CN111948389A - Time-resolved fluorescence immunochromatographic test strip for detecting influenza A and B viruses and novel coronavirus antigens and preparation method thereof - Google Patents
Time-resolved fluorescence immunochromatographic test strip for detecting influenza A and B viruses and novel coronavirus antigens and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a time-resolved fluorescence immunochromatographic test strip for simultaneously detecting influenza A virus, influenza B virus and novel coronavirus antigens in a throat swab and a preparation method thereof. Sample pads, time-resolved fluorescent microsphere combination pads marked by influenza A virus, influenza B virus and novel coronavirus antibodies, a reaction membrane and a water absorption pad are sequentially stuck on a PVC (polyvinyl chloride) base plate in a staggered way by about 2mm, and then a test card is assembled by fixing test paper strips by upper and lower plastic card shells which are matched. The reaction membrane is pre-coated with an influenza A virus capture antibody (T1 detection line), an influenza B virus capture antibody (T2 detection line), a novel coronavirus capture antibody (T3 detection line) and a goat anti-mouse polyclonal antibody (C control line). The invention introduces the time-resolved fluorescence immunochromatography technology into the detection of the pharyngeal swab influenza A virus, influenza B virus and the novel coronavirus, thereby greatly saving the detection time, improving the stability and the sensitivity of the detection, having simple and convenient operation and being used for field detection; meanwhile, the method also has the advantages of low cost and high cost performance.
Description
Technical Field
The invention belongs to the technical field of medical examination, and particularly relates to a time-resolved fluorescence immunochromatographic test strip and a kit for simultaneously detecting influenza A virus and influenza B virus and novel coronavirus antigens, and a preparation method thereof.
Background
Influenza (influenza for short) is an acute respiratory infection caused by influenza virus, and is a disease with strong infectivity and high transmission speed. It is transmitted primarily by airborne droplets, human-to-human contact, or contact with contaminated items. Typical clinical symptoms are: high fever, general soreness, general weakness and mild respiratory symptoms. Generally, the autumn and winter season is the high-incidence period of the disease, and the complications and death phenomena caused by the disease are very serious. The disease is caused by influenza virus and can be divided into three types of A, B and C, wherein influenza caused by the A type is most prevalent and serious, the B type often causes outbreak, and the C type often causes infantile sporadic disease. The A-type virus often has antigen variation, is high in infectivity and rapid in transmission, and is very easy to cause pandemic. The symptoms after infection are mainly manifested as high fever, cough, watery nasal discharge, myalgia, etc., and most of them are accompanied by severe pneumonia, and severe cases cause death due to failure of various organs such as heart, kidney, etc. Among influenza a viruses, avian influenza virus subtypes that have been found to be able to directly infect humans are: H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N8, wherein H1, H5, H7, H9 are highly pathogenic. Influenza b virus is a disease with strong infectivity and rapid transmission speed. It is transmitted primarily by airborne droplets, human-to-human contact, or contact with contaminated items. Typical clinical symptoms are: acute high fever, general pain, marked weakness and mild respiratory symptoms. The common autumn and winter season is a high-incidence period, which can cause local outbreak of influenza but can not cause the pandemic of the worldwide influenza.
The novel coronavirus was first discovered in 12 months in 2019, and named by the world health organization "2019-nCoV" in 12 months in 2020. The novel coronavirus (2019-nCoV) is a pathogen mainly causing respiratory tract and intestinal tract diseases. Clinical presentation of 2019-nCoV: based on current epidemiological investigations, the latency of 2019-nCoV is generally 3-7 days, and the longest latency is not more than 14 days. The clinical manifestations of 2019-nCoV are: fever, hypodynamia, dry cough as main respiratory symptoms and dyspnea gradually appear; severe cases manifest as acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, and hemorrhagic coagulation dysfunction; some patients have slight illness symptoms and can have no fever; the prognosis is good for most patients, and the disease is critical or even death for a few patients. The 2019-nCoV condition can manifest as simple infection, mild pneumonia and severe pneumonia, ARDS, sepsis, and septic shock. The clinical syndrome is classified into mild, moderate and severe according to the severity, and the latter includes severe pneumonia, ARDS, sepsis and septic shock. The existing laboratory test method has long detection time, complex operation, high cost and certain limitation in clinical use.
The traditional detection methods for influenza A virus and/or influenza B virus and/or novel coronavirus mainly comprise the following steps.
a. Fluorescence PCR method-this method has high requirements for template RNA quality, but needs special laboratory, and the detection instrument is expensive, and the detection time is longer, and the detection sensitivity is high.
b. Gold-labeled method- -the method has the characteristics of rapidness, simplicity, convenience and easy observation, but the sensitivity is not high.
c. Virus isolation-the method is accurate, long-lived, and requires a special laboratory for operation.
d. The enzyme linked immunosorbent assay is accurate and high in sensitivity, but is complex to operate and long in period.
The fluorescence immunochromatographic assay has the advantages of safety, low cost, simple and convenient operation, capability of realizing bedside rapid detection and the like, wherein the time-resolved fluorescence immunochromatographic assay has no radioactive pollution and no background fluorescence interference, and the principle is as follows: and realizing quantitative detection on the object to be detected by utilizing the fluorescence intensity of the time-resolved fluorescent microspheres. The method is one of new directions for POCT development, has the characteristics of high accuracy and good sensitivity of detection results, and can meet the requirements of clinical bedside rapid detection and primary community hospitals. Becomes the current research hotspot and has wide development prospect in clinical detection.
Disclosure of Invention
The invention aims to provide the time-resolved fluorescent test strip for the influenza A and the influenza B viruses, which has the advantages of high sensitivity, simple operation, low cost and short detection time, so that the time-resolved fluorescent test strip can meet the requirements of clinical rapid detection and the requirements of domestic markets.
In order to realize the purpose of the invention, the invention adopts the following technical scheme: the invention provides a time-resolved fluorescence test strip for simultaneously detecting influenza A virus, influenza B virus and novel coronavirus antigens, which is shown in figure 1 and comprises a sample pad, a combination pad, a reaction membrane, a water absorption pad and a bottom plate. The reaction membrane is positioned in the middle, and the sample pad and the water absorption pad are respectively positioned at the left end and the right end of the reaction membrane. The test strip combination pad is loaded with a time-resolved fluorescent microsphere labeled influenza A virus monoclonal antibody, influenza B virus monoclonal antibody and a novel coronavirus monoclonal antibody. The reaction membrane is provided with a T1 detection line, a T2 detection line, a T3 detection line and a C contrast line. T1 detecting the envelope of monoclonal antibody of influenza A virus; the T2 detection line is coated with influenza B virus monoclonal antibody; a T3 detection line is coated with a novel coronavirus monoclonal antibody; the C-line was coated with rabbit anti-mouse antibody. The method is characterized in that the sample is detected according to the principle of the conventional immunochromatography, and the detection is carried out by combining a simple and feasible dry-type fluorescence immunoassay analyzer, so that the detection window period can be greatly shortened while the high-sensitivity detection is realized, various defects of the detection methods can be avoided, the measurement result is accurate, the measuring instrument is simple and reliable, the operation is simple and convenient, and the method is convenient and practical.
In an embodiment of the present invention, the sample pad, the conjugate pad, the reaction membrane and the absorbent pad are arranged in order on the base plate, wherein the sample pad coincides with the conjugate portion, and the length of the coincident portion is about 1-2 mm; the combination pad is partially overlapped with the reaction film, and the length of the overlapped part is about 1-2 mm; the reaction film and the water absorption pad are partially overlapped, and the length of the overlapped part is about 1-2 mm; the length of the detection line and the control line is about 0.5-1.5 mm; the distance between the T1, T2 and T3 detection lines is about 1-3 mm; the distance between the test line and the control line of T3 was about 3-5 mm.
In one embodiment of the invention, the length of the sample pad is 20mm, the length of the conjugate pad is 6mm, the length of the reaction membrane is 25mm, and the length of the bibulous pad is 20 mm.
In some embodiments, the coating concentration of the influenza a, b virus and novel coronavirus monoclonal capture antibodies recognizing a single epitope is 1-1.5 mg/ml; the coating concentration of the goat anti-mouse polyclonal antibody is 0.5-1.0mg/ml, and the coating amount on the coating film is 1-1.5 ul/cm.
In one embodiment of the invention, the diameter of the fluorescent microsphere is 100-300 nm. In a more preferred embodiment of the present invention, the fluorescent microspheres have a diameter of 200 nm.
In one embodiment of the invention, the reaction membrane is a nitrocellulose membrane. Cellulose nitrate, also known as nitrocellulose, cellulose nitrate, is the product of the esterification reaction of cellulose and nitric acid. Cellulose nitrate using cotton fiber as a raw material is called nitrocellulose. Cellulose nitrate is a white fibrous polymer that is resistant to water, dilute acids, weak bases and various oils. Is particularly suitable for being used as a substrate membrane material of the antibody bearing membrane of the invention.
In some of these embodiments, the conjugate pad is a glass fiber that can carry sufficient time-resolved fluorescent microsphere-labeled monoclonal detection antibodies for influenza a, b and the novel coronavirus and that can rapidly release the microspheres upon exposure to a sample.
In one embodiment of the present invention, the time-resolved fluorescent microspheres are loaded with lanthanide or its chelate, preferably samarium, europium or terbium. The surface of the microsphere is provided with active groups which can be connected with biological substances such as protein, saccharides and the like, and the microsphere contains fluorescein. In a more preferred embodiment of the invention, the lanthanide is Eu3+ element.
The invention also provides a preparation method of the time-resolved immunochromatographic test strip for detecting the influenza A and B viruses and the novel coronavirus antigens, which comprises the following steps.
1. Preparation of conjugate pad: selecting a fluorescent microsphere with the diameter of 200nm, and labeling influenza A and B virus and a novel coronavirus antibody on the fluorescent microsphere in a covalent coupling mode by using carbodiimide (EDC) and N-hydroxysuccinimide (NHS); the prepared monoclonal detection antibodies of the influenza A virus, the influenza B virus and the novel coronavirus marked by the time-resolved fluorescent microspheres are detected according to the mass ratio of 1: 1:1 ratio mixing was sprayed on the treated conjugate pad using a quantitative liquid spray device in an amount of 1-4. mu.L/cm.
2. Treatment of conjugate pad: the conjugate pad treatment solution was sprayed onto the conjugate pad in an amount of 1 to 4. mu.L/cm using a quantitative spray device and then dried at 50 ℃ for 2 hours.
3. Preparation of a reaction film: respectively diluting the influenza A virus, the influenza B virus and the novel coronavirus capture antibody to 1.5mg/mL by using a coating buffer solution; the goat anti-mouse polyclonal antibody was diluted to 1.0 mg/mL. And respectively spraying and printing the four detection lines on the nitrocellulose membrane at intervals of 2mm between the detection lines and 4mm between the detection lines and the comparison line by using a quantitative liquid spraying device, drying for 12 hours at 42 ℃, adding a drying agent, and sealing for later use.
4. Assembling the test strip: and (3) sequentially attaching a sample pad, a conjugate pad, a reaction membrane and a water absorption pad on the white PVC base plate in a staggered manner by 2mm to obtain the time-resolved fluorescence immunochromatographic test strip. The test paper strip still includes the shell, the shell includes epitheca and inferior valve, and the epitheca compresses tightly sample pad, reaction membrane and absorption of water pad on the PVC bottom plate, and the epitheca is equipped with application of sample hole and observation window respectively in the part that corresponds sample pad and reaction membrane.
In some embodiments, the preparation method of the fluorescent microsphere-labeled monoclonal detection antibodies for influenza a and b viruses and novel coronaviruses in step 1 comprises the following steps.
(1) Washing the microspheres with 0.01-0.05mol/L MES activating buffer solution with pH of 6.0, adding carbodiimide and N-hydroxysuccinimide to make EDC, NHS and microspheres with final concentrations of 100mg/mL and 1.0-1.5mg/mL respectively, reacting at room temperature for 15-30 min, washing the microspheres sufficiently, and re-dissolving with 0.02-0.05mol/L boric acid buffer solution with pH of 7.8-8.0.
(2) Respectively adding influenza A virus, influenza B virus and novel coronavirus monoclonal detection antibodies into the redissolved microspheres according to the mass ratio of 1:5-10, reacting at room temperature for 1-2 hours, adding boric acid buffer solution with the pH value of 7.8-8.0 and the concentration of 3% BSA0.02mol/L, reacting at room temperature for 1-2 hours, washing, redissolving to the original volume by using 0.02mol/L boric acid buffer solution with the pH value of 7.4-7.6 and the concentration of 0.3% BSA and 0.05% Tween-20, and finally, preparing the influenza A virus, the influenza B virus and the novel coronavirus monoclonal detection antibodies marked by the time-resolved fluorescent microspheres according to the mass ratio of 1: 1: mixing at a ratio of 1, spraying onto glass fiber membrane at a ratio of 1-4ul/cm by using quantitative membrane spraying instrument, keeping out of the sun, and oven drying.
The invention also provides a time-resolved fluorescence immunochromatographic kit for simultaneously detecting the influenza A virus, the influenza B virus and the novel coronavirus antigens, which comprises a plastic card shell, the test strip and a sample buffer solution.
In some of these embodiments, the sample buffer is PBS buffer containing 0.5% BSA, 0.05% Tween-20, pH7.4.
When the time-resolved fluorescence immunochromatographic kit for simultaneously detecting influenza A and B viruses and novel coronavirus antigens is used, a throat swab sample to be detected is added into a sample buffer solution, 60-80 mu L of sample diluent is respectively dripped on a sample pad, and the sample is conveyed to a combination pad through capillary action. When the throat swab sample contains influenza A and/or B virus and/or novel coronavirus antigen, an antigen-antibody complex is formed by the throat swab sample and the monoclonal antibody on the fluorescent microsphere, and the complex moves forwards along with chromatography to reach T1, T2 and T3 detection areas coated with monoclonal capture antibodies of the influenza A and B viruses and the novel coronavirus which recognize single epitope, an antibody-antigen-antibody sandwich complex is formed and is gathered at the detection areas. The labeled microspheres (Eu3+ lanthanide element) which are not combined with influenza A and/or B virus and/or novel coronavirus monoclonal antibodies continue to advance, and when reaching the control area C, the labeled microspheres are combined with goat anti-mouse polyclonal antibodies on the control area, and the aggregation of the labeled microspheres appears at the C line. The whole reaction is completed within 10-15 minutes, and the card is read on a machine. The fluorescence intensity generated under the excitation light source is in direct proportion to the content of the conjugate on the test strip, when the light source irradiates the detection area and the control area of the test strip, the attached fluorescent substance is excited, the emitted light is collected and converted into an electric signal, the strength of the electric signal is related to the number of fluorescent molecules, and the detector calculates the content of the substance to be detected in the sample.
Compared with the existing rapid detection test strip, the invention has the following advantages.
1) The time-resolved fluorescence immunochromatographic assay is introduced into the detection of influenza A and B viruses and novel coronavirus antigens, and is combined with a dry fluorescence immunoassay analyzer, so that the influenza A and B viruses and the novel coronavirus antigens are detected simultaneously, and great convenience is provided for clinical use.
2) The detection of influenza A virus, influenza B virus and novel coronavirus antigens on one detection card is realized, and the clinical diagnosis is greatly facilitated.
The invention has simple operation, is suitable for large-scale production, and portable equipment required by detection is also on the market, so the invention can be widely used in hospitals, rural areas, primary clinics and the like.
Drawings
FIG. 1 shows a schematic structural diagram of a test strip for detecting influenza A and B viruses and novel coronavirus antigens by using a time-resolved fluorescence immunochromatography method.
FIG. 2 is a diagram of a test card housing.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
Example 1 a time resolved immunochromatographic test strip for simultaneous detection of influenza a and b viruses and novel coronaviruses.
The time-resolved immunochromatographic test strip for simultaneously detecting influenza A and influenza B viruses and novel coronavirus antigens comprises a base plate, and a sample pad, a combination pad, a reaction membrane and a water absorption pad which are sequentially arranged on the base plate. The combination pad is coated with a monoclonal detection antibody for influenza A virus, influenza B virus and novel coronavirus marked by fluorescent microspheres in equal quantity; the reaction membrane comprises T1, T2 and T3 detection areas and control areas which are arranged in parallel and are spaced by 2mm and 4 mm; the T1 detection zone is adjacent to the conjugate pad, the control zone is adjacent to the absorbent pad, the T2 and T3 detection zones are intermediate to the T1 detection zone and the control zone, the T2 detection zone is adjacent to the T1 detection zone, and the T3 detection zone is adjacent to the control zone. The T1, T2 and T3 detection areas are respectively coated with influenza A virus, influenza B virus and novel coronavirus monoclonal capture antibodies for identifying single epitope, and the control area is coated with goat anti-mouse polyclonal antibody.
In this embodiment, the reaction membrane is a nitrocellulose membrane of chemically cross-linked polycarbonate and polystyrene acrylonitrile (polymer), and the polycarbonate and polystyrene acrylonitrile polymer has a light transmittance of 10% or less at a wavelength of less than 450nm and a light transmittance of 95% or more at a wavelength of 500nm or more. The material can allow most visible light to pass through, and the photodetector can capture fluorescence signals on the surface and in the multilayer porous membrane, so that the detection result is more accurate.
In this embodiment, the conjugate pad is a glass fiber membrane, which can carry sufficient monoclonal detection antibodies for influenza a virus, influenza b virus and novel coronavirus labeled with time-resolved fluorescent microspheres, and can rapidly release the microspheres after encountering a sample.
In this embodiment, the fluorescent microsphere is Eu3+ lanthanide fluorescent microsphere for labeling antibody, which is known in the art, and the surface of the microsphere has active groups capable of connecting with biological substances such as protein and saccharide and contains fluorescein. The diameter of the fluorescent microsphere is 200 nm.
The preparation method of the time-resolved fluorescence immunochromatographic test strip for simultaneously detecting influenza a and influenza b virus and novel coronavirus antigens in the embodiment comprises the following steps.
(1) And influenza A and B viruses recognizing single epitope, novel coronavirus monoclonal capture antibody and goat anti-mouse polyclonal antibody are respectively fixed on a reaction membrane to form a T1 detection area, a T2 detection area, a T3 detection area and a C control area. The specific method comprises the following steps: respectively diluting the influenza A virus, influenza B virus and novel coronavirus monoclonal capture antibodies which can identify single epitope to 1.5mg/ml and the goat anti-mouse polyclonal antibody to 1.0mg/ml by using a phosphate buffer solution containing 0.05% Tween-20, 0.02mol/L and pH7.4, spraying the influenza A virus, influenza B virus and novel coronavirus monoclonal capture antibodies on a nitrocellulose membrane at an interval of 2mm by using a quantitative membrane spraying instrument, spraying the goat anti-mouse polyclonal antibody on the nitrocellulose membrane (at an interval of 4mm with the novel coronavirus monoclonal capture antibodies) and drying at 42 ℃ for 12h, and adding a drying agent for sealing and reserving for later use.
(2) The prepared fluorescent microsphere labeled monoclonal detection antibody for influenza A virus, influenza B virus and novel coronavirus is prepared according to the following steps of 1: 1: mixing and spraying the mixture on a bonding pad according to the proportion of 1.
(a) Washing the microspheres by using 0.02mol/L MES activated buffer solution with pH6.0, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to ensure that the final concentrations of EDC/NHS and the microspheres are 100mg/mL and 1.0mg/mL respectively, reacting for 15 minutes at room temperature, fully washing the microspheres, and re-dissolving by using 0.02mol/L boric acid buffer solution with pH 8.0; (b) respectively adding influenza A virus, influenza B virus and novel coronavirus monoclonal detection antibodies into the redissolved microspheres according to the mass ratio of 1:10, reacting at room temperature for 2 hours, adding boric acid buffer solution containing 3% BSA, 0.02mol/L and pH7.4, reacting at room temperature for 1 hour, washing, redissolving to the original volume by using boric acid buffer solution containing 0.3% BSA, 0.05% Tween-20, 0.02mol/L and pH7.4, and then preparing the prepared time-resolved fluorescence microsphere labeled influenza A virus, influenza B virus and novel coronavirus monoclonal detection antibodies according to the mass ratio of 1: 1: mixing at a ratio of 1, spraying onto glass fiber membrane at a rate of 4ul/cm by using a quantitative membrane spraying instrument, keeping out of the sun, drying at 42 deg.C for 12 hr, adding desiccant, and sealing for use.
(3) And sequentially adhering a sample pad, a combination pad, a reaction membrane and a water absorption pad on the bottom plate, and cutting into the size of 3.5mm in width to obtain the test strip.
A test paper strip for simultaneously detecting influenza A and B viruses and novel coronavirus antigens is assembled in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, wherein a sample adding hole and an observation window are arranged on the plastic upper shell, the sample adding hole corresponds to a sample pad of the influenza A and B viruses and the novel coronavirus antigen detection test paper strip, and a result observation window corresponds to T1, T2, T3 detection lines and C comparison lines of the influenza A and B viruses and the novel coronavirus antigen detection test paper strip.
Example 2 time resolved immunochromatography kit for simultaneous detection of influenza a, b virus and novel coronavirus antigens.
The time-resolved fluorescence immunochromatography kit for simultaneously detecting influenza a and b viruses and novel coronavirus antigens of the embodiment comprises: the test strip, plastic card shell, sample buffer described in example 1.
In this example, the sample buffer is PBS buffer containing 0.5% BSA and 0.05% Tween-20, pH7.4, 0.02 mol/L.
When the time-resolved fluorescence immunochromatographic kit for simultaneously detecting influenza A and B viruses and novel coronavirus antigens is used, a throat swab sample to be detected is added into a sample buffer, 60 mu L of sample diluent is dropwise added onto a sample pad, and the sample is conveyed to a combination pad through capillary action. When the pharyngeal swab sample contains influenza A and/or B virus and/or novel coronavirus antigens, an antigen-antibody complex is formed with the monoclonal antibody on the fluorescent microsphere. With chromatography, the complex moves forward, reaches the T1, T2 or T3 detection zone coated with monoclonal capture antibodies for influenza a and/or b viruses and/or novel coronaviruses recognizing a single epitope, forms an antibody-antigen-antibody sandwich complex, and accumulates at the detection zone. The labeled microspheres (Eu3+ lanthanide element) which are not combined with influenza A and/or B virus and/or novel coronavirus monoclonal antibodies continue to advance, and when reaching the control area C, the labeled microspheres are combined with goat anti-mouse polyclonal antibodies, and the aggregation of the labeled microspheres appears at the C line. The whole reaction is completed within 15 minutes, and the card is read on a machine. The fluorescence intensity generated under the excitation light source is in direct proportion to the content of the conjugate on the test strip, when the light source irradiates the detection area and the control area of the test strip, the attached fluorescent substance is excited, the emitted light is collected and converted into an electric signal, the strength of the electric signal is related to the number of fluorescent molecules, and the detector calculates the content of the substance to be detected in the sample.
Performance measurements of the kit of example 3.
The kit is subjected to performance measurement, including minimum detection limit, precision, sensitivity, specificity and the like.
1. The lowest detection limit.
The detection results are shown in Table 1, and the lowest detection limits of 0.5ng/mL can be seen for H1N 1A, H5N 1A, H3N 2A and H7N 9A; the lowest detection limit of B/Victoria and B/Yamagata is 1.0 ng/mL; the lowest detection limit of the novel coronavirus is 0.5 ng/mL.
2. Internal precision: 10 parts of time-resolved immunochromatography kit of the same batch are randomly extracted, and influenza A and B viruses and novel coronavirus antigen reference products with the same concentration are respectively detected, wherein the coefficient of variation CV (%) value is less than or equal to 10%.
3. Batch precision: randomly extracting three continuous batches of time-resolved immunochromatography kits, taking 3 parts of each batch of time-resolved immunochromatography kits to respectively detect influenza A, influenza B and novel coronavirus reference products with the same concentration, wherein the CV (%) value of the variation coefficient is less than or equal to 10%.
4. Accuracy and specificity: the reference substances of the syncytium virus, the mycoplasma pneumoniae, the adenovirus, the cytomegalovirus, the parainfluenza virus and the measles virus are used as the interferents to be detected, a sample to be detected is dripped into the sample adding end of the detection kit obtained in the embodiment 1, and a fluorescence detector is used for detecting after 15 minutes.
The results show that the time-resolved immunochromatography kit for the influenza A and B viruses and the novel coronavirus antigens has strong specificity with the corresponding influenza A viruses, influenza B viruses and novel coronavirus, and has no cross reaction with other pathogens.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Table 1.
Table 2.
Claims (11)
1. A time-resolved immunochromatographic test strip for detecting influenza A, B and novel coronavirus antigens is characterized in that the test strip comprises a PVC base plate, and a sample pad, a binding pad, a reaction membrane and a water absorption pad which are sequentially arranged on a bottom liner, wherein the binding pad is coated with anti-influenza A, B and novel coronavirus monoclonal detection antibodies marked by time-resolved fluorescent microspheres, the reaction membrane comprises a detection zone and a control zone which are arranged in parallel and mutually spaced, the T1 detection zone, the T2 detection zone and the T3 detection zone are respectively coated with influenza A virus monoclonal capture antibodies, influenza B virus monoclonal capture antibodies and novel coronavirus monoclonal capture antibodies for identifying single epitope, the control zone is coated with goat anti-mouse polyclonal antibodies, the reaction membrane is a nitrocellulose membrane combined with a polymer, and the polymer has light transmittance of less than 10% at a wavelength of less than 450nm, a material having a light transmittance of 95% or more at a wavelength of 500nm or more.
2. The time-resolved immunochromatographic test strip for detecting influenza a, influenza b and novel coronavirus antigens according to claim 1, wherein the time-resolved fluorescent microspheres carry lanthanide or its chelate, and the lanthanide is preferably samarium, europium or terbium.
3. The time-resolved immunochromatographic test strip for detecting influenza a, b and novel coronavirus antigens according to claim 1 or 2, wherein the final concentration of the anti-influenza a, b and novel coronavirus antibody, time-resolved fluorescent microspheres, is 1.0-1.5 mg/ml.
4. The time-resolved immunochromatographic test strip for detecting influenza A and B virus and novel coronavirus antigens according to claim 1 or 2, wherein the final concentrations of the influenza A and B virus and novel coronavirus monoclonal capture antibodies and goat anti-mouse polyclonal antibody recognizing a single epitope are 1 to 1.5mg/ml and 0.5 to 1.0mg/ml, respectively, and the coating amounts on the reaction membrane are 1 to 1.5 ul/cm, respectively.
5. The time-resolved immunochromatographic test strip for detecting influenza a and b virus and novel coronavirus antigens according to claim 1 or 2, wherein the sample pad and the conjugate pad are glass fibers.
6. The time-resolved immunochromatographic test strip for detecting influenza a, b virus and novel coronavirus antigens according to claim 1 or 2, wherein the fluorescent microspheres have a diameter of 100nm to 300 nm.
7. The time-resolved immunochromatographic strip for detecting influenza A and B virus and novel coronavirus antigens according to claim 1 or 2, wherein the T1 detection zone is close to the conjugate pad, the control zone is close to the absorbent pad, the T2 and T3 detection zones are located between the T1 detection zone and the control zone, the T2 detection zone is close to the T1 detection zone, the T3 detection zone is close to the control zone, and the T1, the T2, the T3 detection zone and the control zone are spaced at 2-4 mm intervals.
8. The method for preparing a time-resolved immunochromatographic test strip for detecting influenza A and B virus and novel coronavirus antigens according to any one of claims 1 to 7, which comprises the steps of:
(1) respectively fixing monoclonal capture antibodies of influenza A virus, influenza B virus, novel coronavirus and goat anti-mouse polyclonal antibody for recognizing single epitope on a reaction membrane to form a T1 detection zone, a T2 detection zone, a T3 detection zone and a control zone C;
(2) preparing monoclonal detection antibodies of influenza A virus, influenza B virus and novel coronavirus marked by time-resolved fluorescent microspheres, and preparing the monoclonal detection antibodies of influenza A virus, influenza B virus and novel coronavirus marked by the prepared time-resolved fluorescent microspheres according to the ratio of 1: 1:1 volume ratio and spraying on a bonding pad;
(3) paste sample pad, combination pad, reaction membrane and the pad that absorbs water in proper order on the bottom plate, obtain time-resolved fluorescence immunochromatography test paper strip, the test paper strip still includes the shell, the shell includes epitheca and inferior valve, and the epitheca compresses tightly sample pad, reaction membrane and the pad that absorbs water on the PVC bottom plate, and the epitheca is equipped with appearance hole and observation window respectively in the part that corresponds sample pad and reaction membrane.
9. The method for preparing a time-resolved immunochromatographic test strip for detecting influenza A and B virus and novel coronavirus antigens according to claim 8, wherein the method for preparing the fluorescently-labeled monoclonal detection antibodies for influenza A and B viruses and novel coronavirus in the step (2) comprises the following steps:
(1) washing the microspheres with 0.01-0.05mol/L MES (methyl methacrylate) activated buffer solution with pH of 5.0-6.0, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to make EDC/NHS and microspheres final concentration of 80-120mg/mL and 0.5-1.0mg/mL respectively, reacting at room temperature for 15-30 min, fully washing the microspheres, and re-dissolving with 0.02-0.05mol/L boric acid buffer solution with pH of 8.0-8.5;
(2) respectively adding influenza A virus, influenza B virus and novel coronavirus monoclonal detection antibodies into the redissolved microspheres according to the mass ratio of 1:5-10, reacting at room temperature for 1-2 hours, adding 0.02mol/L boric acid buffer solution with pH of 8.0-8.5 and containing 5% BSA, reacting at 37 ℃ for 1 hour, washing, redissolving to the original volume by using 0.02mol/L boric acid buffer solution with pH of 7.4-7.6 and containing 0.3% BSA and 0.05% Tween-20, storing at 4 ℃ in a dark place, and carrying out time-resolved fluorescence microsphere labeled influenza A virus, influenza B virus and novel coronavirus monoclonal detection antibodies according to the mass ratio of 1: 1:1, when in use, the mixture is sprayed on a glass fiber membrane by a quantitative membrane spraying instrument in an amount of 1-4ul/cm, the glass fiber membrane is placed into an oven, and is dried in the dark at 40-60 ℃, and a drying agent is added for sealing and storage for later use.
10. A time-resolved fluorescence immunochromatography kit for simultaneously detecting influenza A and B viruses and novel coronavirus antigens, which is characterized by comprising a plastic card shell, the test strip of any one of claims 1 to 7 and a sample reaction solution.
11. The time-resolved fluoroimmunoassay kit for detecting influenza A and B viruses and novel coronaviruses according to claim 10, wherein the sample reaction solution is PBS buffer containing 0.5% BSA, 0.05% Tween-20, pH 7.4.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112904001A (en) * | 2021-01-22 | 2021-06-04 | 厦门市波生生物技术有限公司 | New corona and influenza A and B virus antigen joint inspection kit and preparation method and application thereof |
| CN113777299A (en) * | 2021-09-15 | 2021-12-10 | 杭州宝临生物科技有限公司 | Kit containing immunochromatography detection reagent strip and application thereof |
| CN117451992A (en) * | 2023-11-06 | 2024-01-26 | 中国人民解放军军事科学院军事医学研究院 | Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof |
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2020
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112904001A (en) * | 2021-01-22 | 2021-06-04 | 厦门市波生生物技术有限公司 | New corona and influenza A and B virus antigen joint inspection kit and preparation method and application thereof |
| CN112904001B (en) * | 2021-01-22 | 2022-08-19 | 厦门市波生生物技术有限公司 | New corona and influenza A and B virus antigen joint inspection kit and preparation method and application thereof |
| CN113777299A (en) * | 2021-09-15 | 2021-12-10 | 杭州宝临生物科技有限公司 | Kit containing immunochromatography detection reagent strip and application thereof |
| WO2023040026A1 (en) * | 2021-09-15 | 2023-03-23 | 杭州宝临生物科技有限公司 | Immunochromatographic detection reagent strip and kit comprising same, and applications of both |
| CN117451992A (en) * | 2023-11-06 | 2024-01-26 | 中国人民解放军军事科学院军事医学研究院 | Fluorescent immunochromatography kit for detecting respiratory virus antigen and preparation method thereof |
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