Colloidal gold immunochromatographic test paper box, colloidal gold immunochromatographic test paper and preparation method thereof

Abstract

The present invention relates to the field of immunoassay analysis technology, and in particular to a colloidal gold immunochromatographic test paper box, a colloidal gold immunochromatographic test paper, and a preparation method thereof. The colloidal gold immunochromatographic test paper box includes a sample diluent to be tested and a colloidal gold immunochromatographic test paper; the conjugate pad colloidal gold immunochromatographic test paper box includes a card box, and a sample addition area and a color development area are provided on the conjugate pad card shell; the conjugate pad sample diluent to be tested includes: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20, and 0.1%-0.2% fish gelatin; the conjugate pad is coated with colloidal gold-labeled influenza virus B antibodies, the particle size of the conjugate pad colloidal gold is 50nm-70nm, the antibody at the conjugate pad detection line includes a first antibody for identifying influenza virus B antigens, and the antibody at the conjugate pad quality control line includes a second antibody for identifying conjugate pad influenza virus B antibodies.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay analysis, and in particular to a colloidal gold immunochromatographic test paper box, a colloidal gold immunochromatographic test paper and a preparation method thereof. Background Art

[0002] Colloidal gold immunochromatography combines the antigen-antibody immune reaction with colloidal gold labeling and tracing technology for the qualitative and quantitative detection of antigen and antibody content. Colloidal gold labeling of antibodies primarily relies on the electrostatic interaction between colloidal gold and proteins, creating a strong bond. Due to its rapidity, simplicity, low cost, and excellent stability, it has opened a new door to rapid on-site testing.

[0003] Influenza viruses are commonly referred to as influenza viruses. They are divided into three types: A, B, and C. Influenza viruses can cause infection and illness in a variety of animals, including humans, poultry, pigs, horses, and bats. They are the causative agents of human and animal diseases, including human influenza, avian influenza, swine influenza, and equine influenza. Human influenza viruses can be divided into three categories based on the antigenicity of their nucleoproteins: influenza A virus (also known as influenza A virus); influenza B virus (also known as influenza B virus); and influenza C virus (also known as influenza C virus). Influenza B virus is more likely to cause complications than influenza A virus. Summary of the Invention

[0004] The main purpose of the present invention is to provide a colloidal gold immunochromatographic test paper box to improve the detection sensitivity of viruses.

[0005] To achieve the above-mentioned object, the present invention provides a colloidal gold immunochromatographic test paper kit, comprising a sample diluent to be tested and a colloidal gold immunochromatographic test paper;

[0006] The colloidal gold immunochromatographic test paper box comprises a card box, which comprises a card cover and a card bottom. The card cover and the card bottom enclose a space for accommodating the colloidal gold immunochromatographic test paper, and the card box is provided with a sample addition area and a color development area.

[0007] The sample diluent to be tested includes: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20 and 0.1%-0.2% fish gelatin;

[0008] The colloidal gold immunochromatographic test paper comprises a base plate, and a sample pad, a binding pad, a chromatography membrane and a water-absorbing pad sequentially arranged on the base plate, wherein the chromatography membrane is provided with a detection line and a quality control line;

[0009] The conjugate pad is coated with colloidal gold-labeled influenza virus B antibodies, the particle size of the colloidal gold is 50nm-70nm, the antibodies at the detection line include a first antibody for identifying influenza virus B antigens, and the antibodies at the quality control line include a second antibody for identifying the influenza virus B antibodies.

[0010] Optionally, the influenza virus B antibody comprises the M1304 clone antibody.

[0011] Optionally, the first antibody comprises M1303 influenza virus B antibody.

[0012] Optionally, the conjugate pad is coated with colloidal gold-labeled influenza virus B antibody at a coating concentration of 10 ul / cm.

[0013] Optionally, the concentration of the first antibody at the detection line is 1 ul / cm.

[0014] Optionally, the sample pad, the conjugate pad, the chromatographic membrane and the absorbent pad are overlapped with each other in sequence on the bottom plate, and the overlap width of the sample pad and the conjugate pad is 1mm-2mm; the overlap width of the conjugate pad and the chromatographic membrane is 1mm-2mm; and the overlap width of the chromatographic membrane and the absorbent pad is 1mm-2mm.

[0015] Optionally, the colloidal gold has a particle size of 60 nm;

[0016] And / or, the minimum detection limit of the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen is 1×10 4 , unit: number of virus particles / ml.

[0017] The present application also provides a method for preparing a colloidal gold immunochromatographic test paper, comprising the following steps:

[0018] A conjugate pad pretreatment solution is prepared, and the conjugate pad is immersed in the conjugate pad pretreatment solution. After soaking for a preset time, the conjugate pad is taken out and dried for standby use, wherein the conjugate pad pretreatment solution comprises 10mM-20mM Tris-HCl, 100mM-150mM NaCl, a mass volume concentration of 1%-1.5% casein, a mass volume concentration of 3%-5% sucrose, a volume concentration of 0.1%-5% Tween-20, a mass volume concentration of 0.1%-0.2% PEG 20000, and a volume concentration of 0.1%-0.2% proclin 300;

[0019] Prepare a colloidal gold solution composed of colloidal gold particles with a mass volume concentration of 0.4 parts per million 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 influenza virus B antibody at a concentration of 10 ug / mL-30 ug / mL, shake and react at room temperature for 4 h-5 h, then add BSA solution with a mass volume concentration of 0.5%-1.0% to cover the exposed gold surface sites not coated with antibodies, block for 30 min-60 min, centrifuge at a speed of 6000 r / min-8000 r / min for 10 min-15 min, remove the supernatant to obtain a 10-fold concentrated immune label, resuspend the immune label with a redissolving solution to obtain a 5-10-fold diluted immune label redissolving solution, apply the immune label redissolving solution to the conjugate pad, dry at 4542° C. for 16 h-18 h, and set aside;

[0020] Prepare a sample pad pretreatment solution, place the sample pad in the sample pad pretreatment solution and soak it. After soaking for a preset time, take out the sample pad and dry it for use. The sample pad pretreatment solution comprises 10mM-20mM PBS, 1%-1.5% Tween-20 by volume, 0.2%-0.5% S9 by mass volume, 4%-6% sucrose by mass volume, 0.5%-0.8% PEG6000 by mass volume, 1.5%-2% BSA by mass volume, and 0.1%-0.2% proclin 300 by volume.

[0021] Setting a detection line and a quality control line on the chromatographic membrane, immobilizing a first antibody for recognizing influenza virus B antigen on the detection line, and immobilizing a second antibody for recognizing influenza virus B antibody on the quality control line for later use;

[0022] The sample pad, the binding pad fixed with the immune label, the chromatography membrane fixed with the first antibody and the second antibody, and the absorbent pad are sequentially arranged on the bottom plate to form a colloidal gold immunochromatographic test paper for detecting influenza virus B antigen.

[0023] Optionally, the pH of the colloidal gold solution is 7.5;

[0024] and / or, the concentration of the influenza virus B antibody is 20 μg / ml;

[0025] And / or, the coating concentration of the immunolabel on the conjugate pad is 10 ul / cm.

[0026] The present application also provides a colloidal gold immunochromatographic test paper, which includes the colloidal gold immunochromatographic test paper prepared by the preparation method of the colloidal gold immunochromatographic test paper.

[0027] The colloidal gold immunochromatographic test paper box of the present application includes a sample diluent to be tested and a colloidal gold immunochromatographic test paper; the colloidal gold immunochromatographic test paper box includes a card box, which includes a card cover and a card bottom, the card cover and the card bottom are arranged to form a space for accommodating the colloidal gold immunochromatographic test paper, and the card is provided with a sample addition area and a color development area; the sample diluent to be tested includes: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20% and 0.1%-0.2% fish gelatin; the colloidal gold immunochromatographic test strip includes a base plate, a sample pad, a conjugate pad, a chromatographic membrane, and a water-absorbing pad sequentially arranged on the base plate. The chromatographic membrane is provided with a test line and a quality control line. The conjugate pad is coated with colloidal gold-labeled influenza virus B antibodies, with the colloidal gold particles having a diameter of 50nm-70nm. The antibodies at the test line include a primary antibody that recognizes influenza virus B antigens, and the antibodies at the quality control line include a secondary antibody that recognizes influenza virus B antibodies. Including fish gelatin in the diluent can prevent false positives and shield interfering substances in the sample. The colloidal gold particles have a diameter of 50nm-70nm. The antibodies at the test line include a primary antibody that recognizes influenza virus B antigens, and the antibodies at the quality control line include a secondary antibody that recognizes influenza virus B antibodies. The use of colloidal gold with a particle size of 50nm-70nm can increase the effective collision between antibody molecules and colloidal gold, increase the binding efficiency of antibodies and colloidal gold, increase the effective labeled amount of nanogold, and coat the highly efficiently labeled nanogold onto the conjugate pad, thereby increasing the amount of effectively labeled nanogold on the conjugate pad, thereby facilitating improved detection sensitivity of influenza virus B. Moreover, the volume-to-surface ratio of colloidal gold with a particle size of 50nm-70nm is small, so the amount of antibody used is small, thereby reducing the amount of antibody used. That is, the present application not only improves the labeling efficiency of colloidal gold, but also saves the cost of antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0029] Figure 1 It is the cartridge of the colloidal gold immunochromatography kit of the present application;

[0030] Figure 2 This is a schematic diagram of the bottom plate structure of the colloidal gold immunochromatographic test paper of the present application;

[0031] Figure 3 It is a schematic structural diagram of the colloidal gold immunochromatographic test paper of the present application;

[0032] Figure 4 It is a schematic diagram of the process of preparing the colloidal gold immunochromatographic test paper of the present application.

[0033] Description of Figure Numbers:

[0034] Label name Label name 100 Colloidal gold immunochromatographic test strips 40 Chromatography membrane 10 baseplate 41 Testing line 20 Sample pad 43 Quality control line 30 Conjugate pad 50 absorbent pads

[0035] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0036] Below, embodiments of the colloidal gold immunochromatographic test paper for detecting influenza virus B antigens disclosed herein are described in detail, with appropriate reference to the accompanying drawings. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of substantially identical structures may be omitted. This is to avoid unnecessary length in the following description and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0037] The "ranges" disclosed herein are defined in terms of lower and upper limits, where a given range is defined by selecting a lower limit and an upper limit, and the selected lower and upper limits define the boundaries of the particular range. Ranges defined in this manner may be inclusive or exclusive of the end values ​​and may be combined arbitrarily, i.e., any lower limit may be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a particular parameter, it is understood that ranges of 60 to 110 and 80 to 120 are also contemplated. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise indicated, the numerical range "ab" is a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0038] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0039] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0040] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), indicating that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0041] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0042] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0043] Immunogold technology is a new type of immunolabeling technology that uses colloidal gold as a tracer marker for antigens and antibodies. Colloidal gold is formed by the polymerization of chloroauric acid (HAuCl4) into gold particles of a specific size under the action of a reducing agent. Due to electrostatic effects, it becomes a stable colloidal state, called colloidal gold. Colloidal gold carries a negative charge in a weakly alkaline environment and can form a strong bond with the positively charged groups of protein molecules. Since this bond is electrostatic, it does not affect the biological properties of the protein. Due to some physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, coupled with the immune and biological properties of the conjugate, colloidal gold is widely used in fields such as immunology, histology, pathology and cell biology.

[0044] Influenza B virus is more likely to cause complications than influenza A virus, and the disease burden it causes in a certain season may even exceed that of influenza A.

[0045] Improving the sensitivity of influenza virus B detection can timely and effectively detect influenza virus B infection, provide early treatment, and effectively protect human health and safety.

[0046] Improving labeling efficiency is conducive to improving detection sensitivity. To this end, the present application provides a colloidal gold immunochromatographic test strip kit, in which the conjugate pad in the colloidal gold immunochromatographic test strip used in the test strip kit is coated with colloidal gold-labeled influenza virus B antibodies. The colloidal gold-labeled influenza virus B has a high labeling efficiency to improve detection sensitivity.

[0047] The colloidal gold immunochromatographic test strip box includes a sample diluent to be tested and a colloidal gold immunochromatographic test strip; the colloidal gold immunochromatographic test strip box includes a card box, which includes a card cover and a card bottom. The card cover and the card bottom are arranged to form a space for accommodating the colloidal gold immunochromatographic test strip, and the card is provided with a sample addition area and a color development area; the sample diluent to be tested includes: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20 and 0.1%-0.2% fish gelatin; the colloidal gold immunochromatographic test paper includes a base plate, and a sample pad, a conjugation pad, a chromatographic membrane and a water absorbent pad arranged in sequence on the base plate, the chromatographic membrane is provided with a detection line and a quality control line; the conjugation pad is coated with influenza virus B antibodies labeled with colloidal gold, the particle size of the colloidal gold is 50nm-70nm, the antibodies at the detection line include a first antibody for identifying influenza virus B antigens, and the antibodies at the quality control line include a second antibody for identifying influenza virus B antibodies.

[0048] like Figure 1 As shown, the cartridge of the colloidal gold immunochromatographic kit of the present application comprises a cartridge cover and a cartridge bottom. The cartridge cover and the cartridge bottom enclose a space for accommodating a colloidal gold immunochromatographic test strip for detecting influenza virus B antigen. The cartridge is provided with a sample addition area and a color development area. When testing a sample, the diluted sample is dripped into the sample addition area. After a reaction time, the color is observed in the color development area to determine the test result.

[0049] PBS buffer serves as a buffer matrix, stabilizing the basic physicochemical properties of the sample. A mixture of Triton X-100 solution and Tween-20 is added to the diluent as a surfactant, effectively removing contaminants and exposing viral antigens. Furthermore, the surfactants selected in the present invention are all mild, which helps maintain protein stability. Fish gelatin is a common biopolymer extracted from fish skin and bones and then processed into a high-molecular-weight polypeptide polymer. Fish gelatin generally consists of more than 20 amino acids, primarily glycine, proline, hydroxyproline, and alanine. Fish gelatin has poor antigenicity and is distantly related to mammalian species, which underlies its superiority in replacing BSA in ELISA kits. Including fish gelatin in the diluent can prevent false positives and shield against interfering substances in the sample. Fish gelatin has excellent emulsifying properties, as well as foaming and film-forming properties, manifested by increasing the solution's phase viscosity to reduce the surface tension of the gas-liquid phase. This protects the protein structure from damage and enhances its stability.

[0050] The sample diluent is a key component of the colloidal gold immunochromatographic test strip for detecting influenza virus B antigen. It can be composed of 10mM PBS, 0.05% Triton X100, 0.05% Tween 20, and 0.2% fish gelatin. Dispense the sample diluent into an extraction tube. Use a sampling swab to collect the sample and add it to the extraction tube. Tilt the dropper of the extraction tube toward the sample well and add 5-6 drops of the diluent. After 15 minutes, observe the test results. This sample diluent is used with the colloidal gold immunochromatographic test strip for detecting influenza virus B antigen, effectively lysing the influenza virus B and increasing the sensitivity of the viral antigen.

[0051] The concentration range of 10mM-20mM PBS includes the minimum and maximum values ​​of this range, as well as every value between such minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 10mM, 12mM, 14mM, 16mM, 18mM, and 20mM. The concentration range of 0.05%-0.1% Triton X100 includes the minimum and maximum values ​​of this range, as well as every value between such minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.05%, 0.07%, 0.09%, and 0.1%. The concentration range of 0.05%-0.1% Tween 20 includes the minimum and maximum values ​​of this range, as well as every value between such minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.05%, 0.07%, 0.09%, and 0.1%. The concentration range of 0.1%-0.2% fish gelatin includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 0.1%, 0.15%, and 0.2%.

[0052] The colloidal gold immunochromatographic test paper for detecting influenza virus B antigen includes a base plate, and a sample pad, a conjugation pad, a chromatographic membrane and a water absorbent pad arranged in sequence on the base plate. The chromatographic membrane is provided with a detection line and a quality control line. The conjugation pad is coated with influenza virus B antibodies labeled with colloidal gold, and the particle size of the colloidal gold is 50nm-70nm. The antibodies at the detection line include a first antibody for identifying influenza virus B antigen, and the antibodies at the quality control line include a second antibody for identifying influenza virus B antibodies.

[0053] like Figure 2 and Figure 3As shown, the colloidal gold immunochromatographic test paper 100 for detecting influenza virus B antigen is generally composed of a base plate 10, a sample pad 20, a conjugation pad 30, a chromatographic membrane 40, a water-absorbing pad 50, etc. The chromatographic membrane is marked with a detection line 41 and a quality control line 43. After the antigen or antibody-labeled solid phase carrier fixed on the conjugation pad undergoes a specific immune reaction with the analyte in the sample, a complex is formed and passes through the chromatography on the chromatographic membrane. A specific immune reaction occurs again at the detection 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.

[0054] like Figure 3 As shown, the sample pad, conjugation pad, chromatography membrane, and absorbent pad are sequentially attached to the base plate in an overlapping manner. The overlap width between the sample pad and conjugation pad is 1mm-2mm; the overlap width between the conjugation pad and chromatography membrane is 1mm-2mm; and the overlap width between the chromatography membrane and absorbent pad is 1mm-2mm. This means that D1 has a width of 1mm-2mm. By sequentially attaching the sample pad, conjugation pad, chromatography membrane, and absorbent pad to the base plate in an overlapping manner, the sample to be tested can be effectively passed from the sample pad to the conjugation pad, chromatography membrane, and absorbent pad.

[0055] In the present application, the conjugate pad is coated with colloidal gold-labeled influenza virus B antibodies, the antibodies at the test line include a first antibody for recognizing influenza virus B antigens, and the antibodies at the control line include a second antibody for recognizing influenza virus B antibodies. When the sample to be tested is dropped on the sample pad, the influenza virus B antigen in the sample flows on the sample pad. After reaching the conjugate pad, the influenza virus B antigen specifically binds to the colloidal gold-labeled influenza virus B antibody on the conjugate pad to form an "antigen-antibody-gold conjugate". At the same time, the "antigen-antibody-gold conjugate" is released from the conjugate pad and flows toward the chromatographic membrane through 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 to form an "antibody-antigen-antibody-gold conjugate" at the test line, thereby causing the nanogold to aggregate and show color at the test line. At the same time, the colloidal gold-labeled influenza virus B antibody that has not specifically bound to the influenza virus B antibody continues to flow toward the quality control line and binds to the second antibody after reaching the quality control line to form a "secondary antibody-antibody-gold conjugate". The nanogold aggregates and shows color at the quality control line. The color of the quality control line indicates that the test paper is valid.

[0056] The present application uses colloidal gold with a particle size of 50nm-70nm, so that when preparing colloidal gold-labeled influenza virus B antibodies, the effective collision between antibody molecules and colloidal gold can be increased, the binding efficiency of antibodies and colloidal gold is increased, and the effective labeled amount of nanogold is increased. The highly efficiently labeled nanogold is coated on the binding pad, and the amount of effectively labeled nanogold on the binding pad is increased, which is conducive to improving the detection sensitivity of influenza virus B. In addition, the volume specific surface area of ​​colloidal gold with a particle size of 50nm-70nm is small, and the amount of antibody used is small, which reduces the amount of antibody used. That is, the present application not only improves the labeling efficiency of colloidal gold, but also saves the cost of antibodies.

[0057] In the above-mentioned 50nm-70nm, the values ​​include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 50nm, 55nm, 58nm, 59nm, 60nm, 61nm, 62nm, 65nm, 68nm, 70nm, etc.

[0058] Furthermore, the particle size of the colloidal gold is 60 nm; and / or, the influenza virus B antibody comprises the M1304 clone antibody, and the first antibody comprises the M1303 influenza virus B antibody; and / or, the second antibody comprises goat anti-mouse IgG; and / or, the minimum detection limit of the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen is 1×10 4 , unit is: number of virus particles / ml; and / or, the first antibody is a monoclonal antibody or a polyclonal antibody; and / or, the streaking concentration of the first antibody at the detection line is 1 ul / cm; and / or, the coating concentration of the conjugate pad coated with the colloidal gold-labeled influenza virus B antibody is 10 ul / cm; and / or, the material of the base plate includes at least one of polyvinyl chloride, polyethylene and glass; and / or, the material of the sample pad includes glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964; and / or, the material of the conjugate pad includes glass fiber membrane and / or polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980; and / or, the material of the chromatographic membrane includes nitrocellulose membrane, and the nitrocellulose membrane includes Sartorius 140; and / or, the sample pad, conjugate pad, chromatographic membrane and absorbent pad are sequentially overlapped and attached to the base plate.

[0059] The particle size of the colloidal gold used in this application is 60 nm. The volume-to-surface area of ​​60 nm colloidal gold is small, and the amount of antibody used is relatively small.

[0060] The influenza virus B antibody in this application includes the M1304 clone antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd., and the first antibody includes the M1303 influenza virus B antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd. The above-mentioned influenza virus B antibody pair has high detection sensitivity.

[0061] The secondary 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 Longi Biotechnology Co., Ltd., and Ningbo Maiyue Biotechnology Co., Ltd. The screened goat anti-mouse antibody was used as the quality control line antibody from Ningbo Maiyue Biotechnology Co., Ltd., and the batch number of the goat anti-mouse antibody is EC00102.

[0062] The minimum detection limit of the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen is 1×10 4 , unit: number of virus particles / ml; using a conjugate pad coated with colloidal gold labeled influenza virus B antibody, the particle size of the colloidal gold is 50nm-70nm, which can effectively improve the detection sensitivity of the test paper. The specific steps of the detection limit test are as follows: preparing the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen of the present application, the detection antigen is influenza virus B culture, and the concentration is 1×10 9 The inactivated influenza virus B culture was diluted to different concentrations using a sample diluent for testing. The minimum detection limit obtained by the colloidal gold immunochromatographic test paper for detecting influenza virus B antigens of the present application was 1×10 4 , unit: number of virus particles / ml.

[0063] The first antibody is a monoclonal antibody or a polyclonal antibody. In the present application, the first antibody is an antibody fixed at the detection line for identifying influenza virus B antigen, which can be a monoclonal antibody or a polyclonal antibody.

[0064] The streaking concentration of the first antibody at the detection line is 1 ul / cm. The steps for fixing the first antibody at the detection line are: preparing a first antibody solution with a concentration of 0.5 mg / mL-2 mg / mL for identifying influenza virus B antigen, streaking the first antibody solution on the chromatographic membrane using a three-dimensional streaking instrument to form a detection line on the chromatographic membrane, drying it, and setting it aside. At this time, the streaking concentration of the first antibody at the detection line is 1 ul / cm.

[0065] The conjugate pad is coated with colloidal gold-labeled influenza virus B antibodies at a coating concentration of 10 ul / cm. During the coating of the conjugate pad with the colloidal gold-labeled influenza virus B antibodies, the colloidal gold-labeled influenza virus B antibodies are applied to the conjugate pad by spraying, and the concentration of the colloidal gold-labeled influenza virus B antibodies applied to the conjugate pad is 10 ul / cm. Using the colloidal gold immunochromatographic test paper, the resulting immune label has high labeling efficiency, and the concentration of the colloidal gold-labeled influenza virus B antibodies applied to the conjugate pad is 10 ul / cm. The application of the high concentration of colloidal gold-labeled influenza virus B antibodies facilitates the effective detection of low-concentration influenza virus B antigens.

[0066] The base plate is made of at least one of polyvinyl chloride, polyethylene, and glass, without limitation. The sample pad is made of glass fiber membrane, such as ahlstrom 8964. The conjugation pad is made of glass fiber membrane and / or polyester fiber membrane, such as ahlstrom 8980. The chromatography membrane is made of nitrocellulose membrane, such as Sartorius 140. The sample pad, conjugation pad, chromatography membrane, and absorbent pad are attached to the base plate in an overlapping manner.

[0067] Furthermore, the present application provides a method for preparing a colloidal gold immunochromatographic test paper for detecting influenza virus B antigen, comprising the following steps: combining influenza virus B antibodies with colloidal gold particles with a particle size of 50nm-70nm to form an immune label, and fixing the immune label to a conjugate pad for later use; setting a detection line and a quality control line on a chromatographic membrane, fixing a first antibody for identifying influenza virus B antigen on the detection line, and fixing a second antibody for identifying influenza virus B antibody on the quality control line for later use; and sequentially arranging a sample pad, a conjugate pad fixed with the immune label, a chromatographic membrane fixed with the first antibody and the second antibody, and a water-absorbing pad on a bottom plate to form a colloidal gold immunochromatographic test paper for detecting influenza virus B antigen.

[0068] like Figure 4 The figure shows a process flow diagram of a method for preparing a colloidal gold immunochromatographic test paper for detecting influenza virus B antigen. Influenza virus B antibodies are combined with colloidal gold particles with a particle size of 50nm-70nm to form an immune label, which is then fixed to a conjugate pad for later use. A test line and a quality control line are set on the chromatographic membrane. A first antibody for identifying influenza virus B antigen is fixed to the test line, and a second antibody for identifying influenza virus B antibody is fixed to the quality control line for later use. A sample pad, a conjugate pad with the immune label fixed to it, a chromatographic membrane with the first and second antibodies fixed to it, and a water-absorbing pad are sequentially arranged on a base plate to form a colloidal gold immunochromatographic test paper for detecting influenza virus B antigen.

[0069] By using colloidal gold with a particle size of 50nm-70nm, when preparing colloidal gold-labeled influenza virus B antibodies, the effective collision between antibody molecules and colloidal gold can be increased, the binding efficiency of the antibody and colloidal gold is increased, and the effective labeled amount of nanogold is increased. The highly efficiently labeled nanogold is coated on a conjugate pad, and the amount of effectively labeled nanogold on the conjugate pad is increased, thereby facilitating improved influenza virus B detection sensitivity. In addition, the volume-to-surface ratio of colloidal gold with a particle size of 50nm-70nm is small, and the amount of antibody used is small, thereby reducing the amount of antibody used. That is, the present application not only improves the labeling efficiency of colloidal gold, but also saves the cost of the antibody.

[0070] Furthermore, in the step of combining influenza virus B antibodies with colloidal gold particles with a particle size of 50nm-70nm to form an immune label, and fixing the immune label on a conjugate pad for standby use, the following steps are included: preparing a colloidal gold solution composed of colloidal gold particles with a mass volume concentration of 0.4 parts per million and a particle size of 50nm-70nm, and adjusting the pH of the colloidal gold solution, and then adding influenza virus B antibodies with a concentration of 10ug / mL-30ug / mL, reacting for a preset time, then adding a blocking agent, blocking for a preset time, obtaining a reacted solution and concentrating it to obtain an immune label, resuspending the immune label with a rehydration solution to obtain an immune label rehydration solution, applying the immune label rehydration solution to the conjugate pad, drying it, and setting it aside.

[0071] Mass volume concentration refers to the mass of the solute divided by the volume of the solvent. For example, the mass of gold nanoparticles divided by the volume of the solvent gives the concentration of the gold nanoparticle solution.

[0072] A colloidal gold solution of 0.4 parts per million is used, and the particle size of the nanogold is 50nm-70nm. Compared with the conventional colloidal gold solution of 0.1 parts per million, the present application adopts a high-concentration colloidal gold solution, which can increase the effective collision between the antibody molecules and the colloidal gold, thereby increasing the binding efficiency of the antibody and the colloidal gold. Moreover, compared with the colloidal gold of 40nm, the volume specific surface area of ​​the 50nm-70nm colloidal gold is smaller, and the amount of antibody used is less. Therefore, by preparing the immune label with high-concentration large-particle colloidal gold, the amount of antibody used is reduced, the labeling efficiency of the colloidal gold is improved, and the cost of the antibody is saved.

[0073] In the process of preparing the immune label, the pH of the colloidal gold solution is adjusted, and then influenza virus B antibodies are added at a concentration of 10ug / mL-30ug / mL, preferably at a concentration of 20ug / mL-25ug / mL, so that the antibodies are positively charged in the colloidal gold solution and adsorbed on the negatively charged colloidal gold surface. The reaction is carried out for a preset time, and then a blocking agent is added and blocked for a preset time to cover the exposed gold surface sites not coated with the antibody to obtain a reacted solution. The reacted solution is then concentrated, for example, by centrifugation to remove the supernatant to obtain the immune label, and the immune label is resuspended with a rehydration solution to obtain an immune label rehydration solution. The immune label rehydration solution is applied to a conjugate pad, dried, and set aside.

[0074] In the above-mentioned 10ug / mL-30ug / mL influenza virus B antibody, the values ​​include the minimum and maximum values ​​of the range, as well as each value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 10ug / mL, 15ug / mL, 20ug / mL, 21ug / mL, 22ug / mL, 23ug / mL, 24ug / mL, 25ug / mL, 30ug / mL, etc.

[0075] Furthermore, in the step of combining influenza virus B antibodies with colloidal gold particles with a particle size of 50nm-70nm to form an immune label, and fixing the immune label on a conjugate pad for standby use, the steps include: preparing a colloidal gold solution composed of colloidal gold particles with a mass volume concentration of 0.4 parts per million and a particle size of 50nm-70nm, adjusting the pH of the colloidal gold solution, then adding influenza virus B antibodies with a concentration of 10ug / mL-30ug / mL, reacting for a preset time, then adding a blocking agent, blocking for a preset time, obtaining a reacted solution and concentrating it to obtain a 10-fold concentrated immune label, resuspending the immune label with a reconstitution solution to obtain a 5-fold-10-fold diluted immune label reconstitution solution, applying the immune label reconstitution solution to the conjugate pad, drying it, and setting it aside.

[0076] During the preparation of the immune label, the obtained post-reaction solution is concentrated to obtain a 10-fold concentrated immune label, and the immune label is resuspended in a reconstitution solution to obtain a 5- to 10-fold diluted immune label reconstitution solution, which is applied to a conjugate pad, dried, and set aside.

[0077] In the above 5 times to 10 times, the values ​​include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0078] Furthermore, in the step of combining influenza virus B antibodies with colloidal gold particles with a particle size of 50nm-70nm to form an immune label, and fixing the immune label on a binding pad for standby use, the following steps are included: preparing a colloidal gold solution composed of colloidal gold particles with a mass volume concentration of 0.4 parts per million and a particle size of 50nm-70nm, and adjusting the pH of the colloidal gold solution to 7.0-9.0, and then adding influenza virus B antibodies with a concentration of 10ug / mL-30ug / mL, shaking the reaction at room temperature for 4h-5h, and then adding the mass Cover the exposed gold surface sites not coated with antibodies with a BSA solution having a volume concentration of 0.5%-1.0%, block for 30-60 minutes, centrifuge at a speed of 6000-8000 r / min for 10-15 minutes, remove the supernatant to obtain a 10-fold concentrated immune label, resuspend the immune label with a rehydration solution to obtain a 5-10-fold diluted immune label rehydration solution, apply the immune label rehydration solution to the conjugate pad, dry at 45±2°C for 16-18 hours, and set aside.

[0079] When the pH value of the colloidal gold solution is 7.0-9.0, the colloidal gold is negatively charged, which is conducive to the binding of the antibody to the colloidal gold. The reaction is shaken at room temperature for 4-5 hours to complete the reaction. Then, a 0.5%-1.0% BSA solution is added to cover the exposed gold surface sites not coated with the antibody. The solution is blocked for 30-60 minutes. The solution is centrifuged at a speed of 6000-8000 rpm for 10-15 minutes. The supernatant is removed to obtain a 10-fold concentrated immune label. The immune label is resuspended in a redissolving solution to obtain a 5-10-fold diluted immune label redissolving solution. The immune label redissolving solution is applied to the conjugate pad and dried at 45±2°C for 16-18 hours before use.

[0080] The above pH value is 7.0-9.0, and the values ​​include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 7.0, 7.5, 8.0, 8.5, 9.0, etc.

[0081] In the above-mentioned oscillation reaction at room temperature for 4h-5h, the values ​​include the minimum and maximum values ​​of the range, and every value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 4h, 4.5h, 5h, etc.

[0082] In the above 0.5%-1.0% BSA solution, the values ​​include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 0.5%, 0.7%, 0.9%, 1.0%, etc.

[0083] Furthermore, the pH of the colloidal gold solution is 7.5; and / or the concentration of the influenza virus B antibody is 20 μg / ml; and / or the reconstitution solution includes 50 mM NaCl, 0.1% BSA by mass volume, 1.5% sucrose by mass volume and 1.5% sodium caseinate by mass volume; and / or the influenza virus B antibody includes the M1304 clone antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; and / or the first antibody includes the M1303 influenza virus B antibody of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; and / or the second antibody includes goat anti-mouse IgG; and / or the line concentration of the first antibody at the detection line is 1 ul / cm; and / or the coating concentration of the immune label on the conjugate pad is 10 ul / 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 includes at least one of polyvinyl chloride, polyethylene and glass; and / or the material of the sample pad includes glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964; and / or, the material of the conjugate pad includes a glass fiber membrane, which includes ahlstrom 8980; and / or, the material of the chromatography membrane includes a nitrocellulose membrane, which includes sartorius 140.

[0084] 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 efficiency of antibody labeling 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 adsorbed on the negatively charged colloidal gold surface. The labeling efficiency is the highest at this time. Therefore, the preferred pH value is 7.5.

[0085] The concentration of influenza virus B antibody is 20 μg / ml to avoid incomplete labeling caused by too low influenza virus B antibody concentration and waste of antibody caused by too high concentration.

[0086] The reconstitution solution includes 50 mM NaCl, 0.1% BSA by volume, 1.5% sucrose by volume, and 1.5% sodium caseinate by volume.

[0087] The influenza virus B antibody includes the M1304 clone antibody produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; the first antibody includes the M1303 influenza virus B antibody produced by 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 bottom plate includes at least one of polyvinyl chloride, polyethylene and glass; the material of the sample pad includes glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964; the material of the conjugate pad includes glass fiber membrane, and the glass fiber membrane includes ahlstrom 8980; the material of the chromatographic membrane includes nitrocellulose membrane, and the nitrocellulose membrane includes Sartorius 140.

[0088] The step of fixing the first antibody at the test line is as follows: preparing a first antibody solution with a concentration of 0.5mg / mL-2mg / mL for identifying influenza virus B antigens, streaking the first antibody solution on the chromatographic membrane with a three-dimensional streaking instrument, forming a test line on the chromatographic membrane, drying, and setting aside. At this time, the streaking concentration of the first antibody at the test line is 1ul / cm. The first antibody solution with a concentration of 0.5mg / mL-2mg / mL avoids too low a concentration, insufficient fixed amount of the first antibody, and unclear color development at the test line. At the same time, it also avoids too high a concentration, resulting in waste of antibody. In the above-mentioned 0.5mg / mL-2mg / mL, the values ​​include the minimum and maximum values ​​of the range, as well as each value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 0.5mg / mL, 0.8mg / mL, 1mg / mL, 1.5mg / mL, 1.8mg / mL, 2mg / mL, etc.

[0089] Furthermore, before the step of combining influenza virus B antibodies with colloidal gold particles with a particle size of 50nm-70nm to form an immune label and fixing the immune label to a conjugate pad for standby use, the method further includes the following steps: preparing a conjugate pad pretreatment solution, soaking the conjugate pad in the conjugate pad pretreatment solution, soaking the conjugate pad for a preset time, taking out the conjugate pad, drying it, and setting it aside, wherein the conjugate pad pretreatment solution comprises 10mM-20mM Tris-HCl, 100mM-150mM NaCI, a mass volume concentration of 1%-1.5% casein, a mass volume concentration of 3%-5% sucrose, a volume concentration of 0.1%-5% Tween-20, a mass volume concentration of 0.1%-0.2% PEG 20000, and a volume concentration of 0.1%-0.2% proclin 300.

[0090] The Tris-HCl buffer system stabilizes the solution's pH. The salt ion, sodium chloride, removes sticky materials from the sample, causing them to settle without affecting sample flow and penetration on the reagent strip. Tween-20, a surfactant, wets the conjugate pad more quickly, facilitating the attachment and resolubilization of the immunolabel, thereby promoting better binding of the analyte to the immunolabel. Sucrose protects the antibody protein labeled with the colloidal gold, stabilizing the antibody. Casein protects the antibody protein labeled with the colloidal gold, stabilizing the protein and inhibiting nonspecific reactions, helping to prevent nonspecific binding and ensure complete release. PEG 20000, a macromolecular substance, serves as a framework for the uniform distribution of the colloidal gold after drying, facilitating rapid dissolution and release of the gold label. Proclin 300, a preservative, stabilizes the conjugate pad treatment solution and extends its shelf life. These components work synergistically, making the buffer system of the conjugate pad treatment solution conducive to the binding and release of the immunolabel from the conjugate pad.

[0091] Among them, the concentration range of 10mM-20mM Tris-HCl includes the minimum and maximum values ​​of the range, as well as each value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 10mM, 12mM, 14mM, 16mM, 18mM, and 20mM. The concentration range of 100mM-150mM NaCI includes the minimum and maximum values ​​of the range, as well as each value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 100mM, 120mM, 140mM, and 150mM. The concentration range of casein with a mass volume concentration of 1%-1.5% includes the minimum and maximum values ​​of the range, as well as each value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 1%, 1.2%, 1.4%, and 1.5%. The concentration range of 3%-5% sucrose by volume includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 3%, 3.5%, 4%, and 5%. The concentration range of 0.1%-5% Tween-20 by volume includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.1%, 0.5%, 0.7%, 0.9%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0%. The concentration range of 0.1%-0.2% PEG20000 by volume includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.1%, 0.15%, and 0.2%. The concentration range of 0.1%-0.2% volume concentration of proclin 300 includes the minimum and maximum values ​​of the range, and each value between the minimum and maximum values. Specific examples include but are not limited to the point values ​​in the embodiments and 0.1%, 0.15%, and 0.2%.

[0092] Among them, the soaking time is 20min-30min, the drying temperature is 45℃-50℃, and the drying time is 16h-18h.

[0093] The conjugate pad treatment procedure involves preparing a conjugate pad pretreatment solution consisting of 10mM-20mM Tris-HCl, 100mM-150mM NaCl, 1%-1.5% casein, 3%-5% sucrose, 0.1%-5% Tween-20, 0.1%-0.2% PEG 20000, and 0.1%-0.2% proclin 300. The solution is then poured into an acrylic or stainless steel container approximately 30cm x 30cm x 10cm. The conjugate pad is made of a glass cellulose membrane (Ahlstrom 8980) and placed in the solution, ensuring that the membrane is submerged. The solution is allowed to soak for approximately 20-30 minutes. After soaking, the glass cellulose membrane was fished out, spread on the sieve, and placed in a blast drying oven set at 45-50°C for about 16-18 hours. After drying, it was used as a pre-treated binding pad and sealed and stored at room temperature for later use.

[0094] Furthermore, before the step of sequentially arranging the sample pad, the binding pad fixed with the immune label, the chromatography 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 paper for detecting influenza virus B antigen, the method further includes the following steps: preparing a sample pad pretreatment solution, soaking the sample pad in the sample pad pretreatment solution, soaking the sample pad for a preset time, taking out the sample pad, drying it, and setting it aside, wherein the sample pad pretreatment solution comprises 10mM-20mM PBS, a volume concentration of 1%-1.5% Tween-20, a mass volume concentration of 0.2%-0.5% S9, a mass volume concentration of 4%-6% sucrose, a mass volume concentration of 0.5%-0.8% PEG6000, a mass volume concentration of 1.5%-2% BSA, and a volume concentration of 0.1%-0.2% proclin 300.

[0095] PBS, used as a buffer system, stabilizes the system's pH. The blocking agent BSA, the hydrophilic macromolecular substance PEG6000, and the surfactants Tween-20 and S9 effectively prevent nonspecific adsorption of sample substances onto the immunochromatographic test strip. Treating the sample pad with the aforementioned treatment solution increases its hydrophilicity, facilitating rapid wetting and promoting chromatography. Furthermore, due to its suspension and dispersion properties and stable pH environment, the sample pad protects antibodies in the sample and reduces nonspecific binding, thus facilitating the effective detection of low-concentration influenza virus B and avoiding false positives.

[0096] The concentration range of 10mM-20mM PBS includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 10mM, 12mM, 14mM, 16mM, 18mM, and 20mM. The concentration range of 1%-1.5% Tween-20 includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 1%, 1.3%, and 1.5%. The concentration range of 0.2%-0.5% S9 includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.2%, 0.3%, 0.4%, and 0.5%. The concentration range of 4%-6% sucrose includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 4%, 5%, and 6%. The concentration range of 0.5%-0.8% PEG6000 includes the minimum and maximum values ​​of this range, as well as every value between such minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.5%, 0.6%, 0.7%, and 0.8%. The concentration range of 1.5%-2% BSA sucrose includes the minimum and maximum values ​​of this range, as well as every value between such minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 1.5%, 1.8%, and 2%. The concentration range of 0.1%-0.2% proclin 300 includes the minimum and maximum values ​​of this range, as well as every value between such minimum and maximum values. Specific examples include, but are not limited to, the point values ​​in the Examples, as well as 0.1%, 0.15%, and 0.2%.

[0097] Among them, the soaking time is 20min-30min, the drying temperature is 45℃-50℃, and the drying time is 16h-18h.

[0098] The sample pad treatment procedure is as follows: Prepare a sample pad treatment solution consisting of 10mM-20mM 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 solution into an acrylic or stainless steel container. The sample pad is made of a glass cellulose membrane (Ahlstrom 8964). Place it in the solution until it is completely submerged in the liquid for approximately 20-30 minutes. After soaking, remove the glass cellulose membrane, lay it flat on a mesh, and place it in a forced-air drying oven set at 45°C-50°C for approximately 16-18 hours. After drying, store the pretreated sample pad in a sealed container at room temperature until ready for use.

[0099] In the above method, both the conjugate pad and the sample pad are treated with corresponding pretreatment liquids, which helps to reduce nonspecific binding during the detection process and reduce false positives. Based on the above-mentioned improvements in various aspects of performance, the test paper prepared by the preparation method of colloidal gold immunochromatographic test paper has high sensitivity and low false positives.

[0100] The present application also provides a detection method, which uses the above-mentioned colloidal gold immunochromatographic test paper for detecting influenza virus B antigen to detect a sample to be tested, comprising the following steps: adding the sample to be tested to the sample pad of the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen, and allowing the immunochromatographic reaction to proceed for 10-15 minutes; observing the colors of the test line and the quality control line on the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen to obtain the test result.

[0101] Adopt above-mentioned detection method, colloidal gold test strip measures the antigen of influenza virus B according to the specific reaction of antigen and antibody, can detect influenza virus B antigen by disposable operation, and reading result is quick and intuitive, can obtain test result within 15 minutes.And detection influenza virus B antigen method of the present invention does not need special instrument equipment, does not need the operation of professional personnel, gets rid of the dependence on professional instrument.Compared with traditional double antibody sandwich method, detection method of the present invention improves the gold colloidal gold labeling efficiency by gold concentration (1 / 10,000 is increased to 4 / 10,000) and particle diameter (being increased to 60nm by 40nm), improves detection sensitivity.The overall compliance rate of test result of the present invention is higher, and specificity is 97%, and sensitivity is 96%, is suitable for on-site detection.

[0102] Example

[0103] 1) Preparation and characterization of colloidal gold

[0104] First, take 200mL of ultrapure water into a 500mL round-bottom spherical distillation flask, add 2.0mL of 1% HAuCl4 solution, and place it in a digital constant temperature magnetic heating mantle for heating. After boiling, add 1.5mL of 1% trisodium citrate solution while stirring. After the color changes, continue heating for 15 minutes until the color does not change. Take out the distillation flask, wait for the gold liquid to cool to room temperature, centrifuge at 6000r, remove the supernatant, add ultrapure water to make up to 50mL, and then place it at 4℃ for use.

[0105] Take out 2 mL of the reserved colloidal gold solution and test it using a UV-visible spectrophotometer. Scan it in the wavelength range of 400 nm to 700 nm with a scanning interval of 1 nm. The highest absorption peak is at 541 nm ± 2 nm, which is qualified.

[0106] According to the above-mentioned method for preparing colloidal gold, the results showed that the colloidal gold solution was wine red. The colloidal gold sample was tested by UV-visible spectrum: it showed that the prepared colloidal gold peak was located at 539nm, and the peak was narrow, indicating that the prepared gold particles had a uniform particle size of 60nm and good dispersion.

[0107] 2) Pretreatment of bonding pad

[0108] Prepare a conjugate pad pretreatment solution consisting of 15mM Tris-HCl, 125mM NaCl, 1.25% casein, 4% sucrose, 3% Tween-20, 0.15% PEG 20000, and 0.15% proclin 300. Pour the solution into an acrylic or stainless steel container approximately 30cm × 30cm × 10cm. Use an ahlstrom 8980 glass cellulose membrane as the conjugate pad. Place the membrane in the solution until it is submerged in the liquid for approximately 25 minutes. After soaking, remove the membrane, lay it flat on a mesh, and dry it in a forced-air drying oven set at 45°C-50°C for approximately 17 hours. Once dried, seal and store the pretreated conjugate pad at room temperature until ready for use.

[0109] 3) Sample pad processing

[0110] Prepare a sample pad treatment solution consisting of 15 mM PBS, 1.25% Tween-20, 0.3% S9, 5% sucrose, 0.7% PEG 6000, 1.75% BSA, and 0.15% Proclin 300. Pour the solution into an acrylic or stainless steel container. Use a glass cellulose membrane (Ahlstrom 8964) as the sample pad. Place it in the solution until it is submerged in the liquid for approximately 20-30 minutes. After soaking, remove the glass cellulose membrane, lay it flat on a mesh, and place it in a forced-air drying oven set at 45°C-50°C for approximately 17 hours. Once dry, store the pretreated sample pad in a sealed container at room temperature until ready for use.

[0111] 4) Preparation of gold-labeled antibody conjugate pad

[0112] Prepare a colloidal gold solution consisting of colloidal gold particles with a mass volume concentration of 0.4 parts per million and a particle size of 60 nm, and adjust the pH of the colloidal gold solution to 7.5. Then, add influenza virus B antibody (M1304 clone antibody, Hangzhou Huakui Jinpei Biotechnology Co., Ltd.) at a concentration of 20 ug / mL, and oscillate at room temperature for 5 hours. Then, add a BSA solution with a mass volume concentration of 1.0% to cover the exposed gold surface sites not coated with the antibody, block for 60 minutes, and centrifuge at a centrifugal speed of 7000 r / min for 10-15 minutes. Remove the supernatant to obtain a 10-fold concentrated immune label, resuspend the immune label with a redissolving solution to obtain an 8-fold diluted immune label redissolving solution, apply the immune label redissolving solution to the conjugate pad, dry at 4542°C for 17 hours, and set aside.

[0113] 5) Assembly of colloidal gold immunochromatographic test strips for detecting influenza virus B antigen

[0114] The goat anti-mouse IgG (secondary antibody) with the batch number EC00102DE from Ningbo Maiyue Biotechnology Co., Ltd. and the influenza virus B antibody (primary antibody) (M1303 influenza virus B antibody from Hangzhou Huakui Jinpei Biotechnology Co., Ltd.) were coated on the nitrocellulose membrane (chromatographic membrane) as the quality control line and the detection line, respectively. Then, the sample pad, gold label-antibody binding pad, nitrocellulose membrane and absorbent paper were attached to the bottom plate in sequence. Figure 1 Just assemble it.

[0115] The prepared test paper is used to detect the test object. The sample diluent is 15mM PBS, 0.05% Triton X100, 0.05% Tween 20, and 0.15% fish gelatin. The sample diluent is dispensed into an extraction tube. The collected sample is added to the extraction tube using a sampling swab. The dropper of the extraction tube is tilted toward the sample well, and 5-6 drops of diluent are added thereto. After 15 minutes, the test results are observed. When both the test line and the quality control line show red strips, it indicates that the test sample contains influenza virus B antigen. If the sample does not contain influenza virus B antigen, no red strip will appear when the sample moves to the test line, and only a red strip will appear at the quality control line. As long as the quality control line does not show color, the test strip is invalid and the sample needs to be retested.

[0116] 6) Screening of influenza virus B antibody pairs

[0117] Screening steps: With other experimental conditions unchanged, different influenza virus B antibody pairs (influenza virus B antibody and first antibody) were used to prepare different gold label-antibody binding pads and chromatographic membranes, and assembled into different test strips. Under the same conditions, different test strips were used to test the analytes, and the sensitivity of each test strip was tested. The results are shown in the table below. The test strips prepared with the M1303 influenza virus B antibody (first antibody) of Hangzhou Huakui Jinpei Biotechnology Co., Ltd. and the M1304 clone antibody (influenza virus B antibody) of Hangzhou Huakui Jinpei Biotechnology Co., Ltd. have higher sensitivity. Therefore, this influenza virus B antibody was selected.

[0118] Table 1 Screening list of different antibody pairs

[0119]

[0120] 7) Stability test of colloidal gold immunochromatographic test paper for detecting influenza virus B antigen

[0121] Three batches of influenza virus B antigen test strips were produced continuously, using a concentration of 1×10 9 Inactivated influenza B virus culture was tested and diluted to 1 × 10 7 , 1×10 5 , 1×10 4 The test was repeated 10 times and the results showed no difference. The product was subjected to accelerated destruction at 50℃ for 45 days. The product was treated with a concentration of 1×10 7 , 1×10 5 , 1×10 4Influenza virus B culture was tested, and the test results were no different. The long-term stability was converted using the Nieuw's equation, and it can be stably stored at 4°C-30°C for a long time, and the storage time is 18 months. The test card will not show abnormal results, indicating that the prepared influenza virus B antigen test strip has good stability.

[0122] 8) Specificity test of colloidal gold immunochromatographic test paper for detecting influenza virus B antigen

[0123] The prepared test strips were used to detect other pathogens (including rotavirus, influenza virus A, novel coronavirus, Escherichia coli, Candida albicans, Gardnerella vaginalis, Legionella pneumophila, Mycobacterium tuberculosis, group A Streptococcus, group B Streptococcus, and norovirus), and at least 5 different samples of influenza virus B positive reference materials, and the specificity of the test strips was analyzed by observing the results. The experimental results showed that the test strips were negative for other pathogens, indicating that the test strips of the present application had no cross-reaction with other pathogens.

[0124] Table 2 List of different pathogens detected

[0125] pathogens Test results Rotavirus Negative Influenza virus A Negative Novel Coronavirus Negative Escherichia coli Negative Candida albicans Negative Gardnerella vaginalis Negative Legionella pneumophila Negative Mycobacterium tuberculosis Negative Group A Streptococcus Negative Group B Streptococcus Negative Norovirus Negative Influenza virus B positive reference material 1 Positive Influenza virus B positive reference 2 Positive Influenza virus B positive reference 3 Positive Influenza virus B positive reference 4 Positive Influenza virus B positive reference material 5 Positive

[0126] 9) Detection limit comparison experiment

[0127] The influenza virus B antigen test strip prepared in this application was compared with the test kit of Guangzhou Wondfo Biotechnology Co., Ltd., where the detection antigen was influenza virus B culture, and the concentration was 1×10 9 Inactivated influenza B virus culture was tested and diluted to 1 × 10 7 , 1×10 5 , 2×10 5 , 1×10 4 The test results are shown in the table below. The minimum detection limit of the colloidal gold immunochromatographic test strip for detecting influenza virus B antigen prepared in this application is 1×10 4 The minimum detection limit of the colloidal gold immunochromatographic test strip used by Guangzhou Wondfo Biotechnology Co., Ltd. to detect influenza virus B antigen is 1×10 5 The sensitivity of the colloidal gold immunochromatographic test strip for detecting influenza virus B antigen prepared by this method is higher than that of the colloidal gold immunochromatographic test strip for detecting influenza virus B antigen produced by Guangzhou Wondfo Biotechnology Co., Ltd.

[0128]

[0129]

[0130] 10) Comparative experiment of different colloidal gold particle sizes

[0131] Under the same experimental conditions, the particle size of colloidal gold was changed to 40nm, 50nm, 58nm, 60nm, 62nm, 70nm, and 80nm to prepare colloidal gold immunochromatographic test strips for detecting influenza virus B antigen. The prepared different test strips were used to detect influenza virus B positive analytes with different concentrations. Each test group was tested in parallel 3 times. The results are shown in the following table.

[0132] Colloidal gold particle size Detection limit 40nm <![CDATA[4×10 4 ]]> 50nm <![CDATA[1×10 4 ]]> 58nm <![CDATA[1×10 4 ]]> 60nm <![CDATA[1×10 4 ]]> 62nm <![CDATA[1×10 4 ]]> 70nm <![CDATA[1×10 4 ]]> 80nm <![CDATA[1×10 4 ]]>

[0133] 11) Under the same experimental conditions, the concentration of colloidal gold was changed to 1 / 10,000, 4 / 10,000, and 5 / 10,000 by mass volume to prepare colloidal gold immunochromatographic test strips for detecting influenza virus B antigen. The prepared test strips were used to detect influenza virus B-positive analytes at different concentrations. Each test group was tested in parallel three times. The results are shown in the table below.

[0134] Colloidal gold concentration Detection limit One in ten thousand <![CDATA[4×10 4 ]]> 0.4% <![CDATA[1×10 4 ]]> 50,000ths <![CDATA[1×10 4 ]]>

[0135] 12) While other experimental conditions remained unchanged, the formulations of the sample pad pretreatment solution, conjugate pad pretreatment solution, and test sample diluent were varied to prepare different colloidal gold immunochromatographic test strips for detecting influenza virus B antigen. These different test strips were then used to test 200 negative analytes of the same concentration.

[0136]

[0137]

[0138] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's description and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A colloidal gold immunochromatographic test paper kit, characterized in that: It includes a diluent for the sample to be tested and a colloidal gold immunochromatographic test paper; The colloidal gold immunochromatographic test paper box comprises a card box, which comprises a card cover and a card bottom. The card cover and the card bottom enclose a space for accommodating the colloidal gold immunochromatographic test paper, and the card box is provided with a sample addition area and a color development area. The sample diluent to be tested is: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20, and 0.1%-0.2% fish gelatin; The colloidal gold immunochromatographic test paper comprises a base plate, and a sample pad, a binding pad, a chromatography membrane and a water-absorbing pad sequentially arranged on the base plate, wherein the chromatography membrane is provided with a detection line and a quality control line; The conjugate pad is coated with a colloidal gold-labeled influenza virus B antibody, which is the M1304 clone of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; the colloidal gold-labeled influenza virus B antibody is prepared using a colloidal gold solution with a mass volume concentration of 0.4 parts per million, and the particle size of the colloidal gold is 50 nm to 70 nm. The antibody at the test line includes a first antibody for recognizing influenza virus B antigen, which is the M1303 clone of Hangzhou Huakui Jinpei Biotechnology Co., Ltd.; the antibody at the quality control line includes a second antibody for recognizing the influenza virus B antibody; The material of the sample pad is a glass fiber membrane of model ahlstrom 8964 from Ahlstrom; the material of the conjugate pad is a glass fiber membrane of model ahlstrom 8980 from Ahlstrom; The conjugate pad is treated with a conjugate pad pretreatment solution, wherein the conjugate pad pretreatment solution comprises: 10 mM-20 mM Tris-HCl, 100 mM-150 mM NaCl, 1%-1.5% casein by volume, 3%-5% sucrose by volume, 0.1%-5% Tween-20 by volume, 0.1%-0.2% PEG 20000 by volume, and 0.1%-0.2% proclin 300 by volume; The sample pad is treated with a sample pad pretreatment solution, wherein the components of the sample pad pretreatment solution are: 10mM-20mM PBS, 1%-1.5% Tween-20 by volume, 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.

2. The colloidal gold immunochromatographic test paper kit according to claim 1, wherein The conjugate pad was coated with colloidal gold-labeled influenza virus B antibody at a coating concentration of 10 μl / cm.

3. The colloidal gold immunochromatographic test paper kit according to claim 1 or 2, wherein: The streaking concentration of the first antibody at the detection line is 1 μl / cm.

4. The colloidal gold immunochromatographic test paper kit according to claim 1 or 2, wherein: The sample pad, the conjugate pad, the chromatographic membrane and the absorbent pad are overlapped with each other in sequence on the bottom plate, and the overlap width between the sample pad and the conjugate pad is 1mm-2mm; the overlap width between the conjugate pad and the chromatographic membrane is 1mm-2mm; and the overlap width between the chromatographic membrane and the absorbent pad is 1mm-2mm.

5. The colloidal gold immunochromatographic test paper kit according to claim 1 or 2, wherein: The particle size of the colloidal gold is 60 nm; And / or, the minimum detection limit of the colloidal gold immunochromatographic test paper for detecting influenza virus B antigen is 1×10 4 , unit: number of virus particles / ml.

6. The colloidal gold immunochromatographic test paper kit according to claim 1 or 2, wherein: The preparation method of the colloidal gold immunochromatographic test paper comprises the following steps: A conjugate pad pretreatment solution is prepared, and the conjugate pad is immersed in the conjugate pad pretreatment solution. After soaking for a preset time, the conjugate pad is taken out and dried for standby use, wherein the conjugate pad pretreatment solution comprises: 10mM-20mM Tris-HCl, 100mM-150mM NaCl, a mass volume concentration of 1%-1.5% casein, a mass volume concentration of 3%-5% sucrose, a volume concentration of 0.1%-5% Tween-20, a mass volume concentration of 0.1%-0.2% PEG 20000, and a volume concentration of 0.1%-0.2% proclin 300; Prepare a colloidal gold solution consisting of colloidal gold particles with a mass volume concentration of 0.4 parts per million and a particle size of 50 nm-70 nm, adjust the pH of the colloidal gold solution to 7.0-9.0, then add influenza virus B antibody at a concentration of 10 μg / mL-30 μg / mL, shake and react at room temperature for 4 h-5 h, then add BSA solution with a mass volume concentration of 0.5%-1.0% to cover the exposed gold surface sites not coated with antibodies, block for 30 min-60 min, centrifuge at a speed of 6000 r / min-8000 r / min for 10 min-15 min, remove the supernatant to obtain a 10-fold concentrated immune label, resuspend the immune label with a redissolving solution to obtain a 5-10-fold diluted immune label redissolving solution, apply the immune label redissolving solution to the conjugate pad, dry at 45 ± 2 ° C for 16 h-18 h, and set aside; Prepare a sample pad pretreatment solution, place the sample pad in the sample pad pretreatment solution and soak it. After soaking for a preset time, take out the sample pad and dry it for use. The sample pad pretreatment solution comprises: 10mM-20mM PBS, 1%-1.5% Tween-20 by volume, 0.2%-0.5% S9 by mass volume, 4%-6% sucrose by mass volume, 0.5%-0.8% PEG6000 by mass volume, 1.5%-2% BSA by mass volume, and 0.1%-0.2% proclin300 by volume; Setting a detection line and a quality control line on the chromatographic membrane, immobilizing a first antibody for recognizing influenza virus B antigen on the detection line, and immobilizing a second antibody for recognizing influenza virus B antibody on the quality control line for later use; The sample pad, the binding pad fixed with the immune label, the chromatography membrane fixed with the first antibody and the second antibody, and the absorbent pad are sequentially arranged on the bottom plate to form a colloidal gold immunochromatographic test paper for detecting influenza virus B antigen.

7. The colloidal gold immunochromatographic test paper kit according to claim 6, wherein: The pH of the colloidal gold solution is 7.5; and / or, the concentration of the influenza virus B antibody is 20 μg / ml; And / or, the coating concentration of the immunolabel on the conjugate pad is 10 μl / cm.

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