Colloidal gold immunochromatographic test paper for detecting adenovirus antigen, preparation method thereof, test paper box, and detection method

Abstract

The present invention relates to the field of immunoassay analysis technology, and in particular to a colloidal gold immunochromatographic test paper for detecting adenovirus antigens, a preparation method thereof, a test paper box, and a detection method. The test paper includes a base plate, and a sample pad, a conjugation pad, a chromatographic membrane, and a water-absorbing pad sequentially arranged on the base plate, the chromatographic membrane being provided with a detection line and a quality control line; the conjugation pad is coated with adenovirus antibodies labeled with colloidal gold, the particle size of the colloidal gold being 58-62 nm, the antibodies at the detection line comprising a first antibody for identifying adenovirus antigens, and the antibodies at the quality control line comprising a second antibody for identifying adenovirus antibodies. Using colloidal gold with a particle size of 58-62 nm can increase the effective collision between antibody molecules and colloidal gold, increase the efficiency of antibody-colloidal gold binding, and increase the effective labeled quantity of nanogold, thereby facilitating improvement in the detection sensitivity of adenovirus; and the volume-to-surface area of ​​colloidal gold with a particle size of 58-62 nm is small, the amount of antibody used is small, and the amount of antibody used is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection and analysis, and in particular to a colloidal gold immunochromatographic test paper for detecting adenovirus antigens, a preparation method thereof, a test paper box, and a detection method. 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] Human adenovirus is an important viral pathogen in humans that can cause a variety of diseases, mainly including acute respiratory infections, conjunctivitis, gastrointestinal diseases, etc.

[0004] In the process of preparing immune labels, nanogold solution needs to be used to react with antibodies to label the antibodies on the nanogold surface. During the labeling process, the problem of low labeling efficiency will occur. 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 colloidal gold immunochromatographic test paper for detecting adenovirus antigens, aiming to improve labeling efficiency.

[0006] To achieve the above objectives, the present invention provides a colloidal gold immunochromatographic test paper for detecting adenovirus antigens, comprising a base plate, and a sample pad, a conjugation pad, a chromatographic membrane, and a water-absorbing pad sequentially arranged on the base plate, wherein the chromatographic membrane is provided with a detection line and a quality control line;

[0007] The conjugate pad is coated with colloidal gold-labeled adenovirus antibodies, the particle size of the colloidal gold is 58nm-62nm, the antibodies at the detection line include a first antibody for identifying adenovirus antigens, and the antibodies at the quality control line include a second antibody for identifying the adenovirus antibodies.

[0008] Optionally, the colloidal gold has a particle size of 60 nm;

[0009] And / or, the adenovirus antibody includes the antibody with clone number 7B5M, and the first antibody includes the antibody with clone number 2B10;

[0010] and / or, the second antibody comprises goat anti-mouse IgG;

[0011] And / or, the minimum detection limit of the colloidal gold immunochromatographic test paper for detecting adenovirus antigen is 4×10 4, unit: number of virus particles / ml;

[0012] and / or, the first antibody is a monoclonal antibody or a polyclonal antibody;

[0013] and / or, the streaking concentration of the first antibody at the detection line is 1 ul / cm;

[0014] and / or, the conjugate pad is coated with colloidal gold-labeled adenovirus antibodies at a coating concentration of 10 ul / cm;

[0015] And / or, the material of the base plate includes at least one of polyvinyl chloride, polyethylene and glass;

[0016] And / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom8964;

[0017] And / or, the material of the bonding pad includes glass fiber membrane and / or polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980;

[0018] And / or, the material of the chromatography membrane includes nitrocellulose membrane, and the nitrocellulose membrane includes sartorius140;

[0019] And / or, the sample pad, the conjugate pad, the chromatography membrane and the absorbent pad are sequentially overlapped and attached to the bottom plate.

[0020] The present application also provides a method for preparing a colloidal gold immunochromatographic test paper for detecting adenovirus antigens, comprising the following steps:

[0021] Combining adenovirus antibodies with colloidal gold particles with a particle size of 58 nm to 62 nm to form an immune label, and fixing the immune label on a conjugate pad for later use;

[0022] Setting a detection line and a quality control line on the chromatographic membrane, fixing a first antibody for recognizing adenovirus antigens on the detection line, and fixing a second antibody for recognizing adenovirus antibodies on the quality control line for later use;

[0023] 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 adenovirus antigens.

[0024] Optionally, the step of combining the adenovirus antibody with colloidal gold particles having a particle size of 58 nm to 62 nm to form an immune label and fixing the immune label on a conjugate pad for standby use includes the following steps:

[0025] A colloidal gold solution consisting of colloidal gold particles having a mass volume concentration of 0.4 parts per million and a particle size of 58 nm to 62 nm is prepared, and the pH of the colloidal gold solution is adjusted. Then, an adenovirus antibody having a concentration of 10 μg / mL to 30 μg / mL is added, and the reaction is carried out for a preset time. A blocking agent is then added and blocked for a preset time to obtain a reacted solution and concentrate it to obtain an immune label. The immune label is resuspended in a rehydration solution to obtain an immune label rehydration solution, and the immune label rehydration solution is applied to a conjugate pad, dried, and set aside.

[0026] Optionally, the step of combining the adenovirus antibody with colloidal gold particles having a particle size of 58 nm to 62 nm to form an immune label and fixing the immune label on a conjugate pad for standby use includes the following steps:

[0027] 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 58nm-62nm, and adjust the pH of the colloidal gold solution to 7.0-9.0, then add adenovirus antibodies at a concentration of 10ug / mL-30ug / mL, and oscillate at room temperature for 4h-5h, then add a BSA solution with a mass volume concentration of 0.1%-0.3% to cover the exposed gold surface sites not coated with antibodies, block for 30min-60min, centrifuge at a centrifugal speed of 6000r / min-8000r / min for 10min-15min, remove the supernatant, and obtain a 10-fold concentrated immune label. Use a redissolving solution to resuspend the immune label 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 16h-18h, and set aside.

[0028] Optionally, before the step of combining the adenovirus antibody with colloidal gold particles having a particle size of 58 nm to 62 nm to form an immune label and fixing the immune label on a conjugate pad for standby use, the following steps may also be included:

[0029] Prepare a conjugate pad pretreatment solution, place the conjugate pad in the conjugate pad pretreatment solution and soak it for a preset time, then take out the conjugate pad and dry it for later use;

[0030] The components of the conjugate pad pretreatment solution include 10mM-20mM Tris-HCl, 100mM-150mM NaCI, a mass volume concentration of 1%-1.5% BSA, a mass volume concentration of 3%-5% sucrose, a volume concentration of 0.5%-1% Tween-20, a mass volume concentration of 0.1%-0.2% PEG 20000, and a volume concentration of 0.1%-0.2% proclin 300.

[0031] Optionally, before the step of sequentially placing 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 the colloidal gold immunochromatographic test paper for detecting adenovirus antigens, the following steps are further included:

[0032] Prepare a sample pad pretreatment solution, place the sample pad in the sample pad pretreatment solution and soak it for a preset time, then take out the sample pad and dry it for later use;

[0033] 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.

[0034] Optionally, the pH of the colloidal gold solution is 8.5;

[0035] and / or, the concentration of the adenovirus antibody is 20 μg / ml;

[0036] And / or, the reconstitution solution comprises 50 mM NaCl, BSA with a mass volume concentration of 0.1%, sucrose with a mass volume concentration of 1.5%, and casein sodium with a mass volume concentration of 1.5%;

[0037] And / or, the adenovirus antibody includes the 7B5M clone antibody of Shenzhen Heavy Chain Biotechnology Co., Ltd.;

[0038] And / or, the first antibody comprises the 2B10 clone antibody of Zhuhai Bomei Biotechnology Co., Ltd.;

[0039] and / or, the second antibody comprises goat anti-mouse IgG;

[0040] and / or, the streaking concentration of the first antibody at the detection line is 1 ul / cm;

[0041] and / or, the coating concentration of the immunolabel on the conjugate pad is 10 ul / cm;

[0042] and / or, the concentration of the first antibody is 0.5 mg / mL-2 mg / mL;

[0043] And / or, the material of the base plate includes at least one of polyvinyl chloride, polyethylene and glass;

[0044] And / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom8964;

[0045] And / or, the material of the bonding pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom8980;

[0046] And / or, the material of the chromatography membrane includes nitrocellulose membrane, and the nitrocellulose membrane includes sartorius140.

[0047] The present application also provides a colloidal gold immunochromatographic test paper kit for detecting adenovirus antigens, comprising a test strip, a sample diluent to be tested, and the colloidal gold immunochromatographic test paper for detecting adenovirus antigens as described above, or a colloidal gold immunochromatographic test paper for detecting adenovirus antigens prepared by the method for preparing the colloidal gold immunochromatographic test paper for detecting adenovirus antigens as described above;

[0048] The colloidal gold immunochromatographic test strip kit for detecting adenovirus antigens 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 strip for detecting adenovirus antigens. The card box is provided with a sample addition area and a color development area.

[0049] The test sample diluent includes: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20 and 0.1%-0.2% fish gelatin.

[0050] The present application also provides a detection method, which uses the colloidal gold immunochromatographic test paper for detecting adenovirus antigens as described above to detect a sample to be tested, comprising the following steps:

[0051] Add the sample to be tested to the sample pad of the colloidal gold immunochromatographic test paper for detecting adenovirus antigens, and perform immunochromatographic reaction for 10-15 minutes;

[0052] The test results are obtained by observing the colors of the test line and the quality control line on the colloidal gold immunochromatographic test paper for detecting adenovirus antigens.

[0053] The colloidal gold immunochromatographic test paper for detecting adenovirus antigens of the present application comprises a base plate, and a sample pad, a conjugation pad, a chromatographic membrane and a water-absorbing pad sequentially arranged on the base plate, wherein a detection line and a quality control line are provided on the chromatographic membrane; the conjugation pad is coated with adenovirus antibodies labeled with colloidal gold, the particle size of the colloidal gold is 58nm-62nm, the antibodies at the detection line include a first antibody for identifying adenovirus antigens, and the antibodies at the quality control line include a second antibody for identifying adenovirus antibodies. The use of colloidal gold with a particle size of 58-62nm can increase the effective collision between antibody molecules and colloidal gold, increase the efficiency of antibody-colloidal gold binding, increase the effective labeling quantity of nanogold, and coat the highly efficiently labeled nanogold to the conjugation pad to increase the number of effectively labeled nanogold on the conjugation pad, thereby facilitating the improvement of the detection sensitivity of adenovirus; and the volume-to-surface area of ​​colloidal gold with a particle size of 58-62nm is small, the amount of antibody used is small, and the amount of antibody used is reduced, 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

[0054] 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.

[0055] Figure 1 This is a schematic structural diagram of a colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to an embodiment of the present application;

[0056] Figure 2 This is a schematic flow chart of the preparation method of the colloidal gold immunochromatographic test paper for detecting adenovirus antigens in the present application;

[0057] Figure 3 This is the colloidal gold immunochromatographic reagent test card of this application;

[0058] Figure 4 This is the UV-visible spectrum of colloidal gold.

[0059] Description of Figure Numbers:

[0060]

[0061]

[0062] 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

[0063] The following describes in detail, with appropriate reference to the accompanying drawings, embodiments of the colloidal gold immunochromatographic test strips for detecting adenovirus antigens disclosed herein. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of substantially identical structures may be omitted. This is to avoid unnecessary redundancy 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 facilitate a thorough understanding of the present application by those skilled in the art and are not intended to limit the subject matter recited in the claims.

[0064] 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.

[0065] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] Adenovirus (HAdV) is a non-enveloped, double-stranded DNA virus originally isolated from human glandular cells. Over 50 serotypes have been identified, often causing a range of symptoms in children, including respiratory infections, conjunctivitis, and gastroenteritis. Children infected with adenovirus are primarily between the ages of 6 months and 2 years old. Without prompt treatment, adenovirus infection can easily develop into a severe illness, affecting multiple organs, including the brain, liver, and kidneys, and can even lead to death.

[0072] Improving the sensitivity of adenovirus detection can timely and effectively detect adenovirus infection, provide early treatment, and effectively protect human health and safety.

[0073] Improving labeling efficiency is beneficial to improving detection sensitivity. To this end, the present application provides a colloidal gold immunochromatographic test paper for detecting adenovirus antigens, which has high labeling efficiency to improve detection sensitivity.

[0074] The colloidal gold immunochromatographic test paper for detecting adenovirus antigens includes a base plate, and a sample pad, a conjugation pad, a chromatographic membrane and a water-absorbing 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 adenovirus antibodies labeled with colloidal gold, and the particle size of the colloidal gold is 58nm-62nm. The antibodies at the detection line include a first antibody for identifying adenovirus antigens, and the antibodies at the quality control line include a second antibody for identifying adenovirus antibodies.

[0075] like Figure 1 As shown, the colloidal gold immunochromatographic test paper 100 for detecting adenovirus antigens 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. The solid phase carrier labeled with the antigen or antibody fixed on the conjugation pad undergoes a specific immune reaction with the analyte in the sample to form a complex that passes through the chromatography on the chromatographic membrane and undergoes a specific immune reaction 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.

[0076] In the present application, the conjugate pad is coated with colloidal gold-labeled adenovirus antibodies, the antibodies at the detection line include a first antibody for identifying adenovirus antigens, and the antibodies at the control line include a second antibody for identifying adenovirus antibodies. When the sample to be tested is dropped on the sample pad, the adenovirus in the sample flows on the sample pad. After reaching the conjugation pad, the adenovirus specifically binds to the colloidal gold-labeled adenovirus antibody on the conjugation pad to form an "antigen-antibody-gold conjugate". At the same time, the "antigen-antibody-gold conjugate" is released from the conjugation pad. Through capillary action, the "antigen-antibody-gold conjugate" flows toward the chromatographic membrane. 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 gold nanoparticles to aggregate and show color at the test line. At the same time, the colloidal gold-labeled adenovirus antibodies that are not specifically bound to the adenovirus antibodies continue to flow toward the quality control line and bind to the second antibody after reaching the quality control line to form a "secondary antibody-antibody-gold conjugate". The gold nanoparticles aggregate and show color at the quality control line. The color of the quality control line indicates that the test paper is valid.

[0077] The present application uses colloidal gold with a particle size of 58nm-62nm, so that when preparing colloidal gold-labeled adenovirus antibodies, the effective collision between antibody molecules and colloidal gold can be increased, the binding efficiency of antibodies and colloidal gold can be 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 beneficial to improving the detection sensitivity of adenovirus; and the volume specific surface area of ​​colloidal gold with a particle size of 58nm-62nm is small, the amount of antibody used is small, and the amount of antibody used is reduced. That is, the present application not only improves the labeling efficiency of colloidal gold, but also saves the cost of antibodies.

[0078] In the above-mentioned 58nm-62nm, 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 58nm, 59nm, 60nm, 61nm, 62nm, etc.

[0079] Furthermore, the particle size of the colloidal gold is 60 nm; and / or, the adenovirus antibody comprises the 7B5M clone antibody, and the first antibody comprises the 2B10 clone 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 adenovirus antigens is 4×10 4 , the 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 adenovirus antibodies labeled with colloidal gold 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 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 bottom plate.

[0080] 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.

[0081] The adenovirus antibodies in this application include the 7B5M clone antibody of Shenzhen Heavy Chain Biotechnology Co., Ltd., and the first antibody includes the 2B10 clone antibody of Zhuhai Bomei Biotechnology Co., Ltd. By screening adenovirus antibodies from different suppliers at home and abroad, including antibody pairs from Shenzhen Heavy Chain Biotechnology Co., Ltd., Zhuhai Bomei Biotechnology Co., Ltd., Shenzhen Heavy Chain Biotechnology Co., Ltd., Hangzhou Xianzhi Biotechnology Co., Ltd., and Xiamen Tongrenxin Biotechnology Co., Ltd., the screened antibody pair is the 2B10 clone antibody of Zhuhai Bomei Biotechnology Co., Ltd. and the 7B5M clone antibody of Shenzhen Heavy Chain Biotechnology Co., Ltd. The above-screened antibody pair has high sensitivity.

[0082] 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.

[0083] The minimum detection limit of the colloidal gold immunochromatographic test paper for adenovirus antigen is 4×10 4 , unit: number of virus particles / ml; a conjugate pad coated with adenovirus antibodies labeled with colloidal gold, the particle size of the colloidal gold is 58nm-62nm, which can effectively improve the detection sensitivity of the test paper. The specific steps of the detection limit test are as follows: prepare the colloidal gold immunochromatographic test paper for detecting adenovirus antigens of the present application, the detection antigen is adenovirus culture, and the concentration is 1×10 9 The inactivated adenovirus 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 adenovirus antigens of the present application was 4×10 4 , unit: number of virus particles / ml.

[0084] 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 adenovirus antigens, which can be a monoclonal antibody or a polyclonal antibody.

[0085] 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 recognizing adenovirus antigens, 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.

[0086] The conjugate pad is coated with colloidal gold-labeled adenovirus antibodies at a coating concentration of 10 ul / cm. In the process of coating the conjugate pad with the colloidal gold-labeled adenovirus antibodies, the colloidal gold-labeled adenovirus antibodies are coated on the conjugate pad by spraying. The concentration of the colloidal gold-labeled adenovirus antibodies coated on the conjugate pad is 10 ul / cm.

[0087] 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 conjugate 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, conjugate pad, chromatography membrane, and absorbent pad are sequentially attached to the base plate in an overlapping manner.

[0088] Furthermore, the present application provides a method for preparing a colloidal gold immunochromatographic test paper for detecting adenovirus antigens, comprising the following steps: combining adenovirus antibodies with colloidal gold particles with a particle size of 58nm-62nm to form an immune label, fixing the immune label to a binding pad for later use; setting a detection line and a quality control line on a chromatographic membrane, fixing a first antibody for identifying adenovirus antigens on the detection line, and fixing a second antibody for identifying adenovirus antibodies on the quality control line for later use; and arranging a sample pad, a binding pad fixed with the immune label, a chromatographic membrane fixed with the first antibody and the second antibody, and a water-absorbing pad in sequence on a bottom plate to form a colloidal gold immunochromatographic test paper for detecting adenovirus antigens.

[0089] like Figure 2 The figure shows a process flow diagram of a method for preparing a colloidal gold immunochromatographic test paper for detecting adenovirus antigens. Adenovirus antibodies are combined with colloidal gold particles with a particle size of 58nm-62nm 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 recognizing adenovirus antigens is fixed to the test line, and a second antibody for recognizing adenovirus antibodies is fixed to the quality control line for later use. A sample pad, a conjugate pad with the immune label fixed, a chromatographic membrane with the first and second antibodies fixed, and a water-absorbing pad are sequentially placed on a base plate to form a colloidal gold immunochromatographic test paper for detecting adenovirus antigens.

[0090] By using colloidal gold with a particle size of 58nm-62nm, when preparing colloidal gold-labeled adenovirus antibodies, the effective collision between antibody molecules and colloidal gold can be increased, the binding efficiency of the antibody and colloidal gold can be increased, and the effective labeled amount of nanogold can be increased. The highly efficiently labeled nanogold is coated on a binding pad, and the amount of effectively labeled nanogold on the binding pad is increased, which is beneficial to improving the detection sensitivity of adenovirus. In addition, the volume specific surface area of ​​colloidal gold with a particle size of 58nm-62nm 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.

[0091] Furthermore, in the step of combining adenovirus antibodies with colloidal gold particles with a particle size of 58nm-62nm 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 58nm-62nm, and adjusting the pH of the colloidal gold solution, then adding adenovirus 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.

[0092] 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.

[0093] A colloidal gold solution of 0.4 parts per million is used, and the particle size of the nanogold is 58nm-62nm. 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 58nm-62nm 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.

[0094] In the process of preparing the immune label, the pH of the colloidal gold solution is adjusted, and then adenovirus 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 allowed to proceed 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.

[0095] In the above-mentioned 10ug / mL-30ug / mL adenovirus antibody, 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 10ug / mL, 15ug / mL, 20ug / mL, 25ug / mL, 21ug / mL, 22ug / mL, 23ug / mL, 24ug / mL, 30ug / mL, etc.

[0096] Furthermore, in the step of combining adenovirus antibodies with colloidal gold particles with a particle size of 58nm-62nm 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 58nm-62nm, and adjusting the pH of the colloidal gold solution, then adding adenovirus 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.

[0097] 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.

[0098] 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.

[0099] Furthermore, in the step of combining adenovirus antibodies with colloidal gold particles with a particle size of 58nm-62nm 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 58nm-62nm, and adjusting the pH of the colloidal gold solution to 7.0-9.0, then adding adenovirus antibodies with a concentration of 10ug / mL-30ug / mL, shaking the reaction at room temperature for 4h-5h, and then adding a colloidal gold solution with a mass volume concentration of 0.1%-0.3% The exposed gold surface sites not coated with antibodies were covered with BSA solution and blocked for 30-60 minutes. The cells were centrifuged at 6000-8000 r / min for 10-15 minutes. The supernatant was removed to obtain a 10-fold concentrated immune label. The immune label was resuspended in a rehydration solution to obtain a 5-10-fold diluted immune label rehydration solution. The immune label rehydration solution was applied to the conjugate pad and dried at 45±2°C for 16-18 hours for later use.

[0100] 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 0.1%-0.3% 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.

[0101] 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.

[0102] 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.

[0103] In the above 0.1%-0.3% BSA solution, 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 0.1%, 0.2%, 0.3%, etc.

[0104] Furthermore, the pH of the colloidal gold solution is 8.5; and / or the concentration of the adenovirus 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 adenovirus antibody includes the 7B5M clone antibody of Shenzhen Heavy Chain Biotechnology Co., Ltd.; and / or the first antibody includes the 2B10 clone antibody of Zhuhai Bomei 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 sartorius140.

[0105] Since different antibodies have different isoelectric points, 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, and 8.5 were explored. When the pH value was 8.5, the antibody was positively charged in the colloidal gold solution and adsorbed on the negatively charged colloidal gold surface. The labeling efficiency was the highest at this time. Therefore, the preferred pH value was 8.5.

[0106] The concentration of adenovirus antibody is 20 μg / ml to avoid incomplete labeling caused by too low adenovirus antibody concentration and waste of antibody caused by too high a concentration.

[0107] The reconstitution solution includes 50 mM NaCl, 0.1% BSA by volume, 1.5% sucrose by volume, and 1.5% sodium caseinate by volume.

[0108] The adenovirus antibody includes the 7B5M clone antibody of Shenzhen Heavy Chain Biotechnology Co., Ltd.; the first antibody includes the 2B10 clone antibody of Zhuhai Bomei Biotechnology Co., Ltd.; the second antibody includes goat anti-mouse IgG; the coating concentration of the immune label on the conjugate pad is 10ul / cm, and the above-mentioned colloidal gold immunochromatographic test paper is used, the obtained immune label has a high labeling efficiency, and the concentration of the colloidal gold-labeled adenovirus antibody coated on the conjugate pad is 10ul / cm. The coating of the high-concentration colloidal gold-labeled adenovirus antibody helps to effectively detect low-concentration adenovirus; 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 ahlstrom8964; the material of the conjugate pad includes glass fiber membrane, and the glass fiber membrane includes ahlstrom8980; the material of the chromatography membrane includes nitrocellulose membrane, and the nitrocellulose membrane includes Sartorius140.

[0109] The steps of fixing the first antibody at the detection line are as follows: preparing a first antibody solution with a concentration of 0.5 mg / mL-2 mg / mL for identifying adenovirus antigens, streaking the first antibody solution on the chromatographic membrane with a three-dimensional streaking instrument, forming a detection line on the chromatographic membrane, drying, and setting aside. At this time, the streaking concentration of the first antibody at the detection line is 1 ul / cm. The first antibody solution with a concentration of 0.5 mg / mL-2 mg / mL avoids too low a concentration, insufficient fixed amount of the first antibody, and unclear color development at the detection line. At the same time, it also avoids too high a concentration, resulting in antibody waste. In the above-mentioned 0.5 mg / mL-2 mg / mL, 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 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, etc.

[0110] Furthermore, before the step of combining adenovirus antibodies with colloidal gold particles with a particle size of 58nm-62nm 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% BSA, a mass volume concentration of 3%-5% sucrose, a volume concentration of 0.5%-1% Tween-20, a mass volume concentration of 0.1%-0.2% PEG 20000, and a volume concentration of 0.1%-0.2% proclin 300.

[0111] 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, promoting better binding of the analyte to the immunolabel. Sucrose protects the antibody protein labeled with the colloidal gold, stabilizing the antibody. BSA also 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.

[0112] 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 BSA 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 0.5%-1% 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 0.5%, 0.7%, 0.9%, and 1%. The concentration range of 0.1%-0.2% PEG 20000 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%.

[0113] Among them, the soaking time is 20min-30min, the drying temperature is 45℃-50℃, and the drying time is 16h-18h.

[0114] The conjugate pad treatment procedure involves preparing a conjugate pad pretreatment solution consisting of 10-20 mM Tris-HCl, 100-150 mM NaCl, 1-1.5% BSA, 3-5% sucrose, 0.5-1% 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 30 cm × 30 cm × 10 cm in size. The conjugate pad is made of a glass cellulose membrane (Ahlstrom 8980) and placed in the solution, ensuring that the membrane is submerged in the solution for approximately 20-30 minutes. After soaking, the glass cellulose membrane was fished out, spread on a sieve, and placed in a forced air drying oven set at 45-50°C for about 16-18 hours. After drying, it was used as a pre-treated conjugate pad and sealed and stored at room temperature for later use.

[0115] Furthermore, before the step of sequentially placing 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 adenovirus antigens, 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.

[0116] 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 stable pH environment, the sample pad protects antibodies in the sample and reduces nonspecific binding, thus facilitating the effective detection of low-concentration adenoviruses and avoiding false positives.

[0117] 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%.

[0118] Among them, the soaking time is 20min-30min, the drying temperature is 45℃-50℃, and the drying time is 16h-18h.

[0119] 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.

[0120] 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.

[0121] The present application also provides a colloidal gold immunochromatographic test paper kit for detecting adenovirus antigens, comprising a test strip, a sample diluent to be tested, and the above-mentioned colloidal gold immunochromatographic test paper for detecting adenovirus antigens, or a colloidal gold immunochromatographic test paper for detecting adenovirus antigens prepared by the above-mentioned method for preparing the colloidal gold immunochromatographic test paper for detecting adenovirus antigens.

[0122] Furthermore, a colloidal gold immunochromatographic test strip kit for detecting adenovirus antigens includes a cartridge and the above-mentioned colloidal gold immunochromatographic test strip for detecting adenovirus antigens, the cartridge including a cartridge cover and a cartridge bottom, the cartridge cover and the cartridge bottom enclosing a space for accommodating the colloidal gold immunochromatographic test strip for detecting adenovirus antigens, the cartridge being provided with a sample addition area and a color development area; and / or, the test sample diluent includes: 10mM-20mM PBS, 0.05%-0.1% Triton X100, 0.05%-0.1% Tween 20, and 0.1%-0.2% fish gelatin.

[0123] like Figure 3 As shown, the colloidal gold immunochromatographic reagent detection card of the present application includes a card cover and a card bottom. The card cover and the card bottom enclose a space for accommodating a colloidal gold immunochromatographic test paper for detecting adenovirus antigens. The card is provided with a sample addition area and a color development area. When testing a sample, the diluted sample is added to the sample addition area. After the reaction time, the color is observed in the color development area to determine the test result.

[0124] 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.

[0125] 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%.

[0126] The present application also provides a detection method, which uses the above-mentioned colloidal gold immunochromatographic test paper for detecting adenovirus antigens 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 paper for detecting adenovirus antigens, and allowing the immunochromatographic reaction to proceed for 10 minutes to 15 minutes; observing the colors of the test line and the quality control line on the colloidal gold immunochromatographic test paper for detecting adenovirus antigens to obtain the test results.

[0127] Using the above-mentioned detection method, the colloidal gold test strips detect adenovirus antigens based on the specific reaction between antigen and antibody. Adenovirus antigens can be detected through a one-time operation, and the reading of the results is quick and intuitive, with the test results available within 15 minutes. Furthermore, the method for detecting adenovirus antigens of the present invention does not require special instruments or equipment, nor does it require the operation of professionals, thus eliminating the reliance on professional instruments. Compared with the traditional double-antibody sandwich method, the detection method of the present invention improves the gold-labeled colloidal gold labeling efficiency and detection sensitivity by increasing the gold concentration (increased from 1 / 10,000 to 4 / 10,000) and the particle size (increased from 40nm to 60nm). The overall compliance rate of the test results of the present invention is high, with a specificity of 97% and a sensitivity of 96%, making it suitable for on-site detection.

[0128] Example

[0129] 1) Preparation and characterization of colloidal gold

[0130] 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.

[0131] 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.

[0132] According to the above-mentioned colloidal gold preparation method, the results showed that the colloidal gold solution was wine red. The colloidal gold sample was tested by UV-visible spectrum: Figure 4 As shown, the prepared colloidal gold peak is located at 539 nm and the peak is narrow, indicating that the prepared gold particles have uniform particle size, the gold particle size is 60 nm, and the dispersion is good.

[0133] 2) Conjugate pad pretreatment

[0134] Prepare a conjugate pad pretreatment solution consisting of 15mM Tris-HCl, 125mM NaCl, 1.25% BSA, 4% sucrose, 0.75% Tween-20, 0.15% PEG 20000, and 0.15% proclin 300. Pour the solution into an acrylic or stainless steel container approximately 30cm x 30cm x 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.

[0135] 3) Sample pad processing

[0136] Prepare a sample pad treatment solution consisting of 15 mM PBS, 1.25% Tween-20, 0.25% S9, 5% sucrose, 0.75% PEG 6000, 1.75% BSA, and 0.15% Proclin 300. Pour the solution into an acrylic or stainless steel container. Use an ahlstrom 8964 glass cellulose membrane as the sample 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 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.

[0137] 4) Preparation of gold label-antibody conjugate pad

[0138] 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 was prepared, and the pH of the colloidal gold solution was adjusted to 8.5. Then, an adenovirus antibody (7B5M clone antibody from Shenzhen Heavy Chain Biotechnology Co., Ltd.) with a concentration of 20 ug / mL was added, and the reaction was shaken at room temperature for 4.5 hours. Then, a BSA solution with a mass volume concentration of 0.15% was added to cover the exposed gold surface sites not coated with the antibody, blocked for 50 minutes, and centrifuged at a centrifugal speed of 7000 r / min for 15 minutes. The supernatant was removed to obtain a 10-fold concentrated immune label, and the immune label was resuspended with a redissolving solution to obtain a 70-fold diluted immune label redissolving solution. The immune label redissolving solution was applied to the conjugate pad and dried at 4542°C for 17 hours for standby use.

[0139] 5) Assembly of colloidal gold immunochromatographic test strips for detecting adenovirus antigens

[0140] The goat anti-mouse IgG (secondary antibody) with the batch number EC00102DE from Ningbo Maiyue Biotechnology Co., Ltd. and the adenovirus 2B10 antibody (primary antibody) (clone 2B10 antibody from Zhuhai Bomei 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 conjugate pad, nitrocellulose membrane and absorbent paper were attached to the bottom plate in sequence. Figure 1 Just assemble it.

[0141] The prepared test strips are used to detect the analyte. The sample diluent consists of 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 the diluent are added. After 15 minutes, the test results are observed. If both the test line and the quality control line display red bands, it indicates that the test sample contains adenovirus antigens. If the test sample does not contain adenovirus antigens, no red band will appear when the sample moves to the test line, and only a red band will appear at the quality control line. If the quality control line does not display color, the test strip is invalid and the sample needs to be retested.

[0142] 6) Screening of adenovirus antibody pairs

[0143] Screening step: With other conditions unchanged, different adenovirus antibody pairs (adenovirus antibody and primary antibody) were used to prepare different gold label-antibody conjugate pads and chromatographic membranes, which were assembled into different test strips. Under the same conditions, different test strips were used to test the analyte, and the sensitivity of each test strip was tested. The results are shown in the table below. The test strips prepared with the 2B10 clone antibody (primary antibody) of Zhuhai Bomei Biotechnology Co., Ltd. and the 7B5M clone antibody (adenovirus antibody) of Shenzhen Heavy Chain Biotechnology Co., Ltd. had higher sensitivity. Therefore, these adenovirus antibodies were selected.

[0144] Table 1 Screening list of different antibody pairs

[0145]

[0146] 7) Stability test of colloidal gold immunochromatographic test paper for detecting adenovirus antigen

[0147] Three batches of adenovirus antigen test strips were produced continuously, using a concentration of 1×10 9 The inactivated adenovirus culture was tested and diluted to 1 × 10 7 , 1×10 5 , 4×104 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 , 4×10 4 Adenovirus culture was tested, and the test results were indistinguishable. The long-term stability was converted using the Nieuw's equation, and it could be stably stored at 4°C-30°C for 18 months, and the test card would not show abnormal results, indicating that the prepared adenovirus antigen test strips had good stability.

[0148] 8) Specificity test of colloidal gold immunochromatographic test paper for detecting adenovirus antigen

[0149] The prepared test strips were used to detect other pathogens (including rotavirus, influenza virus A, influenza virus B, 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 adenovirus positive reference samples, 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 did not cross-react with other pathogens.

[0150] Table 2 List of different pathogens detected

[0151] pathogens Test results Rotavirus Negative Influenza virus A Negative Influenza virus B 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 Adenovirus positive reference 1 Positive Adenovirus positive reference 2 Positive Adenovirus positive reference 3 Positive Adenovirus positive reference 4 Positive Adenovirus positive reference 5 Positive

[0152] 9) Detection limit comparison experiment

[0153] The adenovirus antigen detection test strip prepared in this application was compared with the test kit of Guangzhou Wondfo Biotechnology Co., Ltd., in which the detection antigen was adenovirus culture and the concentration was 1×10 9 The inactivated adenovirus culture was tested and diluted to 1 × 10 7 , 1×10 5 , 2×10 5 , 4×10 4 The test results are shown in the table below. The minimum detection limit of the colloidal gold immunochromatographic test strip for detecting adenovirus antigen prepared in this application is 4×10 4 The minimum detection limit of the colloidal gold immunochromatographic test strip for adenovirus antigen detection produced by Guangzhou Wondfo Biotechnology Co., Ltd. is 2×10 5 The sensitivity of the colloidal gold immunochromatographic test strip for detecting adenovirus antigen prepared by this method is higher than that of the test kit of Guangzhou Wondfo Biotechnology Co., Ltd.

[0154]

[0155] 10) Comparative experiment of different colloidal gold particle sizes

[0156] Under the same experimental conditions, the colloidal gold particle size was changed to 40nm, 58nm, 60nm, 62nm, and 70nm to prepare colloidal gold immunochromatographic test strips for detecting adenovirus antigens. The prepared different test strips were used to detect adenovirus-positive analytes of different concentrations. Each group of tests was tested in parallel 3 times. The results are shown in the following table.

[0157] Colloidal gold particle size Detection limit 40nm <![CDATA[2×10 5 ]]> 58nm <![CDATA[4×10 4 ]]> 60nm <![CDATA[4×10 4 ]]> 62nm <![CDATA[4×10 4 ]]> 70nm <![CDATA[4×10 4 ]]>

[0158] 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 adenovirus antigens. The prepared different test strips were used to detect adenovirus-positive analytes of different concentrations. Each test group was tested in parallel 3 times. The results are shown in the following table.

[0159] Colloidal gold concentration Detection limit One in ten thousand <![CDATA[3×10 5 ]]> 0.4% <![CDATA[4×10 4 ]]> 50,000ths <![CDATA[4×10 4 ]]>

[0160] 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 adenovirus antigens. These different test strips were then used to test 200 negative analytes of the same concentration.

[0161]

[0162]

[0163] 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 for detecting adenovirus antigens, characterized in that: The colloidal gold immunochromatographic test paper for detecting adenovirus antigens 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 colloidal gold-labeled adenovirus antibodies, the colloidal gold having a particle size of 58 nm to 62 nm. The antibodies at the test line include a primary antibody for recognizing adenovirus antigens, and the antibodies at the control line include a secondary antibody for recognizing the adenovirus antibodies. The adenovirus antibodies are clone 7B5M from Shenzhen Heavy Chain Biotechnology Co., Ltd., and the primary antibody is clone 2B10 from Zhuhai Bomei Biotechnology Co., Ltd. The conjugate pad is treated with a conjugate pad pretreatment solution, wherein the conjugate pad pretreatment solution comprises: 15 mM Tris-HCl, 125 mM NaCl, 1.25% BSA by mass volume concentration, 4% sucrose by mass volume concentration, 0.75% Tween-20 by volume concentration, 0.15% PEG 20000 by mass volume concentration, and 0.15% proclin 300 by volume concentration; The sample pad was treated with a sample pad pretreatment solution, wherein the components of the sample pad pretreatment solution were: 15 mM PBS, 1.25% Tween-20 by volume, 0.25% S9 by mass volume, 5% sucrose by mass volume, 0.75% PEG6000 by mass volume, 1.75% BSA by mass volume, and 0.15% proclin 300 by volume.

2. The colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to claim 1, wherein The particle size of the colloidal gold is 60 nm; 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 adenovirus antigen is 4×10 4 , unit: number of virus particles / ml; and / or, the streaking concentration of the first antibody at the detection line is 1 μl / cm; and / or, the conjugate pad is coated with colloidal gold-labeled adenovirus antibodies at a coating concentration of 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 chromatography membrane comprises nitrocellulose membrane, and the nitrocellulose membrane comprises sartorius 140; And / or, the sample pad, the conjugate pad, the chromatography membrane and the absorbent pad are sequentially overlapped and attached to the bottom plate.

3. A method for preparing a colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to claim 1, characterized in that: The following steps are involved: Combining adenovirus antibodies with colloidal gold particles with a particle size of 58 nm to 62 nm to form an immune label, and fixing the immune label on a conjugate pad for later use; Setting a detection line and a quality control line on the chromatographic membrane, fixing a first antibody for recognizing adenovirus antigens on the detection line, and fixing a second antibody for recognizing adenovirus antibodies on the quality control line for later use; The conjugate pad is treated with the conjugate pad pretreatment solution, and the sample pad is treated with the sample pad pretreatment solution; 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 adenovirus antigens.

4. The method for preparing the colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to claim 3, wherein: The steps of combining adenovirus antibodies with colloidal gold particles having a particle size of 58 nm to 62 nm to form an immune label and fixing the immune label on a conjugate pad for standby use include the following steps: A colloidal gold solution consisting of colloidal gold particles having a mass volume concentration of 0.4 parts per million and a particle size of 58 nm to 62 nm is prepared, and the pH of the colloidal gold solution is adjusted. Then, an adenovirus antibody having a concentration of 10 μg / mL to 30 μg / mL is added, and the reaction is carried out for a preset time. A blocking agent is then added and blocked for a preset time to obtain a reacted solution and concentrate it to obtain an immune label. The immune label is resuspended in a rehydration solution to obtain an immune label rehydration solution, and the immune label rehydration solution is applied to a conjugate pad, dried, and set aside.

5. The method for preparing the colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to claim 3 or 4, wherein: The steps of combining adenovirus antibodies with colloidal gold particles having a particle size of 58 nm to 62 nm to form an immune label and fixing the immune label on a conjugate pad for standby use include the following steps: 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 58 nm-62 nm, adjust the pH of the colloidal gold solution to 7.0-9.0, then add adenovirus antibodies at a concentration of 10 ug / mL-30 ug / mL, shake and react at room temperature for 4 h-5 h, then add a BSA solution with a mass volume concentration of 0.1%-0.3% to cover the exposed gold surface sites not coated with antibodies, 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 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.

6. The method for preparing the colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to claim 4 or 5, characterized in that: The pH of the colloidal gold solution is 8.5; and / or, the concentration of the adenovirus antibody is 20 μg / ml; And / or, the reconstitution solution comprises 50 mM NaCl, BSA with a mass volume concentration of 0.1%, sucrose with a mass volume concentration of 1.5%, and casein sodium with a mass volume concentration of 1.5%; and / or, the second antibody comprises goat anti-mouse IgG; and / or, the streaking concentration of the first antibody at the detection 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 base plate includes 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 bonding pad comprises a glass fiber membrane, and the glass fiber membrane comprises ahlstrom 8980; And / or, the material of the chromatography membrane includes nitrocellulose membrane, and the nitrocellulose membrane includes Sartorius 140.

7. A colloidal gold immunochromatographic test kit for detecting adenovirus antigens, characterized in that: The invention comprises a test strip, a sample diluent to be tested and a colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to any one of claims 1 to 2, or a colloidal gold immunochromatographic test paper for detecting adenovirus antigens prepared by the method for preparing a colloidal gold immunochromatographic test paper for detecting adenovirus antigens according to any one of claims 3 to 6; The colloidal gold immunochromatographic test strip kit for detecting adenovirus antigens 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 strip for detecting adenovirus antigens, and the card box is provided with a sample addition area and a color development area. The sample diluent to be tested is: 15 mM PBS, 0.075% Triton X100, 0.075% Tween 20 and 0.15% fish gelatin.

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