Preparation method of colloidal gold immunochromatographic test strip, colloidal gold immunochromatographic test strip and test strip cassette

Abstract

The present invention relates to the technical field of immunoassay analysis, and particularly relates to a preparation method of a colloidal gold immunochromatographic test strip, a colloidal gold immunochromatographic test strip and a test strip cassette. The preparation method of the colloidal gold immunochromatographic test strip comprises the following steps: combining an influenza A virus antibody with colloidal gold particles having a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on a conjugate pad for standby; setting a test line and a control line on a chromatographic membrane, fixing a first antibody for recognizing an influenza A virus antigen on the test line, and fixing a second antibody for recognizing an influenza A virus antibody on the control line for standby; sequentially arranging a sample pad, the conjugate pad fixed with the immunolabel, the chromatographic membrane fixed with the first antibody and the second antibody, and an absorbent pad on a base plate to form a colloidal gold immunochromatographic test strip for detecting an influenza A virus antigen.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay analysis, and particularly to a preparation method of a colloidal gold immunochromatographic test strip, a colloidal gold immunochromatographic test strip and a test strip cassette. Background Art

[0002] The colloidal gold immunochromatographic technique is a technique that combines antigen-antibody immune reaction with colloidal gold labeling tracer technique for qualitative and quantitative detection of antigen and antibody contents. The colloidal gold labeling of antibodies is mainly firmly combined through the electrostatic force between colloidal gold and proteins. Due to its advantages such as rapidity, simplicity, low cost, good stability, etc., it has especially opened up a new door for on-site rapid detection.

[0003] Influenza virus is abbreviated as flu virus. It is divided into three types: A (Influenza A), B (Influenza B), and C (Influenza C). The influenza virus can cause infections and diseases in various animals such as humans, poultry, pigs, horses, bats, etc., and is the pathogen of human and animal diseases such as human influenza, avian influenza, swine influenza, and equine influenza. Among them, human influenza viruses can be divided into three categories according to the antigenicity of their nucleoprotein: Influenza A virus, also known as type A influenza virus; Influenza B virus, also known as type B influenza virus; Influenza C virus, also known as type C influenza virus. Influenza A virus is a common influenza virus and is the most likely to mutate.

[0004] In the prior art, there is a method of using the colloidal gold immunochromatographic technique to detect influenza A virus. During the preparation of the immune label, it is necessary to use a nano-gold solution to react with an antibody to label the antibody on the surface of the nano-gold. However, there will be a problem of low labeling efficiency during the labeling process, and how to improve the labeling efficiency is a problem that needs to be solved. Summary of the Invention

[0005] The main purpose of the present invention is to provide a preparation method of a colloidal gold immunochromatographic test strip, aiming to improve the labeling efficiency.

[0006] To achieve the above purpose, a preparation method of a colloidal gold immunochromatographic test strip proposed by the present invention includes the following steps:

[0007] Combining an influenza A virus antibody with colloidal gold particles having a particle size of 50 nm - 70 nm to form an immune label, and fixing the immune label on a conjugate pad for standby;

[0008] Setting a test line and a quality control line on a chromatographic membrane, fixing a first antibody for recognizing influenza A virus antigen on the test line, and fixing a second antibody for recognizing the influenza A virus antibody on the quality control line for standby;

[0009] A sample pad, a conjugate pad fixed with the immune label, a chromatographic membrane fixed with the first antibody and the second antibody, and an absorbent pad are sequentially arranged on a bottom plate to form a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen.

[0010] Optionally, in the step of binding the influenza A virus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immune label, and fixing the immune label on the conjugate pad for standby, the following steps are included:

[0011] Prepare a colloidal gold solution composed of colloidal gold particles with a mass - volume concentration of four ten - thousandths and a particle size of 50 nm - 70 nm, adjust the pH of the colloidal gold solution, then add an influenza A virus antibody with a concentration of 10 μg / mL - 30 μg / mL, react for a preset time, then add a blocking agent and block for a preset time, obtain the reacted solution and concentrate it to obtain an immune label, resuspend the immune label with a re - solution to obtain an immune label re - solution, coat the immune label re - solution on the conjugate pad, and dry it for standby.

[0012] Optionally, in the step of binding the influenza A virus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immune label, and fixing the immune label on the conjugate pad for standby, the following steps are included:

[0013] Prepare a colloidal gold solution composed of colloidal gold particles with a mass - volume concentration of four ten - thousandths and a particle size of 50 nm - 70 nm, adjust the pH of the colloidal gold solution to 7.0 - 9.0, then add an influenza A virus antibody with a concentration of 10 μg / mL - 30 μg / mL, oscillate and react at room temperature for 4 h - 5 h, then add a BSA solution with a mass - volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with the antibody, block for 30 min - 60 min, centrifuge at a centrifugal speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant to obtain an immune label concentrated 10 times, resuspend the immune label with a re - solution to obtain an immune label re - solution diluted 5 - 10 times, coat the immune label re - solution on the conjugate pad, and dry it at 42 - 45 °C for 16 h - 18 h for standby.

[0014] Optionally, before the step of binding the influenza A virus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immune label, and fixing the immune label on the conjugate pad for standby, the following steps are further included:

[0015] Prepare a binding pad pretreatment solution. Immerse the binding pad in the binding pad pretreatment solution and soak for a preset time. After soaking, take out the binding pad and dry it for standby. Among them, the components of the binding pad pretreatment solution include 10 mM - 20 mM Tris-HCI, 100 mM - 150 mM NaCI, casein with a mass-volume concentration of 1% - 1.5%, sucrose with a mass-volume concentration of 3% - 5%, Tween-20 with a volume concentration of 0.1% - 5%, PEG 20000 with a mass-volume concentration of 0.1% - 0.2%, and proclin 300 with a volume concentration of 0.1% - 0.2%.

[0016] Optionally, before the steps of sequentially arranging the sample pad, the binding pad fixed with the immune label, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen, the following steps are further included:

[0017] Prepare a sample pad pretreatment solution. Immerse the sample pad in the sample pad pretreatment solution and soak for a preset time. After soaking, take out the sample pad and dry it for standby. Among them, the components of the sample pad pretreatment solution include 10 mM - 20 mM PBS, Tween-20 with a volume concentration of 1% - 1.5%, S9 with a mass-volume concentration of 0.2% - 0.5%, sucrose with a mass-volume concentration of 4% - 6%, PEG6000 with a mass-volume concentration of 0.5% - 0.8%, BSA with a mass-volume concentration of 1.5% - 2%, and proclin 300 with a volume concentration of 0.1% - 0.2%.

[0018] Optionally, the pH of the colloidal gold solution is 7.5.

[0019] Optionally, the influenza A virus antibody includes the M1302 influenza virus antibody.

[0020] Optionally, the first antibody includes the M1301 influenza A virus antibody.

[0021] This application also provides a colloidal gold immunochromatographic test strip, and the colloidal gold immunochromatographic test strip includes the colloidal gold immunochromatographic test strip prepared by the preparation method of the colloidal gold immunochromatographic test strip as described above.

[0022] This application also provides a colloidal gold immunochromatographic test strip kit, which includes the colloidal gold immunochromatographic test strip prepared by the preparation method of the colloidal gold immunochromatographic test strip as described above.

[0023] The preparation method of the colloidal gold immunochromatographic test strip of the present application includes the following steps: combining the influenza A virus antibody with colloidal gold particles having a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on a conjugate pad for later use; setting a test line and a control line on a chromatographic membrane, fixing a first antibody for recognizing the influenza A virus antigen on the test line, and fixing a second antibody for recognizing the influenza A virus antibody on the control line for later use; sequentially arranging a sample pad, the conjugate pad fixed with the immunolabel, the chromatographic membrane fixed with the first antibody and the second antibody, and an absorbent pad on a base plate to form a colloidal gold immunochromatographic test strip for detecting the influenza A virus antigen. By using colloidal gold with a particle size of 50 nm - 70 nm, the effective collision between antibody molecules and colloidal gold can be increased, the binding efficiency of the antibody and colloidal gold can be increased, the effective labeling quantity of nanogold can be improved. Coating the highly efficiently labeled nanogold on the conjugate pad can increase the quantity of effectively labeled nanogold on the conjugate pad, thereby facilitating the improvement of the detection sensitivity of the influenza A virus; moreover, the colloidal gold with a particle size of 50 nm - 70 nm has a small volume-specific surface area, requires less antibody usage, reduces the amount of antibody used. That is to say, the present application not only improves the labeling efficiency of colloidal gold but also saves the cost of antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the structures shown in these drawings without creative efforts.

[0025] Figure 1 It is a schematic flow chart of the preparation method of the colloidal gold immunochromatographic test strip of the present application;

[0026] Figure 2 It is a schematic structural diagram of the base plate in an embodiment of the present application;

[0027] Figure 3 It is a schematic structural diagram of the colloidal gold immunochromatographic test strip in an embodiment of the present application.

[0028] Explanation of the reference numerals in the drawings:

[0029] Label Name Label Name 100 Colloidal gold immunochromatographic test strip 40 Chromatographic membrane 10 Bottom plate 41 Test line 20 Sample pad 43 Control line 30 Conjugate pad 50 Absorbent pad

[0030] The realization of the object, functional features and advantages of the present invention will be further described with reference to the embodiments and the drawings. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Hereinafter, embodiments of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen of the present application will be specifically described in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary details are omitted. For example, there are cases where details of well-known matters are omitted and repeated descriptions of actually identical structures are omitted. This is to prevent the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0032] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular range. The ranges defined in this way can include or exclude the end values and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, ranges of 60-110 and 80-120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In the present application, unless otherwise stated, the numerical range "a-b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been fully listed herein, and "0-5" is only an abbreviated representation of these numerical combinations. Additionally, when a certain parameter is expressed as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0033] If there is no special instruction, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0034] If there is no special instruction, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0035] Unless otherwise specified, all steps of this application can be carried out in sequence or randomly, preferably in sequence. For example, when the method includes steps (a) and (b), it means that the method may include steps (a) and (b) carried out in sequence, or may also include steps (b) and (a) carried out in sequence. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method may include steps (a), (b), and (c), or may also include steps (a), (c), and (b), or may include steps (c), (a), and (b), etc.

[0036] Unless otherwise specified, the terms "comprising" and "including" mentioned in this application mean open-ended, and can also be closed-ended. For example, the "comprising" and "including" may mean that other components not listed may also be included or comprised, or may only include or comprise the listed components.

[0037] Unless otherwise specified, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) while B is true (or exists); or both A and B are true (or exist).

[0038] The immunogold technique is a new type of immunolabeling technique that uses colloidal gold as a tracer marker for antigen-antibody reactions. Colloidal gold is formed by the polymerization of chloroauric acid (HAuCl4) under the action of a reducing agent into gold particles of a specific size, and due to electrostatic interaction, it becomes a stable colloidal state, which is called colloidal gold. Colloidal gold carries a negative charge in a weak alkaline environment and can form a firm bond with the positive charge groups of protein molecules. Since this bond is an electrostatic bond, it does not affect the biological properties of the protein. According to some physical properties of colloidal gold, such as high electron density, particle size, shape, and color reaction, combined with the immunological and biological properties of the conjugate, colloidal gold is thus widely used in the fields of immunology, histology, pathology, and cell biology.

[0039] The symptoms of humans infected with influenza virus A mainly include high fever, cough, runny nose, muscle pain, etc. Most cases are accompanied by severe pneumonia, and in severe cases, multiple organ failures such as heart and kidney failure lead to death, with a very high fatality rate.

[0040] Improving the sensitivity of detecting influenza virus A can timely and effectively detect the infection of influenza virus A, enable early treatment, and effectively ensure human health and safety.

[0041] Improving the labeling efficiency is beneficial to improving the detection sensitivity. Therefore, the present application provides a preparation method of a colloidal gold immunochromatographic test strip, in which the immune label on the conjugate pad has a high labeling efficiency to improve the detection sensitivity.

[0042] As Figure 2 and Figure 3 shown, the colloidal gold immunochromatographic test strip 100 for detecting influenza A virus antigen generally consists of a base plate 10, a sample pad 20, a conjugate pad 30, a chromatographic membrane 40, an absorbent pad 50, etc. The chromatographic membrane is marked with a test line 41 and a control line 43. After the solid-phase carrier labeled with the antigen or antibody fixed on the conjugate pad undergoes a specific immune reaction with the analyte in the sample, the formed complex passes through the chromatographic action on the chromatographic membrane and undergoes a specific immune reaction again at the test line. The solid-phase carrier serves as a signal amplification marker, and the test result is obtained by visual observation or detection with corresponding equipment.

[0043] The present application provides a preparation method of a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen, including the following steps: combining the influenza A virus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immune label, fixing the immune label on the conjugate pad for standby; setting a test line and a control line on the chromatographic membrane, fixing the first antibody for recognizing the influenza A virus antigen on the test line, and fixing the second antibody for recognizing the influenza A virus antibody on the control line for standby; sequentially arranging the sample pad, the conjugate pad fixed with the immune label, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the base plate to form a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen.

[0044] As Figure 1 shown, it is a schematic flow chart of the preparation method of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen. Combining the influenza A virus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immune label, fixing the immune label on the conjugate pad for standby; setting a test line and a control line on the chromatographic membrane, fixing the first antibody for recognizing the influenza A virus antigen on the test line, and fixing the second antibody for recognizing the influenza A virus antibody on the control line for standby; sequentially arranging the sample pad, the conjugate pad fixed with the immune label, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the base plate to form a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen.

[0045] Colloidal gold with a particle size of 50 nm - 70 nm is used, so that when preparing colloidal gold-labeled influenza A virus antibodies, the effective collision between antibody molecules and colloidal gold can be increased, the binding efficiency of antibodies and colloidal gold can be increased, the effective labeling quantity of nano-gold can be improved, and the nano-gold with high-efficiency labeling is coated on the conjugate pad to increase the quantity of effectively labeled nano-gold on the conjugate pad, thereby being beneficial to improving the detection sensitivity of influenza A virus; moreover, the volume specific surface area of colloidal gold with a particle size of 50 nm - 70 nm is small, and the amount of antibody used is less, reducing the usage amount of the antibody. That is to say, this application not only improves the labeling efficiency of colloidal gold, but also saves the cost of the antibody.

[0046] Further, in the step of combining influenza A virus antibodies with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel and fixing the immunolabel on the conjugate pad for standby, the following steps are included: preparing a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, adjusting the pH of the colloidal gold solution, then adding influenza A virus antibodies with a concentration of 10 μg / mL - 30 μg / mL, reacting for a preset time, then adding a blocking agent and blocking for a preset time, obtaining the reacted solution and concentrating it to obtain an immunolabel, resuspending the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution, coating the immunolabel reconstitution solution on the conjugate pad, and drying it for standby.

[0047] The mass-volume concentration refers to the mass of the solute divided by the volume of the solvent to obtain the mass-volume concentration. For example, the mass of nano-gold divided by the volume of the solvent gives the concentration of the nano-gold solution.

[0048] Using a colloidal gold solution with a mass-volume concentration of four ten-thousandths and the particle size of nano-gold being 50 nm - 70 nm, compared with the conventional colloidal gold solution with a mass-volume concentration of one ten-thousandth, this application uses a colloidal gold solution with a high concentration, which can increase the effective collision between antibody molecules and colloidal gold, increase the binding efficiency of antibodies and colloidal gold, and compared with colloidal gold with a particle size of 40 nm, the volume specific surface area of colloidal gold with a particle size of 50 nm - 70 nm is small, and the amount of antibody used is less. Therefore, by using high-concentration large-particle colloidal gold to prepare an immunolabel, the usage amount of the antibody is reduced, the labeling efficiency of colloidal gold is improved, and the cost of the antibody is saved.

[0049] In the process of preparing the immunolabel, adjust the pH of the colloidal gold solution, and then add influenza virus A antibody with a concentration of 10 μg / mL - 30 μg / mL, preferably with an influenza virus A antibody concentration of 20 μg / mL - 25 μg / mL, so that the antibody is positively charged in the colloidal gold solution and adsorbs on the negatively charged surface of the colloidal gold. React for a preset time, then add a blocking agent and block for a preset time to cover the exposed gold surface sites not coated with the antibody, obtaining the reacted solution. Concentrate the reacted solution, for example, by centrifugation to remove the supernatant to obtain the immunolabel. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution, and coat the immunolabel reconstitution solution on the conjugate pad and dry it for standby.

[0050] Among the above-mentioned 10 μg / mL - 30 μg / mL influenza virus A antibody, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 10 μg / mL, 15 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 30 μg / mL, etc.

[0051] Further, in the step of binding the influenza virus A antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel and fixing the immunolabel on the conjugate pad for standby, the following steps are included: Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, and adjust the pH of the colloidal gold solution. Then add influenza virus A antibody with a concentration of 10 μg / mL - 30 μg / mL, react for a preset time, then add a blocking agent and block for a preset time to obtain the reacted solution and concentrate it to obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad and dry it for standby.

[0052] In the process of preparing the immunolabel, concentrate the obtained reacted solution to obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad and dry it for standby.

[0053] Among the above-mentioned 5 - 10 times, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0054] Further, in the step of binding the influenza A virus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel and fixing the immunolabel on the conjugate pad for later use, the following steps are included: Prepare a colloidal gold solution composed of colloidal gold particles with a mass - volume concentration of four ten - thousandths and a particle size of 50 nm - 70 nm, and adjust the pH of the colloidal gold solution to 7.0 - 9.0. Then add the influenza A virus antibody with a concentration of 10 μg / mL - 30 μg / mL, oscillate and react at room temperature for 4 h - 5 h. Then add a BSA solution with a mass - volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with the antibody, incubate for 30 min - 60 min, centrifuge at a rotational speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant to obtain an immunolabel concentrated 10 times, resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for later use.

[0055] When the pH value of the colloidal gold solution is 7.0 - 9.0, the colloidal gold is negatively charged, which is beneficial to the binding of the antibody to the colloidal gold. Oscillate and react at room temperature for 4 h - 5 h to complete the reaction. Then add a BSA solution with a mass - volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with the antibody, incubate for 30 min - 60 min, centrifuge at a rotational speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant to obtain an immunolabel concentrated 10 times, resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for later use.

[0056] In the above pH value of 7.0 - 9.0, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 7.0, 7.5, 8.0, 8.5, 9.0, etc.

[0057] In the above oscillating reaction at room temperature for 4 h - 5 h, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 4 h, 4.5 h, 5 h, etc.

[0058] In the above 0.5% - 1.0% BSA solution, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.5%, 0.7%, 0.9%, 1.0%, etc.

[0059] Further, the pH of the colloidal gold solution is 7.5; and / or, the concentration of the influenza virus A antibody is 20 μg / ml; and / or, the reconstitution solution comprises 50 mM NaCl, 0.1% (m / v) BSA, 1.5% (m / v) sucrose and 1.5% (m / v) sodium caseinate; and / or, the influenza virus A antibody comprises the M1302 influenza virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; and / or, the first antibody comprises the M1301 influenza virus A antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; and / or, the second antibody comprises goat anti-mouse IgG; and / or, the scribing concentration of the first antibody at the test line is 1 μl / cm; and / or, the coating concentration of the immunolabel on the conjugate pad is 10 μl / cm; and / or, the concentration of the first antibody is 0.5 mg / mL - 2 mg / mL; and / or, the material of the bottom plate comprises at least one of polyvinyl chloride, polyethylene and glass; and / or, the material of the sample pad comprises a glass fiber membrane, and the glass fiber membrane comprises ahlstrom 8964; and / or, the material of the conjugate pad comprises a glass fiber membrane, and the glass fiber membrane comprises ahlstrom 8980; and / or, the material of the chromatographic membrane comprises a nitrocellulose membrane, and the nitrocellulose membrane comprises sartorius 140.

[0060] The pH of the colloidal gold solution is preferably 7.1 - 8.5. Since the isoelectric points of different antibodies vary, it is necessary to explore the labeling efficiency of antibodies with colloidal gold at different pH values. The pH values of 7.0, 7.1, 7.3, 7.5, 8.0, 8.1, 8.5, and 9.0 were explored. When the pH value is 7.5, the antibody is positively charged in the colloidal gold solution and adsorbs on the negatively charged surface of the colloidal gold. At this time, the labeling efficiency is the highest. Therefore, the preferred pH value is 7.5.

[0061] The concentration of the influenza virus A antibody is 20 μg / ml, which can avoid incomplete labeling caused by too low a concentration of the influenza virus A antibody and waste of the antibody caused by too high a concentration.

[0062] The reconstitution solution comprises 50 mM NaCl, 0.1% (m / v) BSA, 1.5% (m / v) sucrose and 1.5% (m / v) sodium caseinate.

[0063] The influenza virus A antibody includes the M1302 influenza virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; the first antibody includes the M1301 influenza virus A antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; the second antibody includes goat anti-mouse IgG; the coating concentration of the immune label on the conjugate pad is 10 ul / cm; the material of the base plate includes at least one of polyvinyl chloride, polyethylene and glass; the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964; the material of the conjugate pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8980; the material of the chromatography membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius 140.

[0064] The step of immobilizing the first antibody at the test line is as follows: Prepare a first antibody solution with a concentration of 0.5 mg / mL - 2 mg / mL for identifying influenza virus A antigen, and use a three-dimensional membrane scribing instrument to scribe the first antibody solution on the chromatography membrane to form a test line. After drying, it is reserved for use. At this time, the scribing concentration of the first antibody at the test line is 1 ul / cm. The first antibody solution with a concentration of 0.5 mg / mL - 2 mg / mL is used to avoid insufficient immobilization of the first antibody due to too low a concentration, resulting in unclear color development at the test line. At the same time, it also avoids excessive concentration, causing waste of the antibody. Among the above 0.5 mg / mL - 2 mg / mL, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include, but are not limited to, the point values in the examples and 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, etc.

[0065] Further, before the step of combining the influenza virus A antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immune label and immobilizing the immune label on the conjugate pad for standby, the following steps are also included: Prepare a conjugate pad pretreatment solution, soak the conjugate pad in the conjugate pad pretreatment solution, take out the conjugate pad and dry it after soaking for a preset time for standby. Among them, the components of the conjugate pad pretreatment solution include 10 mM - 20 mM Tris-HCI, 100 mM - 150 mM NaCI, 1% - 1.5% (mass / volume) casein, 3% - 5% (mass / volume) sucrose, 0.1% - 5% (volume) Tween-20, 0.1% - 0.2% (mass / volume) PEG 20000, and 0.1% - 0.2% (volume) proclin 300.

[0066] The pH of the Tris-HCI buffer system stabilizes the solution. The salt ion substance is sodium chloride, which can remove the viscous substances in the sample, causing the viscous substances to settle without affecting the flow and penetration of the sample on the test strip. Tween-20 is a surfactant that functions to moisten the conjugate pad faster, facilitating the attachment and reconstitution of the immunolabel and helping the analyte to bind better with the immunolabel. The role of sucrose is to protect the antibody protein labeled on the colloidal gold, making the antibody more stable. The role of casein is to protect the antibody protein labeled on the colloidal gold, making the protein more stable. Additionally, it has an inhibitory effect on non-specific reactions, helping to prevent non-specific binding and enabling complete release. The macromolecular substance PEG 20000 serves as the framework for the uniform dispersion of the colloidal gold after drying, facilitating the rapid dissolution and release of the gold-labeled substance. Proclin 300 acts as a preservative, stabilizing the conjugate pad treatment solution and extending its shelf life. The above components cooperate synergistically, making the buffer system of the conjugate pad treatment solution conducive to the binding and release of the immunolabel on the conjugate pad.

[0067] Among them, the value concentration range of 10 mM - 20 mM Tris-HCI includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. The value concentration range of 100 mM - 150 mM NaCl includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 100 mM, 120 mM, 140 mM, 150 mM. The value concentration range of casein with a mass-volume concentration of 1% - 1.5% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 1%, 1.2%, 1.4%, 1.5%. The value concentration range of sucrose with a mass-volume concentration of 3% - 5% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 3%, 3.5%, 4%, 5%. The value concentration range of Tween-20 with a volume concentration of 0.1% - 5% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.5%, 0.7%, 0.9%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%. The value concentration range of PEG20000 with a mass-volume concentration of 0.1% - 0.2% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%. The value concentration range of proclin 300 with a volume concentration of 0.1% - 0.2% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%.

[0068] Among them, the soaking time is 20 min - 30 min, the drying temperature is 45°C - 50°C, and the drying time is 16 h - 18 h.

[0069] The steps for the treatment of the conjugate pad are as follows: Prepare a conjugate pad pretreatment solution, which consists of 10 - 20 mM Tris-HCI, 100 - 150 mM NaCI, 1 - 1.5% casein, 3 - 5% sucrose, 0.1% - 5% Tween-20, 0.1 - 0.2% PEG20000, and 0.1 - 0.2% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel, with the size of the container being approximately 30 cm × 30 cm × 10 cm. The conjugate pad is made of a glass fiber membrane with the model ahlstrom 8980. Place it into the liquid, ensuring that the liquid submerges the glass fiber membrane, and soak for about 20 min - 30 min. After soaking, take out the glass fiber membrane, lay it flat on a sieve, and put it into a forced-air drying oven set at a temperature of 45°C - 50°C for a drying time of about 16 h - 18 h. After drying, it serves as a pretreated conjugate pad and is stored sealed at room temperature for later use.

[0070] Further, before the steps of sequentially arranging the sample pad, the conjugate pad fixed with an immune label, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen, the following steps are also included: Prepare a sample pad pretreatment solution, soak the sample pad in the sample pad pretreatment solution, and after soaking for a preset time, take out the sample pad and dry it for later use. Among them, the components of the sample pad pretreatment solution include 10 mM - 20 mM PBS, 1% - 1.5% Tween-20 by volume concentration, 0.2% - 0.5% S9 by mass-volume concentration, 4% - 6% sucrose by mass-volume concentration, 0.5% - 0.8% PEG6000 by mass-volume concentration, 1.5% - 2% BSA by mass-volume concentration, and 0.1% - 0.2% proclin 300 by volume concentration.

[0071] PBS serves as a buffer system to stabilize the pH of the system. Through the blocking agent BSA, the hydrophilic macromolecule PEG6000, the surfactant Tween-20, and S9, it effectively prevents sample substances from non-specifically adsorbing onto the immunochromatographic test strip. Using the above treatment solution to treat the sample pad can increase the hydrophilicity of the sample pad, contribute to the rapid wetting of the sample pad, and promote the occurrence of chromatography. At the same time, due to the suspension and dispersion effect and the stable pH environment, the sample pad can also protect the antibodies in the sample and reduce non-specific binding, which helps to effectively detect low-concentration influenza A virus and avoid false positive problems.

[0072] Among them, the value concentration range of 10 mM - 20 mM PBS includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. The value concentration range of 1% - 1.5% Tween-20 includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 1%, 1.3%, 1.5%. The value concentration range of 0.2% - 0.5% S9 includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.2%, 0.3%, 0.4%, 0.5%. The value concentration range of 4% - 6% sucrose includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 4%, 5%, 6%. The value concentration range of 0.5% - 0.8% PEG6000 includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.5%, 0.6%, 0.7%, 0.8%. The value concentration range of 1.5% - 2% BSA sucrose includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 1.5%, 1.8%, 2%. The value concentration range of 0.1% - 0.2% proclin 300 includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%.

[0073] Among them, the soaking time is 20 min - 30 min, the drying temperature is 45°C - 50°C, and the drying time is 16 h - 18 h.

[0074] The processing steps of the sample pad are as follows: Prepare a sample pad treatment solution, which consists of 10 mM - 20 mM PBS, 1% - 1.5% Tween-20, 0.2% - 0.5% S9, 4% - 6% sucrose, 0.5% - 0.8% PEG6000, 1.5% - 2% BSA, and 0.1% - 0.2% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel. The sample pad is made of a glass fiber membrane with the model number ahlstrom 8964. Place it in the liquid, ensuring that the liquid submerges the glass fiber membrane. The soaking time is about 20 min - 30 min. After soaking, take out the glass fiber membrane, lay it flat on a sieve, and put it into a forced-air drying oven set at a temperature of 45°C - 50°C. The drying time is about 16 h - 18 h. After drying, it serves as a pretreated sample pad and is stored sealed at room temperature for later use.

[0075] In this application, the conjugate pad is coated with colloidal gold-labeled influenza A virus antibody. The antibody at the test line includes a first antibody for recognizing influenza A virus antigen, and the antibody at the control line includes a second antibody for recognizing influenza A virus antibody. When a test sample is dropped on the sample pad, the influenza A virus antigen in the sample flows on the sample pad. After reaching the conjugate pad, the influenza A virus antigen specifically binds to the colloidal gold-labeled influenza A virus antibody on the conjugate pad, forming an "antigen-antibody-gold conjugate". At the same time, the "antigen-antibody-gold conjugate" is released from the conjugate pad and flows towards the chromatographic membrane by capillary action. When the "antigen-antibody-gold conjugate" reaches the test line, the "antigen-antibody-gold conjugate" binds to the first antibody at the test line, forming an "antibody-antigen-antibody-gold conjugate" at the test line, thereby causing the aggregation and color development of nanogold at the test line. At the same time, the colloidal gold-labeled influenza A virus antibody that has not specifically bound to the influenza A virus antibody continues to flow towards the control line and binds to the second antibody after reaching the control line, forming a "secondary antibody-antibody-gold conjugate", and the nanogold aggregates and develops color at the control line. Among them, the color development of the control line indicates that the test strip is effective.

[0076] This application uses colloidal gold with a particle size of 50 nm - 70 nm. When preparing the colloidal gold-labeled influenza A virus antibody, it can increase the effective collision between antibody molecules and colloidal gold, increase the binding efficiency of antibody and colloidal gold, and improve the effective labeling quantity of nanogold. Coating the highly efficiently labeled nanogold onto the conjugate pad can increase the quantity of effectively labeled nanogold on the conjugate pad, which is beneficial to improving the detection sensitivity of influenza A virus. Moreover, the colloidal gold with a particle size of 50 nm - 70 nm has a small volume-specific surface area, and less antibody is used, reducing the amount of antibody used. That is to say, this application not only improves the labeling efficiency of colloidal gold but also saves the cost of antibodies.

[0077] Among the above 50 nm - 70 nm, the values include the minimum and maximum values of this range, as well as each value between such minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments and 50 nm, 55 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 65 nm, 68 nm, 70 nm, etc.

[0078] Furthermore, the particle size of the colloidal gold is 60 nm; and / or, the influenza A virus antibody includes the M1302 influenza virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd., and the first antibody includes the M1301 influenza A virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; and / or, the second antibody includes goat anti - mouse IgG; and / or, the minimum detection limit of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen is 1×10 4 , with the unit: number of virus particles / ml; and / or, the first antibody is a monoclonal antibody or a polyclonal antibody; and / or, the scribing concentration of the first antibody at the test line is 1 ul / cm; and / or, the coating concentration of the colloidal gold - labeled influenza A virus antibody on the conjugate pad is 10 ul / cm; and / or, the material of the bottom plate includes at least one of polyvinyl chloride, polyethylene, and glass; and / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom8964; and / or, the material of the conjugate pad includes a glass fiber membrane and / or a polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980; and / or, the material of the chromatographic membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius 140; and / or, the sample pad, conjugate pad, chromatographic membrane, and absorbent pad are sequentially and mutually overlapped and attached to the bottom plate.

[0079] In this application, the particle size of the colloidal gold used is 60 nm. The specific surface area per volume of 60 - nm colloidal gold is small, and the amount of antibody used is less.

[0080] In this application, the influenza A virus antibody includes the M1302 influenza virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd., and the first antibody includes the M1301 influenza A virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd. Using the above - mentioned pair of influenza A virus antibodies, the detection sensitivity is high.

[0081] The second antibody includes goat anti - mouse IgG. Goat anti - mouse antibodies from different domestic and foreign suppliers were screened, including Shanghai Modis Medical Technology Co., Ltd., Zhuhai Bomei Biotechnology Co., Ltd., Hangzhou Xianzhi Biotechnology Co., Ltd., Hangzhou Longji Biotechnology Co., Ltd., and Ningbo Maiyue Biotechnology Co., Ltd. The selected goat anti - mouse antibody uses the product of Ningbo Maiyue Biotechnology Co., Ltd. as the quality control line antibody, and the batch number of the goat anti - mouse antibody is EC00102.

[0082] The minimum detection limit of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen is 1×104 ,Unit: number of virus particles / ml; The conjugate pad is coated with colloidal gold-labeled influenza A virus antibody, and the particle size of the colloidal gold is 50nm - 70nm, which can effectively improve the detection sensitivity of the test strip. The specific steps for detecting the detection limit are as follows: Prepare the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen of the present application, and the detected antigen is the culture of influenza A virus. Use a sample diluent to dilute the culture into different concentrations for testing. The lowest detection limit obtained by using the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen of the present application is 1×10 9 inactivated influenza A virus culture, and use the sample diluent to dilute the culture into different concentrations for testing. The lowest detection limit obtained by using the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen of the present application is 1×10 4 , Unit: number of virus particles / ml.

[0083] The first antibody is a monoclonal antibody or a polyclonal antibody. In the present application, the first antibody is an antibody fixed at the test line for recognizing influenza A virus antigen, which can be a monoclonal antibody or a polyclonal antibody.

[0084] The scribing concentration of the first antibody at the test line is 1ul / cm. The steps for fixing the first antibody at the test line are as follows: Prepare a first antibody solution with a concentration of 0.5mg / mL - 2mg / mL for recognizing influenza A virus antigen, use a three-dimensional membrane scribing instrument to scribe the first antibody solution on the chromatographic membrane to form a test line, and after drying, reserve it. At this time, the scribing concentration of the first antibody at the test line is 1ul / cm.

[0085] The coating concentration of the conjugate pad coated with colloidal gold-labeled influenza A virus antibody is 10ul / cm. During the process of coating the conjugate pad with colloidal gold-labeled influenza A virus antibody, the colloidal gold-labeled influenza A virus antibody is coated on the conjugate pad by a spraying method, and the concentration of the colloidal gold-labeled influenza A virus antibody coated on the conjugate pad is 10ul / cm. By using the above preparation method of the colloidal gold immunochromatographic test strip, the prepared immunolabel has a high labeling efficiency, and the concentration of the colloidal gold-labeled influenza A virus antibody coated on the conjugate pad is 10ul / cm. By coating a high concentration of the colloidal gold-labeled influenza A virus antibody, it helps to detect low-concentration influenza A virus antigen. At the same time, both the conjugate pad and the sample pad are treated with corresponding pretreatment solutions, which helps to reduce non-specific binding during the detection process and reduce false positives. Based on the improvement of the above-mentioned various performances, the test strip prepared by using the preparation method of the colloidal gold immunochromatographic test strip has high sensitivity and low false positives.

[0086] The material of the base plate includes at least one of polyvinyl chloride, polyethylene and glass, and is not specifically limited. The material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964. The material of the conjugate pad includes a glass fiber membrane and / or a polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980. The material of the chromatography membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius 140. The sample pad, the conjugate pad, the chromatography membrane and the absorbent pad are sequentially and mutually overlapped and attached to the base plate.

[0087] The present application also provides a colloidal gold immunochromatographic test strip kit, which includes a colloidal gold immunochromatographic test strip prepared by the above-mentioned preparation method of the colloidal gold immunochromatographic test strip.

[0088] It can be understood that the colloidal gold immunochromatographic test strip kit includes a test strip, a diluent for the sample to be tested, and the above-mentioned colloidal gold immunochromatographic test strip for detecting influenza A virus antigen, or a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen prepared by the preparation method of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen as described above.

[0089] Furthermore, the colloidal gold immunochromatographic test strip kit for detecting influenza A virus antigen includes a card box and the above-mentioned colloidal gold immunochromatographic test strip for detecting influenza A virus antigen. The card box includes a card shell cover and a card shell bottom. The card shell cover and the card shell bottom surround to form a space for accommodating the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen. A sample adding area and a color development area are provided on the card shell; and / or, the diluent for the sample to be tested includes: 10 mM - 20 mM PBS, 0.05% - 0.1% Triton X100, 0.05% - 0.1% Tween 20, and 0.1% - 0.2% fish gelatin.

[0090] The card box includes a card shell cover and a card shell bottom. The card shell cover and the card shell bottom surround to form a space for accommodating the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen. A sample adding area and a color development area are provided on the card shell. When testing a sample, the diluted sample is dropped on the sample adding area, and after a reaction time, the color is observed in the color development area to judge the test result.

[0091] The PBS buffer solution serves as a buffering matrix to keep the basic physical and chemical properties of the sample stable. The Triton X-100 solution and Tween-20 mixture are added to the diluent as surfactants, which can effectively remove dirt and expose the viral antigen. Moreover, the surfactants selected in the present invention are all mild types, which is beneficial to maintaining the stability of the protein. Fish gelatin is a common biopolymer, which is extracted from fish skin and fish bones and then processed into a high-molecular polypeptide polymer. There are generally more than 20 kinds of amino acids that make up fish gelatin, mainly glycine, proline, hydroxyproline, and alanine. On the one hand, fish gelatin has poor antigenicity, and on the other hand, it has a far genetic relationship with mammals, which determines the superiority of fish gelatin replacing BSA in the ELISA kit. The inclusion of fish gelatin in the diluent can avoid false positives and shield interfering substances in the sample. Fish gelatin has good emulsifying properties, as well as foaming and film-forming properties manifested by increasing the phase viscosity of the solution to reduce the surface tension between the gas-liquid phases, which can protect the structure of the protein from being damaged and enhance the stability of the protein.

[0092] The sample diluent is an important component of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen. Its composition can be 10 mM PBS, 0.05% Triton X100, 0.05% Tween20, and 0.2% fish gelatin. The sample diluent is dispensed into the extraction tube, and the collected sample is added to the extraction tube with a sampling swab. The dropper of the extraction tube is tilted to the sample addition hole, and 5-6 drops of the diluent are added. After 15 minutes, the test results are observed. This sample diluent is used in conjunction with the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen, which can effectively lyse influenza A virus sufficiently and improve the sensitivity of the viral antigen.

[0093] The value concentration range of 10 mM - 20 mM PBS includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. The value concentration range of 0.05% - 0.1% Triton X100 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.05%, 0.07%, 0.09%, 0.1%. The value concentration range of 0.05% - 0.1% Tween 20 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.05%, 0.07%, 0.09%, 0.1%. The value concentration range of 0.1% - 0.2% fish gelatin includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%.

[0094] The present application also provides a detection method. The colloidal gold immunochromatographic test strip for detecting influenza A virus antigen described above is used to detect a sample to be tested, including the following steps: adding the sample to be tested to the sample pad of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen, and performing an immunochromatographic reaction for 10 min - 15 min; observing the colors on the test line and the quality control line of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen to obtain the detection result.

[0095] Using the above detection method, the colloidal gold test strip measures the antigen of influenza A virus according to the specific reaction of antigen and antibody. The antigen of influenza A virus can be detected through a one-time operation, and the result can be read quickly and intuitively, and the detection result can be obtained within 15 minutes. Moreover, the method for detecting the antigen of influenza A virus of the present invention does not require special instrument equipment, nor does it require the operation of professional personnel, getting rid of the dependence on professional instruments. Compared with the traditional double antibody sandwich method, the detection method of the present invention improves the labeling efficiency of colloidal gold by improving the gold concentration (from one ten-thousandth to four ten-thousandths) and particle size (from 40 nm to 60 m), and improves the detection sensitivity. The overall coincidence rate of the detection results of the present invention is relatively high, with a specificity of 97% and a sensitivity of 96%, which is suitable for on-site detection.

[0096] Examples

[0097] 1) Preparation and characterization of colloidal gold

[0098] First, take 200 mL of ultrapure water in a 500 mL round-bottom spherical distillation flask, add 2.0 mL of 1% HAuCl4 solution, place it in a digital display constant temperature magnetic heating mantle for heating. After boiling, add 1.5 mL of 1% trisodium citrate solution while stirring. After the color changes, continue heating for 15 min until the color remains unchanged. Take out the distillation flask, wait for the gold solution to cool to room temperature, centrifuge at 6000 r, remove the supernatant, and make up the volume to 50 mL with ultrapure water. Then place it at 4 °C for later use.

[0099] Take out 2 mL of the reserved colloidal gold solution, test it with a UV-visible spectrophotometer, scan in the wavelength range of 400 nm - 700 nm, with a scanning interval of 1 nm. It is qualified if the highest absorption peak is at 541 nm ± 2 nm.

[0100] Operate according to the above method for preparing colloidal gold. The results show that the colloidal gold solution is wine red. Test the colloidal gold sample with a UV-visible spectrogram: It shows that the peak of the prepared colloidal gold is located at 539 nm, and the peak shape is narrow, indicating that the prepared gold particles have a uniform particle size, the gold particle size is 60 nm, and the dispersibility is good.

[0101] 2) Conjugate pad pretreatment

[0102] Prepare the conjugate pad pretreatment solution, which consists of 15 mM Tris-HCI, 125 mM NaCl, 1.25% casein, 4% sucrose, 3% Tween-20, 0.15% PEG 20000, 0.15% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel, and the size of the container is about 30 cm × 30 cm × 10 cm. The conjugate pad material used is a glass fiber membrane, model ahlstrom 8980. Put it into the liquid, and the liquid must submerge the glass fiber membrane. The soaking time is about 25 min. After the soaking is over, take out the glass fiber membrane, lay it flat on the screen, and put it into a forced air drying oven set at a temperature of 45 °C - 50 °C. The drying time is about 17 h. After drying, it is used as a pretreated conjugate pad and stored at room temperature in a sealed manner for later use.

[0103] 3) Sample pad treatment

[0104] Prepare a sample pad treatment solution with the composition of 15 mM PBS, 1.25% Tween-20, 0.3% S9, 5% sucrose, 0.7% PEG6000, 1.7% BSA, and 0.15% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel. The sample pad material used is a glass fiber membrane with the model ahlstrom 8964. Place it in the liquid, ensuring that the liquid submerges the glass fiber membrane. The soaking time is about 20 min - 30 min. After soaking, take out the glass fiber membrane, lay it flat on a sieve, and put it into a forced-air drying oven set at a temperature of 45°C - 50°C. The drying time is about 16 h - 18 h. After drying, it serves as a pretreated sample pad and is stored sealed at room temperature for later use.

[0105] 4) Preparation of the gold-labeled antibody conjugate pad

[0106] Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 60 nm, and adjust the pH of the colloidal gold solution to 7.5. Then add an influenza A virus antibody (the M1302 influenza virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.) with a concentration of 20 μg / mL, and react with oscillation at room temperature for 5 h. Then add a BSA solution with a mass-volume concentration of 0.7% to cover the exposed gold surface sites not coated with the antibody, and incubate for 40 min. Centrifuge at a centrifugal speed of 7000 r / min for 15 min, remove the supernatant, and obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 7 times. Coat the immunolabel reconstitution solution on the conjugate pad and dry it at 45 - 42°C for 17 h for later use.

[0107] 5) Assembly of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen

[0108] Use goat anti-mouse IgG (secondary antibody) with the batch number EC00102DE from Ningbo Maiyue Biotechnology Co., Ltd. and an influenza A virus antibody (primary antibody) (the M1301 influenza A virus antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.) to coat the nitrocellulose membrane (chromatographic membrane) as the quality control line and the test line respectively. Then stick the sample pad, the gold-labeled antibody conjugate pad, the nitrocellulose membrane, and the absorbent paper onto the bottom plate in sequence as follows Figure 3 Just assemble it.

[0109] The prepared test strip was used to detect the analyte. The sample diluent was 15 mM PBS, 0.05% Triton X100, 0.05% Tween 20, and 0.15% fish gelatin. The sample diluent was dispensed into extraction tubes. The collected sample was added to the extraction tubes using a sampling swab. The dropper of the extraction tube was tilted to the sample addition hole, and 5 - 6 drops of the diluent were added. After 15 minutes, the test results were observed. When both the test line and the control line showed red bands, it indicated that the analyte sample contained influenza A virus antigen. If the tested sample did not contain influenza A virus antigen, when the sample moved to the test line, no red band would be shown, and only a red band would be shown at the control line; as long as the control line did not show color, it proved that the test strip was invalid and the sample needed to be retested.

[0110] 6) Screening of influenza A virus antibody pairs

[0111] Screening steps: Under the condition that other experimental conditions remained unchanged, different influenza A virus antibody pairs (influenza A virus antibody and the first antibody) were used to prepare different gold-labeled antibody conjugate pads and chromatographic membranes, and different test strips were assembled. The different test strips were used to test the analyte under the same conditions, and the sensitivities of the test strips were measured. The results are shown in the following table. The test strip prepared with the M1301 influenza A virus antibody (the first antibody) of Hangzhou Huakui Jinpei Biotechnology Co., Ltd. and the M1302 influenza virus antibody (influenza A virus antibody) of Hangzhou Huakui Jinpei Biotechnology Co., Ltd. had higher sensitivity. Therefore, this influenza A virus antibody was selected.

[0112] Table 1 Screening list of different antibody pairs

[0113]

[0114]

[0115] 7) Stability test of the colloidal gold immunochromatographic test strip for detecting influenza A virus antigen

[0116] Three batches of influenza A virus antigen detection test strips were continuously produced and tested using an inactivated influenza A virus culture with a concentration of 1×10 9 . It was diluted to 1×10 7 , 1×10 5 , 1×10 4 for 10 repeated tests, and the test results showed no difference. The product was accelerated and damaged at 50 °C for 45 days. At 0 day, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, and 45 days, it was tested with a concentration of 1×10 7 , 1×10 5 , 1×10 4The culture of influenza virus A was tested, and the test results showed no differences. After conversion to long-term stability using the Nius equation, it could be stored stably for 18 months at 4°C - 30°C, and no abnormal results occurred in the test strips, indicating that the prepared influenza virus A antigen detection test strip had good stability.

[0117] 8) Specificity test of the colloidal gold immunochromatographic test strip for detecting influenza virus A antigen

[0118] The prepared test strips were used to detect other pathogens (including rotavirus, influenza virus B, novel coronavirus, Escherichia coli, Candida albicans, Gardnerella vaginalis, Legionella pneumophila, Mycobacterium tuberculosis, Group A streptococcus, Group B streptococcus, norovirus), and at least 5 different human influenza virus A positive reference samples. The specificity of the test strips was analyzed by observing the results. The experimental results showed that the test results for other pathogens were negative, indicating that the test strips of this application had no cross-reaction with other pathogens.

[0119] Table 2 List of different pathogen detections

[0120]

[0121]

[0122] 9) Detection limit comparison experiment

[0123] The influenza virus A antigen detection test strip prepared in this application was compared with the kit of Guangzhou Wondfo Biotech Co., Ltd. The antigen detected was the culture of influenza virus A. The inactivated culture of influenza virus A was used for testing, and it was diluted to 1×10 9 for testing. It was diluted to 1×10 7 , 1×10 5 , 2×10 5 , 1×10 4 for testing. The results are shown in the following table. The lowest detection limit of the colloidal gold immunochromatographic test strip for detecting influenza virus A antigen prepared in this application is 1×10 4 , while the lowest detection limit of the colloidal gold immunochromatographic test strip for detecting influenza virus A antigen of Guangzhou Wondfo Biotech Co., Ltd. is 1×10 5 . The sensitivity of the colloidal gold immunochromatographic test strip for detecting influenza virus A antigen prepared by this method is higher than that of the colloidal gold immunochromatographic test strip for detecting influenza virus A antigen of Guangzhou Wondfo Biotech Co., Ltd.

[0124]

[0125] 10) Comparison experiment of different colloidal gold particle sizes

[0126] Under the condition that other conditions are the same, change the particle size of colloidal gold to be 40nm, 50nm, 58nm, 60nm, 62nm, 70nm, and 80nm respectively to prepare colloidal gold immunochromatographic test strips for detecting influenza A virus antigen, and use the prepared different test strips to detect influenza A virus positive test substances with different concentrations. Each group of tests is parallel tested 3 times, and the results are shown in the following table.

[0127]

[0128]

[0129] 11) Under the condition that other experimental conditions are the same, change the concentration of colloidal gold to be one ten-thousandth, four ten-thousandths, and five ten-thousandths of the mass-volume concentration respectively to prepare colloidal gold immunochromatographic test strips for detecting influenza A virus antigen, and use the prepared different test strips to detect influenza A virus positive test substances with different concentrations. Each group of tests is parallel tested 3 times, and the results are shown in the following table.

[0130] Colloidal gold concentration Detection limit One in ten thousand <![CDATA[1×10 5 > Four in ten thousand <![CDATA[1×10 4 > Five in ten thousand <![CDATA[1×10 4 >

[0131] 12) Under the condition that other experimental conditions remain unchanged, change the formulations of the sample pad pretreatment solution, the conjugate pad pretreatment solution, and the test sample dilution solution to prepare different colloidal gold immunochromatographic test strips for detecting influenza A virus antigen, and use the prepared different test strips to detect 200 negative test substances with the same concentration.

[0132]

[0133]

[0134] The above is only the preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural transformations made under the inventive concept of the present invention by using the content of the specification and drawings of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A colloidal gold immunochromatographic test strip cassette for detecting influenza A virus antigen, characterized in that, The colloidal gold immunochromatographic test strip kit includes a test sample diluent and a colloidal gold immunochromatographic test strip. The composition of the test sample diluent is: 10 mM - 20 mM PBS, 0.05% - 0.1% Triton X100, 0.05% - 0.1% Tween 20, and 0.1% - 0.2% fish gelatin. The preparation method of the colloidal gold immunochromatographic test strip includes the following steps: Prepare a conjugate pad pretreatment solution, soak the conjugate pad in the conjugate pad pretreatment solution, and after soaking for a preset time, take out the conjugate pad and dry it for standby. Among them, the material of the conjugate pad is glass fiber membrane ahlstrom 8980; the composition of the conjugate pad pretreatment solution is: 10 mM - 20 mM Tris-HCl, 100 mM - 150 mM NaCl, 1% - 1.5% casein by mass-volume concentration, 3% - 5% sucrose by mass-volume concentration, 0.1% - 5% Tween-20 by volume concentration, 0.1% - 0.2% PEG 20000 by mass-volume concentration, 0.1% - 0.2% proclin 300 by volume concentration. Combine the influenza A virus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fix the immunolabel on the conjugate pad for standby, including the following steps: prepare a colloidal gold solution composed of colloidal gold particles with a particle size of 50 nm - 70 nm and a mass-volume concentration of four ten-thousandths, adjust the pH of the colloidal gold solution to 7.0 - 9.0, then add an influenza A virus antibody with a concentration of 10 μg / mL - 30 μg / mL, oscillate and react at room temperature for 4 h - 5 h, then add a BSA solution with a mass-volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with the antibody, incubate for 30 min - 60 min, centrifuge at a centrifugal speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant to obtain an immunolabel concentrated 10 times, resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for standby. Set a test line and a quality control line on the chromatographic membrane, fix the first antibody for recognizing the influenza A virus antigen on the test line, and fix the second antibody for recognizing the influenza A virus antibody on the quality control line for standby. Prepare a sample pad pretreatment solution, soak the sample pad in the sample pad pretreatment solution, and after soaking for a preset time, take out the sample pad and dry it for standby. Among them, the material of the sample pad is glass fiber membrane ahlstrom 8964; the components of the sample pad pretreatment solution are: 10 mM - 20 mM PBS, 1% - 1.5% Tween-20 by volume concentration, 0.2% - 0.5% S9 by mass-volume concentration, 4% - 6% sucrose by mass-volume concentration, 0.5% - 0.8% PEG6000 by mass-volume concentration, 1.5% - 2% BSA by mass-volume concentration, 0.1% - 0.2% proclin 300 by volume concentration; Arrange the sample pad, the conjugate pad fixed with the immune label, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate in sequence to form a colloidal gold immunochromatographic test strip for detecting influenza A virus antigen.

2. The colloidal gold immunochromatographic test strip cassette for detecting influenza A virus antigen according to claim 1, wherein The pH of the colloidal gold solution is 7.

5.

3. The colloidal gold immunochromatographic test strip cassette for detecting influenza A virus antigen according to claim 1, wherein, The influenza A virus antibody is M1302 influenza virus antibody.

4. The colloidal gold immunochromatographic test strip for detecting influenza A virus antigen according to claim 1, characterized in that, The first antibody is M1301 influenza A virus antibody.

Images