T7 has Mycoplasma pneumoniae antigen
  • the solid phase on the eighth detection line T8 There is Chlamydia pneumoniae antigen, and quality control line C is set on each of the two lines.
  • the invention also provides a method for preparing a multiple pathogen joint detection device, which includes the following steps:
  • N-carboxyethyl chitosan 1) Synthesis of N-carboxyethyl chitosan: Add 4.0g chitosan to 200mL water containing 2.80mL acrylic acid, stir continuously for 2 days at 50°C, after the reaction is completed, add 1mol to the reaction mixture /L NaOH solution, adjust the pH value of the solution to 10-12, so that all carboxylic acids are converted into sodium salts, pour the mixed solution into acetone for reprecipitation, and then dialyze in the aqueous solution to remove unreacted Acrylic acid was dialyzed for two days and freeze-dried to obtain N-carboxyethyl chitosan;
  • Zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody take 1mL of zinc oxide-chitosan-colloidal gold suspension, disperse it evenly by ultrasonic, then add the antibody dropwise while stirring, after adding the antibody, After about 1 minute of reaction, ultrasonic for 30 seconds, then react for another 4 hours, add BSA for blocking for 24 hours, and then centrifuge at 12000r/min for 20 minutes. Keep the precipitate after centrifugation for later use;
  • step (c) Use colloidal gold antibody buffer to dilute the precipitated zinc oxide-chitosan-colloidal gold antibody after centrifugation in step (b) separately to disperse it. uniformly, obtain immune colloidal gold antibody solutions respectively, and spray the immune colloidal gold antibody solutions on the glass fiber mat respectively to prepare immune colloidal gold antibody glass fiber membrane;
  • step (a) zinc oxide-chitosan-colloidal gold according to the present invention
  • step (a) preparation of zinc oxide-chitosan-colloidal gold 3) preparation of a mixed solution of N-carboxyethyl chitosan and nano-zinc oxide, the water-soluble chitosan solution and nano-zinc oxide are
  • the mixing ratio is that the mass ratio of N-carboxyethyl chitosan and zinc oxide is 100/5, 100/8 or 100/10.
  • the colloidal gold antibody buffer in step (c) of the present invention includes: a 20mM Tris-HCL solution, a sucrose concentration of 5%, a trehalose concentration of 1%, a proclin concentration of 1%, and a BSA concentration of 1%. , pH is 8.5.
  • step (d) of the present invention the polydopamine treatment solution pretreats the nitrocellulose membrane: the nitrocellulose membrane is soaked in the polydopamine treatment solution for 1 hour, and oscillated slowly, and then washed 3 times with distilled water after taking it out. Dry in a vacuum drying oven.
  • the polydopamine treatment solution has a polydopamine concentration of 1% and is filtered through a 0.22 ⁇ m filter membrane for later use.
  • the sample pad treatment liquid used in the pretreated sample pad in step (e) of the present invention includes a concentration of 0.1 mol/LTris-HCL liquid, a concentration of 1% bovine serum albumin BSA, and a concentration of 1% surfactant. .
  • Dopamine (DA) has also been proven to have super adhesive properties due to its combination of catechol functional groups and amino functional groups of lysine.
  • DA can oxidize and self-polymerize in alkaline solutions, forming a super-viscous polydopamine (PDA) layer on the surface of the material, thereby achieving super-strong adhesion on the surface of various materials.
  • the formation process of PDA is simple and does not require organic solvents.
  • the material only needs to be immersed in the alkaline Tris-Hcl buffer containing DA or other alkaline solutions to form a PDA layer on the surface. Therefore, PDA is widely used on the surface of materials. modified.
  • polydopamine can improve the coating efficiency of NC membrane proteins from both charge and hydrophobic effects.
  • Chitin is the most abundant renewable resource on earth after cellulose.
  • Chitosan is its deacetyl derivative, which is connected by 2-amino2-deoxy- ⁇ -D-glucose through ⁇ -1,4-glycosidic bonds. polymer formed.
  • Chitosan has become a popular tissue engineering scaffold material due to its good biocompatibility, biodegradability, broad-spectrum antibacterial properties, antiseptic properties, non-antigenicity, and special functions such as stopping bleeding and promoting wound healing.
  • Nano zinc oxide (ZnO) white hexagonal crystal or spherical particles, with a particle size less than 100nm, an average particle size of 50nm, and a specific surface area greater than 4m 2 /g.
  • the detection device of the present invention has a novel structure, and is coated with influenza virus A antigen, influenza virus B antigen, rhinovirus antigen, parainfluenza virus antigen, respiratory syncytial virus antigen, adenovirus antigen, Mycoplasma pneumoniae antigen, and Chlamydia pneumoniae antigen.
  • nitrocellulose membrane On nitrocellulose membrane, it has strong specificity and can simultaneously detect IgM antibodies of influenza virus type A, influenza virus type B, rhinovirus, parainfluenza virus, respiratory syncytial virus, and adenovirus in specimens, or influenza virus type A, IgM antibodies for influenza virus type B, rhinovirus, parainfluenza virus, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae without increasing the complexity of production operations.
  • the present invention pretreats the nitrocellulose membrane with polydopamine and modifies the antibody coating the nitrocellulose membrane, thereby increasing the coating efficiency of the antibody, improving the adsorption effect, and making the protein distribution more uniform, thus improving the efficiency of immunochromatography.
  • the detection device of the present invention does not require any special equipment and has low detection cost.
  • the detection device of the present invention is easy to operate and does not require professional operation. Highly practical.
  • FIG. 1 is a schematic structural diagram of the present invention.
  • the detection lines T1, T2, T3, T4, T5 and T6 are respectively arranged horizontally on six lines;
  • Figure 2 is a cross-sectional view along line A-A of Figure 1;
  • Figure 3 is a B-B cross-sectional view of Figure 1;
  • Figure 4 is a C-C cross-sectional view of Figure 1;
  • Figure 5 is a D-D cross-sectional view of Figure 1;
  • Figure 6 is a cross-sectional view along line E-E of Figure 1;
  • Figure 7 is a cross-sectional view along line F-F of Figure 1;
  • FIG. 8 is a schematic structural diagram of the present invention in which the detection lines T1, T2, T3, T4, T5, T6, T7 and T8 are respectively arranged horizontally on eight lines;
  • Figure 9 is a G-G cross-sectional view of Figure 8.
  • Fig. 10 is a cross-sectional view taken along line H-H in Fig. 8 .
  • the sample pad 1, the immune colloidal gold antibody glass fiber membrane 2, the immune nitrocellulose membrane 3, and the absorption pad 4 are respectively pasted on the plastic plate 5.
  • the two ends of the immune nitrocellulose membrane 3 The other end of the immune colloidal gold antibody glass fiber membrane 2 overlaps with the sample pad 1;
  • the immune nitrocellulose membrane 3 is provided with a first detection Line T1, second detection line T2, third detection line T3, fourth detection line T4, fifth detection line T5, sixth detection line T6 and quality control line C, there is influenza in the solid phase on the first detection line T1 Virus A antigen
  • the second detection line T2 has influenza virus B antigen on the solid phase
  • the third detection line T3 has rhinovirus antigen on the solid phase
  • the fourth detection line T4 has parainfluenza virus antigen on the solid phase
  • the fifth detection line T5 There is respiratory syncytial virus antigen on the upper solid phase, there is adenovirus antigen on the solid phase on the sixth detection line T6, and sheep anti-mouse IgG polyclon
  • the first immune nitrocellulose membrane is set on The detection line T1, the second detection line T2, the third detection line T3, the fourth detection line T4, the fifth detection line T5 and the sixth detection line T6 are respectively set on six lines, and quality control is set on each line. Line C.
  • the immune nitrocellulose membrane 3 is also provided with a seventh detection line T7 and an eighth detection line T8.
  • the seventh detection line T7 has a solid phase of Mycoplasma pneumoniae antigen
  • the eighth detection line T7 There is Chlamydia pneumoniae antigen on the solid phase on line T8, and quality control line C is set on each of the two lines.
  • N-carboxyethyl chitosan 1) Synthesis of N-carboxyethyl chitosan: Add 4.0g chitosan to 200mL water containing 2.80mL acrylic acid, stir continuously for 2 days at 50°C, after the reaction is completed, add 1mol to the reaction mixture /L NaOH solution, adjust the pH value of the solution to 10, so that all carboxylic acids are converted into sodium salts, pour the mixed solution into acetone for reprecipitation, and then dialyze in the aqueous solution.
  • the molecular weight intercept of the dialysis membrane is 8000g. /mol, remove unreacted acrylic acid, dialyze for two days, and freeze-dry to obtain N-carboxyethyl Chitosan 3.6g, yield 90%;
  • N-carboxyethyl chitosan and nano-zinc oxide mixed solution Add 5.00g N-carboxyethyl chitosan to 300 mL deionized water, stir vigorously at room temperature for 0.5h, and fully dissolve to obtain a water-soluble shell.
  • the mass ratio of N-carboxyethyl chitosan to zinc oxide is 100/5, and the mixture is stirred vigorously for 3 hours to make it evenly mixed;
  • Zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody take 1mL of zinc oxide-chitosan-colloidal gold suspension, disperse it evenly by ultrasonic, then add the antibody dropwise while stirring, after adding the antibody, After about 1 minute of reaction, ultrasonic for 30 seconds, then react for another 4 hours, add BSA for blocking for 24 hours, and then centrifuge at 12000r/min for 20 minutes. Keep the precipitate after centrifugation for later use;
  • step (c) Use colloidal gold antibody buffer to dilute the precipitated zinc oxide-chitosan-colloidal gold antibody after centrifugation in step (b), and disperse it evenly to obtain immune colloidal gold antibody solutions. Spray on the glass fiber mat to prepare immune colloidal gold antibody glass fiber membrane 2;
  • the colloidal gold antibody buffer includes: a concentration of 20mM Tris-HCL solution, a sucrose concentration of 5%, a trehalose concentration of 1%, a proclin concentration of 1%, a BSA concentration of 1%, and a pH of 8.5;
  • the sample pad treatment liquid used in the pre-processed sample pad includes a concentration of 0.1mol/L Tris-HCL solution and a concentration of 1% bovine serum. Albumin BSA, concentration 1% surfactant.
  • N-carboxyethyl chitosan 1) Synthesis of N-carboxyethyl chitosan: Add 4.0g chitosan to 200mL water containing 2.80mL acrylic acid, stir continuously for 2 days at 50°C, after the reaction is completed, add 1mol to the reaction mixture /L NaOH solution, adjust the pH value of the solution to 11, so that all carboxylic acids are converted into sodium salts, pour the mixed solution into acetone for reprecipitation, and then dialyze in the aqueous solution.
  • the molecular weight intercept of the dialysis membrane is 10000g. /mol, remove unreacted acrylic acid, dialyze for two days, and freeze-dry to obtain N-carboxyethyl chitosan;
  • Zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody take 1mL of zinc oxide-chitosan-colloidal gold suspension, disperse it evenly by ultrasonic, then add the antibody dropwise while stirring, after adding the antibody, After about 1 minute of reaction, ultrasonic for 30 seconds, then react for another 4 hours, add BSA for blocking for 24 hours, and then centrifuge at 12000r/min for 20 minutes. Keep the precipitate after centrifugation for later use;
  • step (c) Use colloidal gold antibody buffer to dilute the precipitated zinc oxide-chitosan-colloidal gold antibody after centrifugation in step (b), and disperse it evenly to obtain immune colloidal gold antibody solutions. Spray on the glass fiber mat to prepare immune colloidal gold antibody glass fiber membrane 2;
  • the colloidal gold antibody buffer includes: a concentration of 20mM Tris-HCL solution, a sucrose concentration of 5%, a trehalose concentration of 1%, a proclin concentration of 1%, a BSA concentration of 1%, and a pH of 8.5;
  • the sample pad treatment liquid used in the pre-processed sample pad includes a concentration of 0.1mol/L Tris-HCL solution and a concentration of 1% bovine serum. Albumin BSA, concentration 1% surfactant.
  • N-carboxyethyl chitosan 1) Synthesis of N-carboxyethyl chitosan: Add 4.0g chitosan to 200mL water containing 2.80mL acrylic acid, stir continuously for 2 days at 50°C, after the reaction is completed, add 1mol to the reaction mixture /L NaOH solution, adjust the pH value of the solution to 12, so that all carboxylic acids are converted into sodium salts, pour the mixed solution into acetone for reprecipitation, and then dialyze in the aqueous solution.
  • the molecular weight intercept of the dialysis membrane is 12000g. /mol, remove unreacted acrylic acid, dialyze for two days, and freeze-dry to obtain N-carboxyethyl chitosan;
  • Zinc oxide-chitosan-colloidal gold cross-linked anti-human IgM antibody take 1mL of zinc oxide-chitosan-colloidal gold suspension, disperse it evenly by ultrasonic, then add the antibody dropwise while stirring, after adding the antibody, After about 1 minute of reaction, ultrasonic for 30 seconds, then react for another 4 hours, add BSA for blocking for 24 hours, and then centrifuge at 12000r/min for 20 minutes. Keep the precipitate after centrifugation for later use;
  • step (c) Use colloidal gold antibody buffer to dilute the precipitated zinc oxide-chitosan-colloidal gold antibody after centrifugation in step (b), and disperse it evenly to obtain immune colloidal gold antibody solutions. Spray on the glass fiber mat to prepare immune colloidal gold antibody glass fiber membrane 2;
  • the colloidal gold antibody buffer includes: a concentration of 20mM Tris-HCL solution, a sucrose concentration of 5%, a trehalose concentration of 1%, a proclin concentration of 1%, a BSA concentration of 1%, and a pH of 8.5;
  • the sample pad treatment liquid used in the pre-processed sample pad includes a concentration of 0.1mol/L Tris-HCL solution and a concentration of 1% bovine serum. Albumin BSA, concentration 1% surfactant.
  • Polydopamine is prepared from distilled water to a concentration of 1%, filtered through a 0.22 ⁇ m filter membrane, and set aside.
  • Untreated and treated nitrocellulose membranes were respectively used to prepare influenza virus type A, influenza virus type B, rhinovirus, parainfluenza virus, respiratory syncytial virus, adenovirus, Mycoplasma pneumoniae, and Chlamydia pneumoniae according to the process flow of the above embodiments.
  • IgM joint detection test paper the test process refers to the test paper instructions, and compares the difference in adsorption capacity and stability index between untreated and treated nitrocellulose membranes.
  • zinc oxide-chitosan-colloidal gold as a carrier, add 200 ⁇ L zinc oxide-chitosan-colloidal gold nanoparticles (1 mg/mL) solution and 25 ⁇ anti-human IgM antibody (40 ⁇ mol/L) solution into ultrapure water. , making the final reaction system 1mL. After thorough mixing, set the shaking table temperature to 25°C and the rotation speed to 200 r/m. The above mixed solution was shaken and cultured in the shaking table in the dark for 2 hours. After the reaction, the mixed solution was thoroughly centrifuged and washed 4 times with ultrapure water at 13000 r/m in an ultracentrifuge to remove excess unreacted antibodies in the supernatant. The resulting precipitate is zinc oxide-chitosan-colloidal gold-anti-human IgM antibody probe complex, which is diluted to 1 mL with ultrapure water and stored at 4°C.
  • the anti-human IgM antibody modified with zinc oxide-chitosan-colloidal gold nanoparticles and the unmodified anti-human IgM antibody were respectively prepared according to the process flow of the above embodiments.
  • the testing process refers to the test paper instructions to compare zinc oxide-chitosan. -The difference in protein adsorption capacity and stability index between colloidal gold nanoparticles treated and untreated.
  • the overall number of consistent cases for the test reagent was 67, with an overall coincidence rate of 100%.
  • the results show that there is no statistically significant difference between the test results of the test reagent and the control reagent between the groups, and the consistency is good.
  • the results are shown in Tables 7 and 8;
  • the number of negative consistent cases was 155, with a coincidence rate of 97.48%.
  • the overall number of consistent cases for the test reagents was 328, with an overall coincidence rate of 98.20%.
  • the results show that there is no statistically significant difference between the test results of the test reagent and the control reagent between the groups, and the consistency is good. The results are shown in Tables 9 and 10;
  • the number of negative consistent cases was 17, with a coincidence rate of 100%.
  • the overall number of consistent cases for the test reagent was 67, and the overall coincidence rate was 100%.
  • the results show that there is no statistically significant difference between the test results of the test reagent and the control reagent between the groups, and the consistency is good. The results are shown in Tables 11 and 12;
  • the number of positive consistent cases for the test reagents was 192, with a coincidence rate of 100%.
  • the number of negative consistent cases was 142, with a coincidence rate of 100%.
  • the overall number of consistent cases for the test reagents was 334, and the overall coincidence rate was 100%.
  • the results show that there is no statistically significant difference between the test results of the test reagent and the control reagent between the groups, and the consistency is good. The results are shown in Tables 13 and 14;
  • the number of negative consistent cases was 28, with a coincidence rate of 100%.
  • the overall number of consistent cases for the test reagents was 67, with an overall coincidence rate of 100%.
  • the results showed that there was no statistically significant difference between the test results of the test reagent and the control reagent, and the consistency was good. The results are shown in Tables 15 and 16;
  • the number of negative consistent cases was 52, with a coincidence rate of 100%.
  • the overall number of consistent cases for the test reagent was 67, and the overall coincidence rate was 100%.
  • the results show that there is no statistically significant difference between the test results of the test reagent and the control reagent between the groups, and the consistency is good. The results are shown in Tables 17 and 18;
  • the number of negative consistent cases was 297, with a coincidence rate of 99.33%.
  • the overall number of consistent cases for the test reagent was 331, with an overall coincidence rate of 99.10%. It means that the difference between the test results of the test reagent and the control reagent is not statistically significant and the consistency is good.
  • the results are shown in Tables 19 and 20;
  • the multiple pathogen joint detection device of the present invention can solve the problems of insufficient adsorbed protein and weak binding force of NC membrane in the existing technology, and can quickly and accurately detect the condition of respiratory tract infections. Risk judgment.

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Abstract

本发明属于医疗检测装置领域，尤其涉及一种多项病原体联合检测装置及其制备方法。所述多项病原体联合检测装置的样品垫、免疫胶体金抗体玻璃纤维膜、免疫硝酸纤维素膜、吸收垫分别粘贴在塑料板上，所述硝酸纤维素膜的两端分别与吸收垫、免疫胶体金抗体玻璃纤维膜搭接，所述免疫胶体金抗体玻璃纤维膜的另一端与样品垫搭接，利用胶体金免疫层析技术以及捕获法原理制备全血、血清、血浆标本中人流感病毒A型、流感病毒B型、鼻病毒、副流感病毒、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体的IgM抗体的联合检测装置，提高了免疫层析方法学的灵敏度、精准度和稳定性，操作简便，实用性强，可实现呼吸道感染性疾病的灵敏、特异、全面快速检测。

Description

一种多项病原体联合检测装置及其制备方法
本申请要求于2022年04月17日提交中国专利局、申请号为CN202210402586.9、发明名称为“一种多项病原体联合检测装置及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于医疗检测装置领域，特别涉及一种多项病原体联合检测装置及其制备方法。
背景技术
流行性感冒是由流感病毒感染引起的一种急性呼吸道传染病，经呼吸道迅速传播，潜伏期短，季节性明显，极易造成大范围暴发流行，控制难度大。流感病毒分为甲(A)、乙(B)、丙(C)3型，其中甲型流感病毒、乙型流感病毒感染较常见，传染性强，传播迅速，对人类健康产生严重的威胁。由于流感病毒传播速度快，且病毒容易发生变异，每年都会发生不同规模的流行，甚至每隔10～15年发生一次全球大流行。流感临床主要表现为发热、头痛、肌痛和全身不适，可伴咳嗽、咳痰等呼吸系统症状，也可伴呕吐、腹泻等消化系统症状。儿童、孕产妇、老年人及患有各种慢性病或体质虚弱者患流感后易出现严重并发症或加重原有的慢性病，甚至引起相应器官功能衰竭而危及生命。因此，了解流感的特征有利于尽早识别、及时治疗。
流感病毒的变异以甲型最为重要，常与世界性大流行有密切联系。一般来讲，流感病毒的抗原性变异就是指H和N抗原结构的改变，在亚型内部经常发生小变异(量变)，称为抗原漂移。抗原变异仅发生于甲型病毒。它可能是由于同一细胞感染了人类和动物的2种病毒，病毒之间发生基因重配而产生的。由此产生的病毒血凝素和神经氨酸酶发生全新结合，而使得人群没有免疫力。抗原转变是造成流感全球大流行的原因。一般新旧亚型之间有明显的交替现象，在新的亚型出现并流行到一个地区后，旧的亚型就不再能分离到。乙型流感染毒间同样有大变异与小变异，但未划分成亚型转变。丙型流感病毒尚未发现抗原变异。流感病毒A型和B型、HRV、HPIV、RSV、HAdV、MP、CP临床治疗方案有所差异，因此需要进行鉴别诊断出究竟是哪种致病因素，然后针对性治疗；因此流感病毒A型和B型、HRV、HPIV、RSV、HAdV、MP、CP的IgM抗体联合定性检测项目能够及时、便捷、精准、全面检测出感染了哪种病毒，给临床诊疗带来极大收益，也给各级医疗机构、疾控中心等场景提供一个有力的、全面的、精准的筛查和诊断工具。其次其检测样本为全血或者血清、血浆，取材方便安全；流感病毒A型和B型、HRV、HPIV、RSV、HAdV、MP、CP的IgM抗体为现症感染指标，不存在假阳性问题，因此更加精准，对临床诊断价值非常大。
病毒的确认一般采用鸡胚或MDCK细胞分离培养和血凝抑制法，操作繁琐且费时费力，难以满足快速诊断需要。随着快速诊断技术的发展和应用，目前市场上流感及2019鼻病毒产品类型基本可以分为3大类：核酸诊断类(PCR法)、酶联免疫吸附法(ELISA法)和胶体金免疫层析法。PCR法准确度最高，是确诊的首选方法，但检测成本较高，检测时间较长；ELISA法是医院检验科和疾控系统实验室的经典方法；胶体金法速度最快、技术成熟稳、简便易行，适合于各级医疗机构、疾控中心等场合的普遍筛查和诊断。
胶体金免疫层析技术(goldimmunochromatography assay，GICA)是一种将胶体金标记技术和蛋白质层析技术结合的以微孔滤膜为载体的固相膜免疫分析技术。胶体金免疫层析技术是一种常用的免疫层析检测方法，由于其操作简单、省时、制造成本较低、结果易判读等特点，非常适合于现场检测，广泛用于生物、医药、食品等领域。由于胶体金免疫层析技术是一步完成检测，因此检测过程的干扰因素较多，其灵敏度低是限制胶体金免疫层析应用范围的主要因素，传统的胶体金免疫层析技术的检测限高于ELISA等方法。
在胶体金免疫层析检测中，蛋白质固着于硝酸纤维素膜(NC膜)作为待测样本的捕获试剂。由于检测结果完全取决于捕获试剂在膜上达到良好的吸附效果，因此蛋白质在膜上均一、良好的吸附对胶体金检测结果非常重要。如果NC膜上结合的蛋白量不足或者蛋白结合力不够强，就会出现相当多的问题，在检测结果的检测线上非常明显。如果膜上结合的蛋白量太低，那么在结果中检测线 显色较弱而且检测灵敏度降低。如果蛋白不能牢固的吸附于NC膜，那么在蛋白吸附于NC膜以前发生扩散，从而导致检测线较宽、显色较弱而不是鲜艳而清晰，使检测结果难以解释。在极端条件下，如果蛋白与NC膜的物理吸附作用太弱，流过的蛋白检测物和表面活性剂溶液可能将固着的蛋白从NC膜上洗掉，从而显示较宽或者根本不清晰的检测线，难以解释检测结果。
发明内容
本发明提供一种多项病原体联合检测装置及其制备方法，以解决现有技术存在的NC膜吸附蛋白量不足、结合力不强的问题，本发明制备的流感病毒A型和B型、HRV、HPIV、RSV、HAdV的IgM抗体六合一，或流感病毒A型和B型、HRV、HPIV、RSV、HAdV、MP、CP的IgM抗体八合一定性联合检测装置，提高了对呼吸道疾病患者进行合理综合、全面判定，能够快速、准确进行呼吸道感染疾病的病情风险判断。
本发明提供了一种多项病原体联合检测装置，样品垫、免疫胶体金抗体玻璃纤维膜、免疫硝酸纤维素膜、吸收垫分别粘贴在塑料板上，所述硝酸纤维素膜的两端分别与吸收垫、免疫胶体金抗体玻璃纤维膜搭接，所述免疫胶体金抗体玻璃纤维膜的另一端与样品垫搭接；所述免疫硝酸纤维素膜上设置第一检测线T1、第二检测线T2、第三检测线T3、第四检测线T4、第五检测线T5、第六检测线T6和质控线C，所述第一检测线T1上固相有流感病毒A抗原，第二检测线T2上固相有流感病毒B抗原，第三检测线T3上固相有鼻病毒抗原，第四检测线T4上固相有副流感病毒抗原，第五检测线T5上固相有呼吸道合胞病毒抗原，第六检测线T6上固相有腺病毒抗原，质控线C上喷点羊抗鼠IgG多克隆抗体，所述免疫硝酸纤维素膜上设置的第一检测线T1、第二检测线T2、第三检测线T3、第四检测线T4、第五检测线T5和第六检测线T6分别设置在六条线上，并在每条线上分别设置质控线C。
本发明所述所述免疫硝酸纤维素膜上还设置第七检测线T7和第八检测线T8，所述的第七检测线T7上固相有肺炎支原体抗原，第八检测线T8上固相有肺炎衣原体抗原，并在两条线的每条线上分别设置质控线C。
本发明还提供了一种多项病原体联合检测装置的制备方法，包括下列步骤：
(a)氧化锌-壳聚糖-胶体金制备
1)N-羧乙基壳聚糖的合成：将4.0g壳聚糖加入到含有2.80mL丙烯酸的200mL水中，在50℃下，持续搅拌2天，反应完成后，在反应混合液中加入1mol/L的NaOH溶液，调节溶液的pH值至10-12，使其所有的羧酸全部转化为钠盐，将混合溶液倒入到丙酮中进行重沉淀，然后在水溶液中透析，除去未反应的丙烯酸，透析两天后，冷冻干燥得到N-羧乙基壳聚糖；
2)纳米氧化锌制备：称取1.3225g ZnAc2·2H2O溶解在20mL的蒸馏水中,缓慢滴加9mL 5mol/L的NaOH溶液，溶液先出现白色浑浊，继续滴加则浑浊完全消失，再加入11mL的蒸馏水，搅拌均匀，然后倒入低压反应釜中，将反应釜放入烘箱中，在170℃下加热12个小时，自然冷却至室温后，得到沉淀后，分别用水和乙醇清洗、离心两次，将沉淀放入烘箱内于60℃烘干备用；
3)N-羧乙基壳聚糖与纳米氧化锌混合溶液的制备：将5.00g N-羧乙基壳聚糖加入到300mL去离子水中，在室温下剧烈搅拌0.5h，充分溶解得到水溶性壳聚糖溶液，将上述水溶性壳聚糖溶液和纳米氧化锌按一定的比例混合，将混合液剧烈搅拌3h，使其混合均匀；
4)胶体金的制备：
将装有90-100mL纯化水的圆底烧瓶放入电热套加热，加热10-12分钟后，待烧瓶中的水沸腾时开启转数控制开关，调至400rpm，并用注射器缓慢将1mL 1％氯化金溶液沿漩涡边缘加入，待溶液开始沸腾后，快速加入1％柠檬酸三钠溶液1-2mL，待溶液冷却到室温后，加水称重补足重量；
5)氧化锌-壳聚糖-胶体金制备：以苯二甲醛作为交联剂，在常温下进行交联；交联结束，将所得的氧化锌-壳聚糖-胶体金洗涤，真空干燥24h；
(b)氧化锌-壳聚糖-胶体金交联记抗人IgM抗体，取1mL氧化锌-壳聚糖-胶体金悬浮液，超声分散均匀，然后边搅拌边滴加抗体，加入抗体后，大约反应1min后，再到超声波上超声30秒，然后再反应4小时，加BSA进行封闭24小时，封闭好后进行离心，速度为12000r/min，20min，保留离心后的沉淀，待用；
(c)采用胶体金抗体缓冲液分别稀释步骤(b)离心后的沉淀氧化锌-壳聚糖-胶体金抗体，使其分散 均匀，分别获得免疫胶体金抗体溶液，分别用免疫胶体金抗体溶液喷涂于玻璃纤维垫，制得免疫胶体金抗体玻璃纤维膜；
(d)将硝酸纤维素膜用聚多巴胺处理液预处理后，喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原作为检测线，或喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原、肺炎支原体抗原、肺炎衣原体抗原作为检测线，喷点羊抗鼠IgG抗体作为质控线，制得免疫硝酸纤维素膜；
(e)将预处理的样品垫、步骤(c)制备的免疫胶体金抗体玻璃纤维膜、步骤(d)制备的免疫硝酸纤维素膜、吸水纸依次粘贴在塑料板上，切裁制得检测试剂条，最后将检测试剂条装入塑料外壳。
本发明所述步骤(a)氧化锌-壳聚糖-胶体金制备中，1)N-羧乙基壳聚糖的合成，在水溶液中透析时，采用的透析膜截取分子量为8000-12000g/mol。
本发明所述步骤(a)氧化锌-壳聚糖-胶体金制备中，2)纳米氧化锌颗粒制备，缓慢滴加9mL5mol/L的NaOH溶液，该NaOH溶液为新配制的溶液。
所述步骤(a)氧化锌-壳聚糖-胶体金制备中，3)N-羧乙基壳聚糖与纳米氧化锌混合溶液的制备，所述水溶性壳聚糖溶液和纳米氧化锌的混合比例是，N-羧乙基壳聚糖与氧化锌的质量之比为100/5、100/8或100/10。
本发明所述步骤(c)所述的胶体金抗体缓冲液包括：浓度为20mM Tris-HCL液、蔗糖浓度为5％、海藻糖浓度为1％、proclin浓度为1％，BSA浓度为1％，pH为8.5。
本发明所述步骤(d)中聚多巴胺处理液预处理硝酸纤维素膜：将硝酸纤维素膜放入聚多巴胺处理液中浸泡1h，并慢速振荡摇晃，取出后用蒸馏水清洗3遍，最后在真空干燥箱中干燥。
所述的聚多巴胺处理液，聚多巴胺浓度为1％，经0.22μm滤膜过滤，备用。
本发明所述步骤(e)所述的预处理的样品垫采用的样品垫处理液包括浓度为0.1mol/LTris-HCL液、浓度为1％牛血清白蛋白BSA、浓度为1％表面活性剂。
多巴胺(dopamine,DA)因其结合儿茶酚官能团和赖氨酸的氨基官能团，也被证明具有超强的黏附性能。DA在碱性溶液中可发生氧化自聚合，在材料表面形成具有超强黏性的聚多巴胺(PDA)层，从而实现在各种材料表面的超强黏附。PDA形成过程简单且不需要有机溶剂，仅需将材料浸入含DA的碱性Tris-Hcl缓冲液或其他碱性溶液中，即可在表面形成PDA层，因此，PDA被广泛应用于材料的表面改性。综上，聚多巴胺可以从电荷作用和疏水作用两方面来提高NC膜蛋白的包被效率。
甲壳素是地球上储量仅次于纤维素的可再生资源，壳聚糖是其脱乙酰基衍生物，由2-氨基2-脱氧-β-D-葡萄糖通过β-1，4-糖苷键连接形成的聚合物。由于壳聚糖具有良好的生物相容性、生物可降解性、广谱抗菌性、防腐性、无抗原性，以及可止血和促进伤口愈合等特殊功能，使得壳聚糖成为组织工程支架材料中最有应用前景的生物大分子之一。纳米氧化锌(ZnO)，白色六方晶系结晶或球形粒子，粒径小于100nm，平均粒径50nm，比表面积大于4m2/g。具有极高的化学活性及优异的催化性和光催化活性。本研究探讨了氧化锌-壳聚糖-胶体金纳米颗粒对NC膜包被抗体的影响，先将划膜用的抗体先与氧化锌-壳聚糖-胶体金纳米颗粒结合，封闭，离心纯化，去掉未结合的抗体，然后复溶到一定比例，然后划膜，这样一个氧化锌-壳聚糖-胶体金粒子可以结合多个抗体，从而增加了包被抗体效率，借助其更大的表面积增加抗体的包被效率，提高胶体金免疫层析实验的灵敏度。
为了提高胶体金免疫层析技术的灵敏度、稳定性和精准度，我们通过对硝酸纤维素膜用聚多巴胺处理液进行了预处理，将包被NC的抗体与氧化锌-壳聚糖-胶体金结合，达到了提高试纸各种性能指标的目的。
本发明的有益效果在于：
1、本发明的检测装置结构新颖，将流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原、肺炎支原体抗原、肺炎衣原体抗原包被于硝酸纤维膜膜上，特异性强，既能同时检测标本中流感病毒A型、流感病毒B型、鼻病毒、副流感病毒、呼吸道合胞病毒、腺病毒的IgM抗体，或流感病毒A型、流感病毒B型、鼻病毒、副流感病毒、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体的IgM抗体，又没有增加生产操作的复杂度。
2、免疫胶体金制备步骤中，通过配合合适的喷金缓冲液和样品垫处理液，可保证免疫胶体金释放完全，同时这样的结合方式，会使标记物与抗体牢牢吸附在一起，从而抗体或抗原不会从标记物表面脱落，提高标记效率及免疫层析方法学的稳定性、灵敏度和精准度；且同样的阈值下，还可降低胶体金的用量，节约成本。
3、本发明对硝酸纤维素膜进行聚多巴胺预处理，对包被硝酸纤维素膜的抗体进行了修饰，增加抗体的包被效率、提高吸附效果、蛋白分布更加均一，从而提高了免疫层析方法学的灵敏度、精准度和稳定性。
4、本发明的检测装置不需要任何特殊仪器设备，检测成本低。
5、本发明的检测装置操作简便，不需要专业人员操作。实用性强。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1是本发明的结构示意图，图中检测线T1、T2、T3、T4、T5和T6分别横向排列在六条线上；
图2是图1的A-A剖视图；
图3是图1的B-B剖视图；
图4是图1的C-C剖视图；
图5是图1的D-D剖视图；
图6是图1的E-E剖视图；
图7是图1的F-F剖视图；
图8是本发明检测线T1、T2、T3、T4、T5、T6、T7和T8分别横向排列在八条线上的结构示意图；
图9是图8的G-G剖视图；
图10是图8的H-H剖视图。
具体实施方式
见图1～7所示，样品垫1、免疫胶体金抗体玻璃纤维膜2、免疫硝酸纤维素膜3、吸收垫4分别粘贴在塑料板5上，所述免疫硝酸纤维素膜3的两端分别与吸收垫4、免疫胶体金抗体玻璃纤维膜2搭接，所述免疫胶体金抗体玻璃纤维膜2的另一端与样品垫1搭接；所述免疫硝酸纤维素膜3上设置第一检测线T1、第二检测线T2、第三检测线T3、第四检测线T4、第五检测线T5、第六检测线T6和质控线C，所述第一检测线T1上固相有流感病毒A抗原，第二检测线T2上固相有流感病毒B抗原，第三检测线T3上固相有鼻病毒抗原，第四检测线T4上固相有副流感病毒抗原，第五检测线T5上固相有呼吸道合胞病毒抗原，第六检测线T6上固相有腺病毒抗原，质控线C上喷点羊抗鼠IgG多克隆抗体，所述免疫硝酸纤维素膜上设置的第一检测线T1、第二检测线T2、第三检测线T3、第四检测线T4、第五检测线T5和第六检测线T6分别设置在六条线上，并在每条线上分别设置质控线C。
如图8～10所示，所述免疫硝酸纤维素膜3上还设置第七检测线T7和第八检测线T8，所述的第七检测线T7上固相有肺炎支原体抗原，第八检测线T8上固相有肺炎衣原体抗原，并在两条线的每条线上分别设置质控线C。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的技术方案进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
包括下列步骤：
(a)氧化锌-壳聚糖-胶体金制备
1)N-羧乙基壳聚糖的合成：将4.0g壳聚糖加入到含有2.80mL丙烯酸的200mL水中，在50℃下，持续搅拌2天，反应完成后，在反应混合液中加入1mol/L的NaOH溶液，调节溶液的pH值至10，使其所有的羧酸全部转化为钠盐，将混合溶液倒入到丙酮中进行重沉淀，然后在水溶液中透析，透析膜截取分子量为8000g/mol，除去未反应的丙烯酸，透析两天后，冷冻干燥得到N-羧乙基 壳聚糖3.6g，产率90％；
2)纳米氧化锌制备：称取1.3225g ZnAc2·2H2O溶解在20mL的蒸馏水中,缓慢滴加9mL新配制的5mol/L的NaOH溶液，溶液先出现白色浑浊，继续滴加则浑浊完全消失，再加入11mL的蒸馏水，搅拌均匀，然后倒入低压反应釜中，将反应釜放入烘箱中，在170℃下加热12个小时，自然冷却至室温后，得到沉淀后，分别用水和乙醇清洗、离心两次，将沉淀放入烘箱内于60℃烘干备用；
3)N-羧乙基壳聚糖与纳米氧化锌混合溶液的制备：将5.00gN-羧乙基壳聚糖加入到300mL去离子水中，在室温下剧烈搅拌0.5h，充分溶解得到水溶性壳聚糖溶液，将N-羧乙基壳聚糖与氧化锌的质量之比为100/5，将混合液剧烈搅拌3h，使其混合均匀；
4)胶体金的制备：
将装有90mL纯化水的圆底烧瓶放入电热套加热，加热10分钟后，待烧瓶中的水沸腾时开启转数控制开关，调至400rpm，并用注射器缓慢将1mL 1％氯化金溶液沿漩涡边缘加入，待溶液开始沸腾后，快速加入1％柠檬酸三钠溶液1mL，待溶液冷却到室温后，加水称重补足重量；
5)氧化锌-壳聚糖-胶体金制备：以苯二甲醛作为交联剂，在常温下进行交联；交联结束，将所得的氧化锌-壳聚糖-胶体金洗涤，真空干燥24h；
(b)氧化锌-壳聚糖-胶体金交联记抗人IgM抗体，取1mL氧化锌-壳聚糖-胶体金悬浮液，超声分散均匀，然后边搅拌边滴加抗体，加入抗体后，大约反应1min后，再到超声波上超声30秒，然后再反应4小时，加BSA进行封闭24小时，封闭好后进行离心，速度为12000r/min，20min，保留离心后的沉淀，待用；
(c)采用胶体金抗体缓冲液分别稀释步骤(b)离心后的沉淀氧化锌-壳聚糖-胶体金抗体，使其分散均匀，分别获得免疫胶体金抗体溶液，分别用免疫胶体金抗体溶液喷涂于玻璃纤维垫，制得免疫胶体金抗体玻璃纤维膜2；
所述的胶体金抗体缓冲液包括：浓度为20mM Tris-HCL液、蔗糖浓度为5％、海藻糖浓度为1％、proclin浓度为1％，BSA浓度为1％，pH为8.5；
(d)将硝酸纤维素膜用聚多巴胺处理液预处理后，喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原作为检测线，或喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原、肺炎支原体抗原、肺炎衣原体抗原作为检测线，喷点羊抗鼠IgG抗体作为质控线，制得免疫硝酸纤维素膜3；其中所述的聚多巴胺处理液，聚多巴胺浓度为1％，经0.22μm滤膜过滤，备用，将硝酸纤维素膜放入聚多巴胺处理液中浸泡1h，并慢速振荡摇晃，取出后用蒸馏水清洗3遍，最后在真空干燥箱中干燥；
(e)将预处理的样品垫1、步骤(c)制备的免疫胶体金抗体玻璃纤维膜2、步骤(d)制备的免疫硝酸纤维素膜3、吸水纸4依次粘贴在塑料板5上，切裁制得检测试剂条，最后将检测试剂条装入塑料外壳；所述的预处理的样品垫采用的样品垫处理液包括浓度为0.1mol/L Tris-HCL液、浓度为1％牛血清白蛋白BSA、浓度为1％表面活性剂。
实施例2
包括下列步骤：
(a)氧化锌-壳聚糖-胶体金制备
1)N-羧乙基壳聚糖的合成：将4.0g壳聚糖加入到含有2.80mL丙烯酸的200mL水中，在50℃下，持续搅拌2天，反应完成后，在反应混合液中加入1mol/L的NaOH溶液，调节溶液的pH值至11，使其所有的羧酸全部转化为钠盐，将混合溶液倒入到丙酮中进行重沉淀，然后在水溶液中透析，透析膜截取分子量为10000g/mol，除去未反应的丙烯酸，透析两天后，冷冻干燥得到N-羧乙基壳聚糖；
2)纳米氧化锌制备：称取1.3225g ZnAc2·2H2O溶解在20mL的蒸馏水中,缓慢滴加9mL新配制的5mol/L的NaOH溶液，溶液先出现白色浑浊，继续滴加则浑浊完全消失，再加入11mL的蒸馏水，搅拌均匀，然后倒入低压反应釜中，将反应釜放入烘箱中，在170℃下加热12个小时，自然冷却至室温后，得到沉淀后，分别用水和乙醇清洗、离心两次，将沉淀放入烘箱内于60℃烘干备用；
3)N-羧乙基壳聚糖与纳米氧化锌混合溶液的制备：将5.00g N-羧乙基壳聚糖加入到300mL去离子水中，在室温下剧烈搅拌0.5h，充分溶解得到水溶性壳聚糖溶液，将上述水溶性壳聚糖溶液和纳米氧化锌按一定的比例混合，使得N-羧乙基壳聚糖与氧化锌的质量之比为100/8，将混合液剧烈搅拌3h，使其混合均匀；
4)胶体金的制备：
将装有95mL纯化水的圆底烧瓶放入电热套加热，加热11分钟后，待烧瓶中的水沸腾时开启转数控制开关，调至400rpm，并用注射器缓慢将1mL 1％氯化金溶液沿漩涡边缘加入，待溶液开始沸腾后，快速加入1％柠檬酸三钠溶液1.5mL，待溶液冷却到室温后，加水称重补足重量；
5)氧化锌-壳聚糖-胶体金制备：以苯二甲醛作为交联剂，在常温下进行交联；交联结束，将所得的氧化锌-壳聚糖-胶体金洗涤，真空干燥24h；
(b)氧化锌-壳聚糖-胶体金交联记抗人IgM抗体，取1mL氧化锌-壳聚糖-胶体金悬浮液，超声分散均匀，然后边搅拌边滴加抗体，加入抗体后，大约反应1min后，再到超声波上超声30秒，然后再反应4小时，加BSA进行封闭24小时，封闭好后进行离心，速度为12000r/min，20min，保留离心后的沉淀，待用；
(c)采用胶体金抗体缓冲液分别稀释步骤(b)离心后的沉淀氧化锌-壳聚糖-胶体金抗体，使其分散均匀，分别获得免疫胶体金抗体溶液，分别用免疫胶体金抗体溶液喷涂于玻璃纤维垫，制得免疫胶体金抗体玻璃纤维膜2；
所述的胶体金抗体缓冲液包括：浓度为20mM Tris-HCL液、蔗糖浓度为5％、海藻糖浓度为1％、proclin浓度为1％，BSA浓度为1％，pH为8.5；
(d)将硝酸纤维素膜用聚多巴胺处理液预处理后，喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原作为检测线，或喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原、肺炎支原体抗原、肺炎衣原体抗原作为检测线，喷点羊抗鼠IgG抗体作为质控线，制得免疫硝酸纤维素膜3；其中所述的聚多巴胺处理液，聚多巴胺浓度为1％，经0.22μm滤膜过滤，备用，将硝酸纤维素膜放入聚多巴胺处理液中浸泡1h，并慢速振荡摇晃，取出后用蒸馏水清洗3遍，最后在真空干燥箱中干燥；
(e)将预处理的样品垫1、步骤(c)制备的免疫胶体金抗体玻璃纤维膜2、步骤(d)制备的免疫硝酸纤维素膜3、吸水纸4依次粘贴在塑料板5上，切裁制得检测试剂条，最后将检测试剂条装入塑料外壳；所述的预处理的样品垫采用的样品垫处理液包括浓度为0.1mol/L Tris-HCL液、浓度为1％牛血清白蛋白BSA、浓度为1％表面活性剂。
实施例3
包括下列步骤：
(a)氧化锌-壳聚糖-胶体金制备
1)N-羧乙基壳聚糖的合成：将4.0g壳聚糖加入到含有2.80mL丙烯酸的200mL水中，在50℃下，持续搅拌2天，反应完成后，在反应混合液中加入1mol/L的NaOH溶液，调节溶液的pH值至12，使其所有的羧酸全部转化为钠盐，将混合溶液倒入到丙酮中进行重沉淀，然后在水溶液中透析，透析膜截取分子量为12000g/mol，除去未反应的丙烯酸，透析两天后，冷冻干燥得到N-羧乙基壳聚糖；
2)纳米氧化锌制备：称取1.3225g ZnAc2·2H2O溶解在20mL的蒸馏水中,缓慢滴加9mL新配制的5mol/L的NaOH溶液，溶液先出现白色浑浊，继续滴加则浑浊完全消失，再加入11mL的蒸馏水，搅拌均匀，然后倒入低压反应釜中，将反应釜放入烘箱中，在170℃下加热12个小时，自然冷却至室温后，得到沉淀后，分别用水和乙醇清洗、离心两次，将沉淀放入烘箱内于60℃烘干备用；
3)N-羧乙基壳聚糖与纳米氧化锌混合溶液的制备：将5.00g N-羧乙基壳聚糖加入到300mL去离子水中，在室温下剧烈搅拌0.5h，充分溶解得到水溶性壳聚糖溶液，将上述水溶性壳聚糖溶液和纳米氧化锌按一定的比例混合，使得N-羧乙基壳聚糖与氧化锌的质量之比为100/10，将混合液剧烈搅拌3h，使其混合均匀；
4)胶体金的制备：
将装有100mL纯化水的圆底烧瓶放入电热套加热，加热12分钟后，待烧瓶中的水沸腾时开启转数控制开关，调至400rpm，并用注射器缓慢将1mL 1％氯化金溶液沿漩涡边缘加入，待溶液开始沸腾后，快速加入1％柠檬酸三钠溶液2mL，待溶液冷却到室温后，加水称重补足重量；
5)氧化锌-壳聚糖-胶体金制备：以苯二甲醛作为交联剂，在常温下进行交联；交联结束，将所得的氧化锌-壳聚糖-胶体金洗涤，真空干燥24h；
(b)氧化锌-壳聚糖-胶体金交联记抗人IgM抗体，取1mL氧化锌-壳聚糖-胶体金悬浮液，超声分散均匀，然后边搅拌边滴加抗体，加入抗体后，大约反应1min后，再到超声波上超声30秒，然后再反应4小时，加BSA进行封闭24小时，封闭好后进行离心，速度为12000r/min，20min，保留离心后的沉淀，待用；
(c)采用胶体金抗体缓冲液分别稀释步骤(b)离心后的沉淀氧化锌-壳聚糖-胶体金抗体，使其分散均匀，分别获得免疫胶体金抗体溶液，分别用免疫胶体金抗体溶液喷涂于玻璃纤维垫，制得免疫胶体金抗体玻璃纤维膜2；
所述的胶体金抗体缓冲液包括：浓度为20mM Tris-HCL液、蔗糖浓度为5％、海藻糖浓度为1％、proclin浓度为1％，BSA浓度为1％，pH为8.5；
(d)将硝酸纤维素膜用聚多巴胺处理液预处理后，喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原作为检测线，或喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原、肺炎支原体抗原、肺炎衣原体抗原作为检测线，喷点羊抗鼠IgG抗体作为质控线，制得免疫硝酸纤维素膜3；其中所述的聚多巴胺处理液，聚多巴胺浓度为1％，经0.22μm滤膜过滤，备用，将硝酸纤维素膜放入聚多巴胺处理液中浸泡1h，并慢速振荡摇晃，取出后用蒸馏水清洗3遍，最后在真空干燥箱中干燥；
(e)将预处理的样品垫1、步骤(c)制备的免疫胶体金抗体玻璃纤维膜2、步骤(d)制备的免疫硝酸纤维素膜3、吸水纸4依次粘贴在塑料板5上，切裁制得检测试剂条，最后将检测试剂条装入塑料外壳；所述的预处理的样品垫采用的样品垫处理液包括浓度为0.1mol/L Tris-HCL液、浓度为1％牛血清白蛋白BSA、浓度为1％表面活性剂。
下边通过实验例来进一步说明本发明。
实验例1聚多巴胺处理液对硝酸纤维素膜抗体吸附能力的比较
1材料与方法
1材料：硝酸纤维素膜，孔径4.5um，购自美国通用电气公司，聚多巴胺购自Sigma公司
2硝酸纤维素膜处理方法
2.1配制聚多巴胺处理液
配制聚多巴胺处理液：聚多巴胺由蒸馏水配置成浓度为1％，经0.22μm滤膜过滤，备用。
2.2硝酸纤维素膜处理方法
将硝酸纤维素膜放入聚多巴胺处理液中浸泡1h，并慢速振荡摇晃，取出后用蒸馏水清洗3遍，最后在真空干燥箱中干燥。
3实验方法
分别将未处理和已处理过的硝酸纤维素膜按照上述实施例工艺流程制备流感病毒A型、流感病毒B型、鼻病毒、副流感病毒、呼吸道合胞病毒、腺病毒、肺炎支原体、肺炎衣原体的IgM联合检测试纸，测试流程参照试纸说明书，比较硝酸纤维素膜未处理和处理后吸附力和稳定性指标差异。
4结果:
4.1蛋白吸附力比较
取聚多巴胺处理组和未处理组试纸，分别加入待检样品，通过观察显色情况判断处理后膜对蛋白吸附能力，结果见表1。结果显示，处理后的硝酸纤维素膜在溶液浸润性方面要明显优于未处理膜，处理后膜阳性条带颜色略深，尤其在浓度较低时，提高了反应的灵敏度，表明蛋白吸附能力明显增强，提高了反应灵敏度。聚多巴胺组吸附效果明显优于未处理组。
表1硝酸纤维素膜吸附能力比较

4.2硝酸纤维素膜稳定性比较
取聚多巴胺处理组和未处理组试纸，通过37℃加速实验观察显色情况来判断处理后硝酸纤维素膜上吸附蛋白的稳定性，结果见表2。表2结果与表1结果比较发现，处理后硝酸纤维素膜颜色变化与10天前基本一致，稳定性好。
表2硝酸纤维素膜加速稳定性比较(37℃放置10天)
实验例2氧化锌-壳聚糖-胶体金纳米颗粒修饰抗人IgM抗体
1材料与方法
1.1材料：硝酸纤维素膜，孔径4.5μm，购自美国通用电气公司
1.2氧化锌-壳聚糖-胶体金纳米颗粒修饰抗人IgM抗体
以氧化锌-壳聚糖-胶体金作为载体，取200μL氧化锌-壳聚糖-胶体金纳米颗粒(1mg/mL)溶液和25μ抗人IgM抗体(40μmol/L)溶液加入到超纯水中，使最终反应体系1mL。充分混匀后，设置摇床温度为25℃，转速为200r/m条件下，将上述混合溶液在摇床内避光震荡培养2h。反应后的混合溶液用超速离心机在13000r/m转速条件下，使用超纯水彻底离心洗涤4次，去除上清液中过量的未反应的抗体。所得沉淀物即为氧化锌-壳聚糖-胶体金-抗人IgM抗体探针复合物，用超纯水将其定容至1mL并在4℃条件储存。
2实验方法
分别将氧化锌-壳聚糖-胶体金纳米颗粒修饰的抗人IgM抗体与未修饰的抗人IgM抗体按照上述实施例工艺流程制备检测试纸，测试流程参照试纸说明书，比较氧化锌-壳聚糖-胶体金纳米颗粒处理和未处理对蛋白吸附力和稳定性指标差异。
3结果
3.1蛋白吸附力比较
取氧化锌-壳聚糖-胶体金纳米颗粒处理组和未处理组试纸，分别加入待检样品，通过观察显色情况判断处理后膜对蛋白吸附能力，结果见表3。结果显示，氧化锌-壳聚糖-胶体金纳米颗粒修饰组膜尤其在浓度较低时，提高了反应的灵敏度，表明蛋白吸附能力明显增强，提高了反应灵敏度。
表3氧化锌-壳聚糖-胶体金纳米颗粒修饰对蛋白吸附能力比较

3.2稳定性比较
取氧化锌-壳聚糖-胶体金纳米颗粒处理组和未处理组试纸，通过37℃加速实验观察显色情况来判断氧化锌-壳聚糖-胶体金修饰后硝酸纤维素膜上吸附蛋白的稳定性，结果见表4。表4结果与表3结果比较发现，处理后硝酸纤维素膜颜色变化与10天前基本一致，稳定性好。
表4加速稳定性比较(37℃放置10天)
实验例3多项病原体联合检测装置在实际应用于临床标本中的检测能力研究
1.研究对象一般资料
此次临床试验吉林省中医药科学院第一临床医院、吉林省人民医院共入选1002例病人的血清，对血样进行检测，并且其中任一一检测线阳性率至少达到30％。本临床试验共检测血样，本中心共收集334例病人的血清样本。所有入选样本均完成了试验，这些数量的样本有足够的统计学意义来评价本检测试纸血清检测结果与对照试纸检测的一致性。
2.流感病毒A型IgM测定结果的一致性分析
在血清的流感病毒A型IgM定性检测过程中，试验试剂与对照试剂的均为阳性的有186例；试验试剂阳性，对照试剂阴性的有0例；试验试剂阴性，对照试剂阳性的0例；试验试剂与对照试剂均为阴性的有148例；P＝1，P＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。
结果见表5，6；
表5两种试剂流感病毒A型IgM抗体定性测定结果
表6考核试剂相对于参比试剂的符合率
3.流感病毒B型IgM测定结果的一致性分析
在血清的流感病毒B型IgM定性检测过程中，试验试剂与对照试剂的均为阳性的有38例；试验试剂阳性，对照试剂阴性的有0例；试验试剂阴性，对照试剂阳性的0例；试验试剂与对照试剂均为阴性的有29例；P＝1，P＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。试验试剂阳性一致例数为38例，符合率为100％，阴性一致例数为29例，符合率为100％，试验试剂的整体一致例数为67例，整体符合率为100％。结果表示试验试剂与对照试剂的检测结果的组间差异无统计学意义，一致性较好。结果见表7、8；
表7两种试剂流感病毒B型IgM抗体定性测定结果
表8考核试剂相对于参比试剂的符合率
4.人鼻病毒(HRV)IgM测定结果的一致性分析
在血清的人鼻病毒(HRV)IgM定性检测过程中，试验试剂与对照试剂的均为阳性的有173例； 试验试剂阳性，对照试剂阴性的有4例；试验试剂阴性，对照试剂阳性的2例；试验试剂与对照试剂均为阴性的有155例；采用McNemar检验，P＝0.6875，P＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。试验试剂阳性一致例数为173例，符合率为98.86％，阴性一致例数为155例，符合率为97.48％，试验试剂的整体一致例数为328例，整体符合率为98.20％。结果表示试验试剂与对照试剂的检测结果的组间差异无统计学意义，一致性较好。结果见表9、10；
表9两种试剂鼻病毒(HRV)IgM抗体定性测定结果
5.人类副流感病毒(HPIV)IgM测定结果的一致性分析
在血清的人类副流感病毒(HPIV)IgM抗体定性检测过程中，试验试剂与对照试剂的均为阳性的有50例；试验试剂阳性，对照试剂阴性的有0例；试验试剂阴性，对照试剂阳性的0例；试验试剂与对照试剂均为阴性的有17例；P＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。试验试剂阳性一致例数为50例，符合率为100％，阴性一致例数为17例，符合率为100％，试验试剂的整体一致例数为67例，整体符合率为100％。结果表示试验试剂与对照试剂的检测结果的组间差异无统计学意义，一致性较好。结果见表11、12；
表11两种试剂人类副流感病毒(HPIV)IgM抗体定性测定结果
表12考核试剂相对于参比试剂的符合率
6.人类呼吸道合胞病毒(RSV)IgM测定结果的一致性分析
在血清的人类呼吸道合胞病毒(RSV)IgM定性检测过程中，试验试剂与对照试剂的均为阳性的有192例；试验试剂阳性，对照试剂阴性的有0例；试验试剂阴性，对照试剂阳性的0例；试验试剂与对照试剂均为阴性的有142例；采用McNemar检验，P＝1，P＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。试验试剂阳性一致例数为192例，符合率为100％，阴性一致例数为142例，符合率为100％，试验试剂的整体一致例数为334例，整体符合率为100％。结果表示试验试剂与对照试剂的检测结果的组间差异无统计学意义，一致性较好。结果见表13、14；
表13两种试剂人类呼吸道合胞病毒(RSV)IgM抗体定性测定结果
表14考核试剂相对于参比试剂的符合率
7.人类腺病毒(HAdV)IgM测定结果的一致性分析
在血清的人类腺病毒(HAdV)IgM抗体定性检测过程中，试验试剂与对照试剂的均为阳性的有39例；试验试剂阳性，对照试剂阴性的有0例；试验试剂阴性，对照试剂阳性的0例；试验试剂与对照试剂均为阴性的有28例；P＝1＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。试验试剂阳性一致例数为39例，符合率为100％，阴性一致例数为28例，符合率为100％，试验试剂的整体一致例数为67例，整体符合率为100％。结果表示试验试剂与对照试剂的检测结果组间差异无统计学意义，一致性较好。结果见表15、16；
表15两种试剂人类腺病毒(HAdV)IgM抗体定性测定结果
表16考核试剂相对于参比试剂的符合率
8.人类肺炎支原体(MP)IgM测定结果的一致性分析
在血清的人类肺炎支原体(MP)IgM抗体定性检测过程中，试验试剂与对照试剂的均为阳性的有15例；试验试剂阳性，对照试剂阴性的有0例；试验试剂阴性，对照试剂阳性的0例；试验试剂与对照试剂均为阴性的有52例；采用McNemar检验，P＝1，P＞0.05，组间差异无统计学意义。Kappa检验系数为1，大于0.75，结果表示试验试剂与对照试剂的一致性较好。试验试剂阳性一致例数为15例，符合率为100％，阴性一致例数为52例，符合率为100％，试验试剂的整体一致例数为67例，整体符合率为100％。结果表示试验试剂与对照试剂的检测结果的组间差异无统计学意义，一致性较好。结果见表17、18；
表17两种试剂人类肺炎支原体(MP)IgM抗体定性测定结果
表18考核试剂相对于参比试剂的符合率
9.人类肺炎衣原体(CP)IgM测定结果的一致性分析
在血清的人类肺炎衣原体(CP)IgM抗体定性检测过程中，试验试剂与对照试剂的均为阳性的有34例；试验试剂阳性，对照试剂阴性的有2例；试验试剂阴性，对照试剂阳性的1例；试验试剂与对照试剂均为阴性的有297例；采用McNemar检验，P＝1，P＞0.05，组间差异无统计学意义。Kappa检验系数为0.95，大于0.75，表示一致性较好。试验试剂阳性一致例数为34例，符合率为97.14％，阴性一致例数为297例，符合率为99.33％，试验试剂的整体一致例数为331例，整体符合率为99.10％。表示试验试剂与对照试剂的检测结果的组间差异无统计学意义，一致性较好。结果见表19、20；
表19两种试剂人类肺炎衣原体(CP)IgM抗体定性测定结果
表20考核试剂相对于参比试剂的符合率
由以上实施例可以得出：本发明所述多项病原体联合检测装置可以解决现有技术存在的NC膜吸附蛋白量不足、结合力不强的问题，且能够快速、准确进行呼吸道感染疾病的病情风险判断。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (15)

  1. 一种多项病原体联合检测装置，其特征在于，所述多项病原体联合检测装置由样品垫、免疫胶体金抗体玻璃纤维膜、免疫硝酸纤维素膜、吸收垫和塑料板组成；所述样品垫、免疫胶体金抗体玻璃纤维膜、免疫硝酸纤维素膜、吸收垫分别粘贴在塑料板上，所述免疫硝酸纤维素膜的两端分别与吸收垫、免疫胶体金抗体玻璃纤维膜搭接，所述免疫胶体金抗体玻璃纤维膜的另一端与样品垫搭接；所述免疫硝酸纤维素膜上设置第一检测线T1、第二检测线T2、第三检测线T3、第四检测线T4、第五检测线T5、第六检测线T6和质控线C，所述第一检测线T1上固相有流感病毒A抗原，第二检测线T2上固相有流感病毒B抗原，第三检测线T3上固相有鼻病毒抗原，第四检测线T4上固相有副流感病毒抗原，第五检测线T5上固相有呼吸道合胞病毒抗原，第六检测线T6上固相有腺病毒抗原，质控线C上喷点羊抗鼠IgG多克隆抗体；所述第一检测线T1、第二检测线T2、第三检测线T3、第四检测线T4、第五检测线T5和第六检测线T6分别设置在六条线上，并在每条线上分别设置质控线C。
  2. 根据权利要求1所述的多项病原体联合检测装置，其特征在于，所述免疫硝酸纤维素膜上还设置第七检测线T7和第八检测线T8，所述的第七检测线T7上固相有肺炎支原体抗原，第八检测线T8上固相有肺炎衣原体抗原，并在所述第七检测线T7和第八检测线T8的每条线上分别设置质控线C。
  3. 根据权利要求1或2所述的多项病原体联合检测装置，其特征在于，所述多项病原体联合检测装置由6个或8个检测单元组成，每一个检测单元由样品垫、免疫胶体金抗体玻璃纤维膜、免疫硝酸纤维素膜、吸收垫和塑料板组成；每一个检测单元上分别设置检测线和质控线C。
  4. 根据权利要求1或2所述的多项病原体联合检测装置，其特征在于，所述免疫胶体金抗体玻璃纤维膜由氧化锌-壳聚糖-胶体金、抗体和玻璃纤维垫组成；所述氧化锌-壳聚糖-胶体金与抗体结合后，吸附于所述玻璃纤维垫上。
  5. 根据权利要求4所述的多项病原体联合检测装置，其特征在于，所述氧化锌-壳聚糖-胶体金中的氧化锌为纳米氧化锌。
  6. 如权利要求1～5任一项所述的多项病原体联合检测装置的制备方法，其特征在于，包括下列步骤：
    (a)氧化锌-壳聚糖-胶体金制备
    1)N-羧乙基壳聚糖的合成：将4.0g壳聚糖加入到含有2.80mL丙烯酸的200mL水中，在50℃下，持续搅拌2天，反应完成后，在反应混合液中加入1mol/L的NaOH溶液，调节溶液的pH值至10-12，使所述丙烯酸所有的羧酸全部转化为钠盐，将混合溶液倒入到丙酮中进行重沉淀，然后在水溶液中透析，除去未反应的丙烯酸，透析两天后，冷冻干燥得到N-羧乙基壳聚糖；
    2)纳米氧化锌制备：称取1.3225g ZnAc2·2H2O溶解在20mL的蒸馏水中,缓慢滴加9mL 5mol/L的NaOH溶液，溶液先出现白色浑浊，继续滴加则浑浊完全消失，再加入11mL的蒸馏水，搅拌均匀，然后倒入低压反应釜中，将反应釜放入烘箱中，在170℃下加热12个小时，自然冷却至室温后，得到沉淀后，分别用水和乙醇清洗、离心两次，将沉淀于60℃烘干，得到纳米氧化锌备用；
    3)N-羧乙基壳聚糖与纳米氧化锌混合溶液的制备：将5.00g N-羧乙基壳聚糖加入到300mL去离子水中，在室温下剧烈搅拌0.5h，充分溶解得到水溶性N-羧乙基壳聚糖溶液，将上述水溶性N-羧乙基壳聚糖溶液和纳米氧化锌按一定的比例混合，将混合液剧烈搅拌3h，使其混合均匀；
    4)胶体金的制备：
    将装有90-100mL纯化水的圆底烧瓶放入电热套加热，加热10-12分钟后，待烧瓶中的水沸腾时开启转数控制开关，调至400rpm，并用注射器缓慢将1mL 1％氯化金溶液沿漩涡边缘加入，待溶液开始沸腾后，快速加入1％柠檬酸三钠溶液1-2mL，待溶液冷却到室温后，加水称重补足重量，得到胶体金；
    5)氧化锌-壳聚糖-胶体金制备：以苯二甲醛作为交联剂，在常温下将所述N-羧乙基壳聚糖与纳米氧化锌混合溶液与胶体金进行交联；交联结束，将所得的氧化锌-壳聚糖-胶体金洗涤，真空干燥24h；
    (b)氧化锌-壳聚糖-胶体金交联记抗人IgM抗体的制备：取1mL氧化锌-壳聚糖-胶体金悬浮液，超声分散均匀，然后边搅拌边滴加抗体，加入抗体后，大约反应1min后，再到超声波上超声30秒， 然后再反应4小时，加BSA进行封闭24小时，封闭好后进行离心，速度为12000r/min，20min，保留离心后的沉淀，待用；
    (c)采用胶体金抗体缓冲液分别稀释步骤(b)离心后的沉淀氧化锌-壳聚糖-胶体金抗体，使其分散均匀，分别获得免疫胶体金抗体溶液，分别用免疫胶体金交联记抗人IgM抗体溶液喷涂于玻璃纤维垫，制得免疫胶体金抗体玻璃纤维膜；
    (d)将硝酸纤维素膜用聚多巴胺处理液预处理后，喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原作为检测线，或喷点流感病毒A抗原、流感病毒B抗原、鼻病毒抗原、副流感病毒抗原、呼吸道合胞病毒抗原、腺病毒抗原、肺炎支原体抗原、肺炎衣原体抗原作为检测线，喷点羊抗鼠IgG抗体作为质控线，制得免疫硝酸纤维素膜；
    (e)将预处理的样品垫、步骤(c)制备的免疫胶体金抗体玻璃纤维膜、步骤(d)制备的免疫硝酸纤维素膜、吸水垫依次粘贴在塑料板上，切裁制得检测试剂条，最后将检测试剂条装入塑料外壳。
  7. 根据权利要求6所述的制备方法，其特征在于，所述步骤1)中透析时，采用的透析膜的截取分子量为8000-12000g/mol。
  8. 根据权利要求6所述制备方法，其特征在于，所述步骤2)中的NaOH溶液为新配制的溶液。
  9. 根据权利要求6所述制备方法，其特征在于，所述步骤3)中所述一定的比例为N-羧乙基壳聚糖与氧化锌的质量之比为100/5、100/8或100/10。
  10. 根据权利要求6所述的制备方法，其特征在于，所述步骤(c)中的胶体金抗体缓冲液包括：浓度为20mM Tris-HCL液、蔗糖浓度为5％、海藻糖浓度为1％、proclin浓度为1％，BSA浓度为1％，pH值为8.5。
  11. 根据权利要求6所述的制备方法，其特征在于，所述步骤(d)中的预处理：将硝酸纤维素膜放入聚多巴胺处理液中浸泡1h，并慢速振荡摇晃，取出后用蒸馏水清洗3遍，最后在真空干燥箱中干燥。
  12. 根据权利要求11所述的制备方法，其特征在于，所述的聚多巴胺处理液中聚多巴胺的浓度为1％，经0.22μm滤膜过滤，备用。
  13. 根据权利要求6所述的制备方法，其特征在于，所述步骤(e)中所述预处理用处理液包括：浓度为0.1mol/LTris-HCL液、浓度为1％牛血清白蛋白BSA和浓度为1％表面活性剂。
  14. 权利要求1～5任一项所述的多项病原体联合检测装置或权利要求6～13任一项所述的制备方法制备得到的多项病原体联合检测装置在临床检测中的应用。
  15. 根据权利要求14所述的应用，其特征在于，所述临床检测的样本包括血清和/或血浆。
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CN117471098A (zh) * 2023-12-27 2024-01-30 北京芯迈微生物技术有限公司 肺炎支原体、肺炎衣原体IgM抗体联合检测的芯片及应用
CN117471098B (zh) * 2023-12-27 2024-03-12 北京芯迈微生物技术有限公司 肺炎支原体、肺炎衣原体IgM抗体联合检测的芯片及应用

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