Test strip, kit and method for detecting monkey pox virus antigen

Abstract

The invention belongs to the field of biological diagnosis, and relates to a monkey pox virus antigen detection test strip, a kit and a method.The test strip comprises a PVC bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, wherein the combination pad is provided with a tracer marker labeled anti-monkey pox virus E8L protein monoclonal antibody, and the tracer marker is colloidal gold; a detection line and a quality control line which are parallel to each other are arranged on the nitrocellulose membrane, the detection line is coated with a monkey pox virus E8L protein monoclonal antibody, and the quality control line is coated with Super C +. The monkey pox virus can be accurately and rapidly detected, the marking method is simple and convenient, the affinity with the monkey pox virus is high, and the sensitivity and specificity of the product are effectively improved.

Description

Technical Field

[0001] The present application relates to the field of biological diagnosis technology, and in particular to a monkeypox virus antigen detection test strip, a kit and a method thereof. Background Art

[0002] Since smallpox was eradicated in the 1980s, monkeypox virus has become the most important orthopoxvirus in public health, primarily endemic in west and central Africa. However, since May 7, 2022, human cases of monkeypox virus infection have been reported in more than 100 countries outside the African epidemic zone.

[0003] Monkeypox virus belongs to the genus Orthopoxvirus of the family Poxviridae. It is one of the four Orthopoxviruses that are lethal to humans, the other three being smallpox virus, vaccinia virus, and cowpox virus. Monkeypox virus is a homogeneous core surrounded by an outer membrane. Like smallpox virus, vaccinia virus, and cowpox virus, it contains soluble antigens, core protein antigens, and hemagglutinins. The antigenic properties are basically the same, and there is cross-immunity between them. Monkeypox virus is an enveloped double-stranded DNA virus. Compared with RNA viruses, DNA viruses are better at detecting and repairing their own mutations. The high-fidelity replication characteristics of DNA mean that monkeypox virus is unlikely to undergo very rapid and continuous mutations in a short period of time like the new coronavirus (a rapidly evolving RNA virus).

[0004] The incubation period of monkeypox virus is long, typically 7 to 14 days, with a maximum of 21 days. The course of monkeypox infection is divided into two main phases: the prodromal phase, which lasts approximately 0 to 2 days and is characterized by fever, fatigue, severe headache, swollen lymph nodes, and muscle aches; and the rash phase, which lasts 7 to 21 days. The rash typically begins within 1 to 5 days of fever, and once the rash appears, the patient is contagious. The rash is concentrated on the face and extremities, affecting the face (95%), palms and soles (75%), oral mucosa (70%), genitals (20%), and conjunctivae (20%). The rash lasts approximately 2 to 4 weeks, evolving from plaques to papules, blisters, pustules, and crusting, which then slough off. Herpes and pustules are often spherical, approximately 0.5 to 1 cm in diameter, firm in texture, and may be accompanied by significant itching and pain. Monkeypox is a zoonotic disease caused by the monkeypox virus, which is mainly transmitted through contact with skin and mucosal lesions, respiratory secretions, blood and other body fluids of infected animals or humans.

[0005] According to the "Technical Guidelines for Monkeypox Prevention and Control (2022 Edition)" issued by the General Office of the National Health Commission, the recommended sample type for laboratory confirmation of monkeypox is skin lesion material, including lesion rash, swabs of blister surface and / or exudate, blister fluid, blister epidermis or scabs. The specimen should be refrigerated (2 to 8°C) or frozen (-20°C or lower) within 1 hour after collection. At present, the main detection method for monkeypox virus at home and abroad is biological real-time fluorescence quantitative PCR, which requires equipment and has high requirements for detection equipment or platform. The detection steps are cumbersome and the detection cycle is long, which is not conducive to rapid screening during a virus pandemic. It cannot quickly, accurately and safely detect a large number of suspected cases, asymptomatic infection cases in the incubation period, and asymptomatic virus carriers at higher risk.

[0006] Colloidal gold labeling technology was developed in the 1980s, following the development of fluorescein, radioisotopes, and enzymes. In 1971, Faulk and Taylor introduced colloidal gold into immunochemistry. Since then, this labeling method has been widely used in drug testing, biomedical testing, agricultural and veterinary drug testing, and clinical disease detection. Colloidal gold labeling, which uses colloidal gold as a tracer, was initially applied in immunoelectron microscopy. With the development of immunology, it has been gradually applied to animal agglutination tests, immunoblotting, light microscopy staining, immunospots, and immunochromatography. Colloidal gold immunochromatography uses colloidal gold as a tracer for antigen-antibody detection. Colloidal gold immunochromatography offers advantages such as low preparation cost, simple operation, direct visual interpretation of results, and short detection time. Currently, kits for detecting monkeypox virus using colloidal gold immunochromatography have poor specificity and low sensitivity, and there are no antigen detection kits for the monkeypox virus E8L protein. Summary of the Invention

[0007] The purpose of this application is to propose a monkeypox virus antigen detection test strip, kit and method thereof to solve the technical problems that the existing kits for detecting monkeypox virus using colloidal gold immunochromatography technology have poor specificity and low sensitivity, and there is no antigen detection kit for monkeypox virus E8L protein.

[0008] In order to solve the above technical problems, the present invention provides a monkeypox virus antigen detection test strip, which adopts the following technical solution:

[0009] The test strip includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad sequentially overlapped on the PVC base plate; wherein the conjugate pad is provided with a tracer marker labeled with an anti-monkeypox virus E8L protein monoclonal antibody, and the tracer marker is colloidal gold; the nitrocellulose membrane is provided with a test line and a quality control line parallel to each other, the test line is coated with the monkeypox virus E8L protein monoclonal antibody, and the quality control line is coated with Super C+.

[0010] Furthermore, the colloidal gold is ordinary colloidal gold or large-particle colloidal gold. The particle size of the ordinary colloidal gold is 40 to 60 nm, and the particle size of the large-particle colloidal gold is 100 to 300 nm.

[0011] Furthermore, the sample pad is prepared by soaking and drying in a sample pad treatment solution, wherein the components of the sample pad treatment solution include 0.2%-1% casein sodium salt, 0.2%-0.5% PVP-K30, 0.5% Tritonx-100, 2% sucrose, 0.3-1.0 mg / mL antibody blocking agent and 0.2M pH 8.0 boric acid borax solution.

[0012] Furthermore, the common colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody is prepared by the following steps:

[0013] Take 1 mL of the prepared ordinary colloidal gold solution, add 5-10 μL of K2CO3 with a mass concentration of 0.2 M to adjust the pH value of the colloidal gold solution to obtain an adjusted ordinary colloidal gold solution;

[0014] adding 10-15 μg of anti-monkeypox virus E8L protein monoclonal antibody to the adjusted ordinary colloidal gold solution and mixing them evenly to obtain a first mixed solution;

[0015] Gradually add 100 μL of 5% sodium casein to the first mixed solution and mix well to obtain a first blocking mixed solution;

[0016] The first blocking mixed solution was centrifuged at a speed of 3000-5000 rpm for 5-8 minutes and at a speed of 10000-12000 rpm for 10-15 minutes, respectively. The supernatant was discarded and the precipitate was fixed to 100 μL with a preservation solution to obtain a conventional colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody.

[0017] The large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody is prepared by the following steps:

[0018] Take 50 μL of large-particle colloidal gold, add 50-75 μL of 0.01M pH 7.0 phosphate buffer or 0.01M pH 8.5 boric acid borax solution, and vortex to mix to obtain a large-particle colloidal gold solution;

[0019] adding 3-6 μg of anti-monkeypox virus E8L protein monoclonal antibody to the large particle colloidal gold solution, and incubating at room temperature to obtain a second mixed solution;

[0020] Gradually add 50 μL of 5% sodium casein to the second mixed solution and mix well to obtain a second blocking mixed solution;

[0021] The second blocking mixed solution was centrifuged at a speed of 8000-10000 rpm for 10-15 min, the supernatant was discarded, and 500-1000 μL of preservation solution was added to the precipitate and vortexed to obtain large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody.

[0022] Furthermore, when the test line is coated with a monoclonal antibody against the monkeypox virus E8L protein, the coating concentration of the monoclonal antibody against the monkeypox virus E8L protein is 1.0-1.5 mg / mL, and the coating volume is 1.0 μL / cm;

[0023] When the quality control line was coated with Super C+, the coating concentration of Super C+ was 0.5 mg / mL and the coating volume was 1.0 μL / cm.

[0024] In order to solve the above technical problems, the present application also provides a monkeypox virus antigen detection kit, which adopts the following technical solution:

[0025] Comprising the monkeypox virus antigen detection test strip and sample extract as described above;

[0026] Each 1000 mL of the sample extract comprises the following components in percentage by mass: 0.3% disodium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 0.5-1% sodium chloride, 1% potassium chloride, 0.1-0.5% casein sodium salt, and 0.1-0.5% Tritonx-100.

[0027] Furthermore, the test kit also includes a cartridge; the monkeypox virus antigen detection test strip is installed in the cartridge, the cartridge is provided with a sample addition hole at a location corresponding to the sample pad of the monkeypox virus antigen detection test strip, and the cartridge is provided with a display area in an area corresponding to the detection line and the quality control line.

[0028] Furthermore, the kit is used in detecting monkeypox virus for purposes other than disease diagnosis and treatment.

[0029] In order to solve the above technical problems, the present application also provides a method using a monkeypox virus antigen detection kit, which adopts the following technical solution:

[0030] The method is for the purpose of non-disease diagnosis and treatment, and includes the following steps:

[0031] placing the sample to be tested into a sample extracting solution to obtain a sample solution;

[0032] The sample solution was added to the sample pad, and the test results were as follows:

[0033] 1) When the test line shows color and the quality control line also shows color, the test result of the sample to be tested is positive;

[0034] 2) If the test line does not show color and the quality control line shows color, the test result of the sample to be tested is negative;

[0035] 3) If the quality control line does not show color, the kit is invalid and needs to be re-measured with a new kit.

[0036] Furthermore, the sample to be tested is a scab fluid or pustular fluid sample.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] The monkeypox virus antigen detection test strips and kits provided in this application use colloidal gold immunochromatography, which can accurately and rapidly detect monkeypox virus. Secondly, the use of colloidal gold to label anti-monkeypox virus E8L protein monoclonal antibodies has a simple labeling method and high affinity for monkeypox virus, effectively improving the sensitivity and specificity of the product. The quality control line on the test strip of this application is coated with Super C+, which is homologous to the monkeypox virus E8L protein. The quality control line can non-specifically bind to the colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody, which can effectively monitor the effectiveness of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the solutions in this application, a brief introduction will be given below to the drawings required for use in the description of the embodiments of this application. Obviously, the drawings described below are some embodiments of this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0040] Figure 1 This is a schematic diagram of the structure of the monkeypox virus antigen detection test strip provided by the present application;

[0041] Figure 2 This is a schematic diagram of the structure of the card in the monkeypox virus antigen detection kit provided by the present application;

[0042] Figure 3 Schematic diagram of the test results of the monkeypox virus antigen detection kit provided by the present application;

[0043] Figure 4 This is a graph showing the test results of the large particle colloidal gold monkeypox virus antigen detection kit provided by the present application for recombinant monkeypox virus antigen;

[0044] Figure 5 This is a graph showing the test results of the ordinary particle colloidal gold monkeypox virus antigen detection kit provided by the present application for recombinant monkeypox virus antigen;

[0045] Figure 6 This is a graph showing the test results of the control kit on the recombinant monkeypox virus antigen. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of the application are for the purpose of describing specific embodiments only and are not intended to limit this application. The terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0047] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0048] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0049] This application provides a monkeypox virus antigen detection test strip, see Figure 1 As shown, the test strip includes a PVC base 7, and a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 4, and a water-absorbing pad 6, which are sequentially attached to the PVC base 7. The conjugate pad 2 is provided with a monoclonal antibody against the monkeypox virus E8L protein, which is labeled with a tracer marker, wherein the tracer marker is colloidal gold. The nitrocellulose membrane 4 is provided with parallel test lines 3 and quality control lines 5. The test line 3 is coated with the monoclonal antibody against the monkeypox virus E8L protein, and the quality control line 5 is coated with Super C+.

[0050] Among them, Super C+ is a histidine quality control system for the Escherichia coli expression system. Using Super C+ can avoid cross-reactions.

[0051] In some embodiments, the sample pad 1 may be selected from a glass cellulose membrane; the conjugate pad 2 may be selected from a glass cellulose membrane; the nitrocellulose membrane 4 may be selected from a CN95 membrane; and the absorption pad 6 may be selected from filter paper fibers.

[0052] In some embodiments, in order to obtain better detection effect, the boundary distance between the absorption pad 6 and one end of the nitrocellulose membrane 4 is 2 mm, the boundary distance between the other end of the nitrocellulose membrane 4 and one end of the binding pad 2 is 1 to 2 mm, and the boundary distance between one end of the sample pad 1 and the other end of the binding pad 2 is 1 to 2 mm.

[0053] In some embodiments, based on testing and cost control requirements, the width of the test strip is set to 3 mm.

[0054] In some embodiments, the colloidal gold is ordinary colloidal gold or large-particle colloidal gold. The particle size of ordinary colloidal gold is 40 to 60 nm, and the particle size of large-particle colloidal gold is 100 to 300 nm.

[0055] In some embodiments, the sample pad 1 is prepared by soaking in a sample pad treatment solution and then drying, wherein the components of the sample pad treatment solution include 0.2%-1% casein sodium salt, 0.2%-0.5% PVP-K30, 0.5% Tritonx-100, 2% sucrose, 0.3-1.0 mg / mL antibody blocking agent and 0.2M pH 8.0 boric acid borax solution.

[0056] The sample pad treatment solution can increase the absorption capacity of the sample pad 1, control the release of the tracer marker, and promote the occurrence of chromatography.

[0057] It should be noted that the units of casein sodium salt, PVP-K30, Triton X-100, and sucrose are mass percentages. PVP-K30 is a non-ionic polymer compound and a type of polyvinylpyrrolidone product with a K value of 30.

[0058] The preparation method of the monkeypox virus antigen detection test strip in this application is as follows:

[0059] 1. Preparation of Sample Pad

[0060] (1) Preparation of sample pad treatment solution:

[0061] Prepare the sample pad treatment solution according to the above composition.

[0062] (2) Sample pad treatment:

[0063] Soak the glass fiber membrane in the treatment solution, take it out after treatment, and place it on a clean, dry, and flat drying rack. Dry it overnight (16-24) hours at a temperature of (18-26)°C and a relative humidity of ≤30% to obtain a sample pad 1.

[0064] 2. Labeling of Monoclonal Antibodies Against Monkeypox Virus E8L Protein

[0065] This application uses a direct labeling method. That is, at room temperature, the prepared anti-monkeypox virus E8L protein monoclonal antibody is directly reacted with a colloidal gold solution, and then centrifuged to obtain the desired colloidal gold label. The specific method is as follows:

[0066] 2.1 Ordinary colloidal gold-labeled monoclonal antibody against monkeypox virus E8L protein

[0067] (1) Take 1 mL of the prepared ordinary colloidal gold solution, add 5-10 μL of K2CO3 with a mass concentration of 0.2 M to adjust the pH value of the colloidal gold solution to obtain an adjusted ordinary colloidal gold solution;

[0068] (2) adding 10-15 μg of anti-monkeypox virus E8L protein monoclonal antibody to the adjusted ordinary colloidal gold solution and mixing uniformly to obtain a first mixed solution;

[0069] (3) gradually adding 100 μL of 5% sodium casein to the first mixed solution and mixing evenly to obtain a first blocking mixed solution;

[0070] (4) The first blocking mixed solution was centrifuged at a speed of 3000-5000 rpm for 5-8 min and at a speed of 10000-12000 rpm for 10-15 min, respectively. The supernatant was discarded and the precipitate was fixed to 100 μL with preservation solution to obtain a conventional colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody.

[0071] The mixing time in step (2) is 30 minutes; the mixing time in step (3) is 15 minutes; and after obtaining the ordinary colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody in step (4), it is stored at 4° C. for future use.

[0072] 2.2 Large-particle colloidal gold-labeled monoclonal antibody against monkeypox virus E8L protein

[0073] (1) Take 50 μL of large-particle colloidal gold, add 50-75 μL of 0.01 M pH 7.0 phosphate buffer or 0.01 M pH 8.5 boric acid borax solution, and vortex to mix to obtain a large-particle colloidal gold solution;

[0074] (2) adding 3-6 μg of anti-monkeypox virus E8L protein monoclonal antibody to the large particle colloidal gold solution and incubating at room temperature to obtain a second mixed solution;

[0075] (3) gradually adding 50 μL of 5% sodium casein to the second mixed solution and mixing well to obtain a second blocking mixed solution;

[0076] (4) The second blocking mixed solution was centrifuged at a speed of 8000-10000 rpm for 10-15 min, the supernatant was discarded, and 500-1000 μL of preservation solution was added to the precipitate and vortexed to mix, thereby obtaining large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibodies.

[0077] The incubation time in step (2) is 30-60 min; the mixing time in step (3) is 15 min.

[0078] From the above preparation method, it can be seen that the monkeypox virus antigen detection kit provided by the present application, which contains large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibodies, uses a small amount of antibody, increases the utilization rate of colloidal gold particles by 10 times or more, and reduces costs by 20%.

[0079] In some embodiments, the preservation solution is prepared as follows:

[0080] Each 100 ml of preservation solution is made of 0.2M pH 8.0 boric acid borax solution, 1% bovine serum albumin, 0.5% casein sodium salt, 8% sucrose, 2% trehalose, 0.1% Tween-20 and 0.05% Proclin 300, wherein the unit mass percentage concentrations of bovine serum albumin, casein sodium salt, sucrose, trehalose, Tween-20 and Proclin 300 are shown.

[0081] 2.3 Preparation of conjugate pad

[0082] Ordinary colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody or large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody was sprayed onto the glass fiber membrane using a gold sprayer at 4-5 μL / cm and dried at 37° C. for 16-24 h to prepare conjugate pad 2.

[0083] 3. Preparation of Nitrocellulose Membrane

[0084] (1) Preparation of coating buffer: The coating buffer is made of 0.01-0.02 M phosphate buffer with a pH of 7.4, 1% trehalose and purified water.

[0085] (2) Preparation of test lines and quality control lines

[0086] Dilute the monkeypox virus E8L protein monoclonal antibody to 1.0-1.5 mg / mL with coating buffer to obtain the test line coating solution; dilute Super C+ to 0.5 mg / mL with coating buffer to obtain the quality control line coating solution.

[0087] Glue the CN95 membrane to a PVC base plate and use a film scribing instrument to draw the test line coating solution and the quality control line coating solution at their specific positions, respectively. The coating amount of the test line is 1.0 μL / cm, and the coating amount of the quality control line is 1.0 μL / cm. After coating, dry it in a constant temperature drying oven at 37°C for 16-24 hours and then take it out to obtain the nitrocellulose membrane for use.

[0088] 4. Assembly of test strips

[0089] First, the sample pad 1, conjugate pad 2, nitrocellulose membrane 4, and absorption pad 6 are sequentially attached to one surface of a PVC base plate 7 to prepare a sample test strip; wherein, the boundary distance between the absorption pad 6 and one end of the nitrocellulose membrane 4 is 2 mm; the boundary distance between the other end of the nitrocellulose membrane 4 and one end of the conjugate pad 2 is 1 to 2 mm; and the boundary distance between one end of the sample pad 1 and the other end of the conjugate pad 2 is 1 to 2 mm.

[0090] Secondly, use a strip cutter to cut the sample test strips into 3mm wide test strips, then pack them in aluminum foil bags, add desiccant, and seal them.

[0091] The present application also provides a monkeypox virus antigen detection kit, comprising the monkeypox virus antigen detection test strip as described above and a sample extract;

[0092] Each 1000 mL of sample extract includes the following components in percentage by mass: 0.3% disodium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 0.5-1% sodium chloride, 1% potassium chloride, 0.1-0.5% casein sodium salt, and 0.1-0.5% Tritonx-100.

[0093] In some embodiments, the kit further comprises a cartridge 8, see Figure 2 As shown, the monkeypox virus antigen test strip is installed in the card, the card is provided with a sample addition hole 9 at the sample pad 1 corresponding to the monkeypox virus antigen test strip, and the card is provided with a display area 10 in the area corresponding to the detection line 3 and the quality control line 5.

[0094] The cut test strips are placed into the card housing 8 to obtain a test card, and the test card and the sample extract are labeled and packed into a box to obtain a finished monkeypox virus antigen detection kit.

[0095] The monkeypox virus antigen detection kit of the present application is used to detect monkeypox virus for the purpose of non-disease diagnosis and treatment.

[0096] The monkeypox virus antigen detection kit of the present application is used to detect monkeypox virus antigen, and the detection method comprises the following steps:

[0097] placing the sample to be tested into a sample extracting solution to obtain a sample solution;

[0098] The sample solution was added to the sample pad and the test results were as follows:

[0099] 1) When the test line shows color and the quality control line also shows color, the test result of the sample to be tested is positive;

[0100] 2) If the test line does not show color and the quality control line shows color, the test result of the sample to be tested is negative;

[0101] 3) If the quality control line does not show color, the kit is invalid and needs to be re-measured with a new kit.

[0102] The specific detection steps are as follows:

[0103] Take out the test card and place it on a dry, clean surface;

[0104] Sampling swabs are used to collect samples of crust and pustular fluid;

[0105] Immerse the sampled swab in the extraction tube containing the sample extract and squeeze and rotate it 10 times;

[0106] Put the lid of the extraction tube on and vertically drip 2-3 drops (about 50μL-80μL) of the mixed liquid in the extraction tube into the sample well of the test card;

[0107] Let it stand at room temperature for 10 to 15 minutes and then read the result;

[0108] According to the displayed results, the detection effect of monkeypox virus antigen is judged.

[0109] like Figure 3 As shown, the colloidal gold labeling test results are interpreted as follows:

[0110] 1) Positive: Two red reaction lines, i.e., one red reaction line appears in the test area (T) and one red reaction line appears in the quality control area (C).

[0111] 2) Negative: One red reaction line, that is, only one red reaction line appears in the quality control area (C).

[0112] 3) Invalid: No red reaction line appears in the control area (C) or bands of other colors appear in the test area (T). The test is invalid. It is recommended to retest with a new test card.

[0113] Note: The color depth of the band is related to the content of the substance being tested in the extracted sample. Regardless of the color intensity, the result should be judged by whether the band is colored. This kit includes a quality control process. The appearance of a red band in Area C indicates that the operation is correct and effective; otherwise, the test is invalid.

[0114] The method and results of the sample detection sensitivity of the above-mentioned monkeypox virus antigen detection kit are as follows:

[0115] (1) Testing the minimum detection limit of the kit

[0116] The monkeypox virus E8L protein antigen was diluted to six concentrations using the prepared sample diluent: 2000 ng / mL, 200 ng / mL, 20 ng / mL, 2 ng / mL, 0.2 ng / mL, and 0.1 ng / mL. Two kits were tested at each concentration, each containing a different tracer marker: large-particle colloidal gold and standard colloidal gold.

[0117] Drop the six solutions of concentration gradients and the control reagent prepared above into the sample wells of the test card, let them stand for 10 to 15 minutes and then observe the results. Figures 4-6 shown.

[0118] (2) Test results

[0119] The sensitivity test results are shown in Table 1 below.

[0120]

[0121]

[0122] In the above results, “+++” indicates a strong positive, “++” indicates a moderate positive, “+” indicates a weak positive, and “-” indicates a negative.

[0123] It should be noted that the control reagent is a large plate purchased from a third party, including the conjugate pad, sample pad, coating membrane, sample extract, etc.

[0124] The above experimental results show that when the sample concentration is greater than or equal to 0.1 ng / mL, the large-particle colloidal gold kit has all positive test results; when the sample concentration is greater than or equal to 0.2 ng / mL, the standard-particle colloidal gold kit has all positive test results; and when the sample concentration is greater than or equal to 2 ng / mL, the control reagent has all positive test results. Therefore, the minimum detection limit of the monkeypox virus antigen detection kit for large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibodies provided in this application is 0.1 ng / mL; the minimum detection limit of the monkeypox virus antigen detection kit for standard-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibodies is 0.2 ng / mL; and the minimum detection limit of the control reagent is 2 ng / mL. It was concluded that the sensitivity of the monkeypox virus antigen detection kit with large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody was 2 times higher than that of the monkeypox virus antigen detection kit with ordinary-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody; compared with the control reagent, the sensitivity of the monkeypox virus antigen detection kit provided in the present application was increased by 10 times, further demonstrating that the monkeypox virus antigen detection kit of the present application has strong specificity, good stability, and high affinity for monkeypox virus.

[0125] Obviously, the embodiments described above are only some of the embodiments of the present application, rather than all of the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification and the accompanying drawings, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.

Claims

1. A monkeypox virus antigen detection test strip, characterized in that: The test strip includes a PVC base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad sequentially overlapped on the PVC base plate; wherein the conjugate pad is provided with a tracer marker labeled with an anti-monkeypox virus E8L protein monoclonal antibody, and the tracer marker is colloidal gold; the nitrocellulose membrane is provided with a test line and a quality control line parallel to each other, the test line is coated with the monkeypox virus E8L protein monoclonal antibody, and the quality control line is coated with Super C+.

2. The monkeypox virus antigen detection test strip according to claim 1, characterized in that: The colloidal gold is ordinary colloidal gold or large-particle colloidal gold. The particle size of the ordinary colloidal gold is 40-60 nm, and the particle size of the large-particle colloidal gold is 100-300 nm.

3. The monkeypox virus antigen detection test strip according to claim 1, characterized in that The sample pad is prepared by soaking in a sample pad treatment solution and then drying it, wherein the components of the sample pad treatment solution include 0.2%-1% casein sodium salt, 0.2%-0.5% PVP-K30, 0.5% Tritonx-100, 2% sucrose, 0.3-1.0 mg / mL antibody blocking agent and 0.2M pH 8.0 boric acid borax solution.

4. The monkeypox virus antigen detection test strip according to claim 2, characterized in that: The common colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody is prepared by the following steps: Take 1 mL of the prepared ordinary colloidal gold solution, add 5-10 μL of K2CO3 with a mass concentration of 0.2 M to adjust the pH value of the colloidal gold solution to obtain an adjusted ordinary colloidal gold solution; adding 10-15 μg of anti-monkeypox virus E8L protein monoclonal antibody to the adjusted ordinary colloidal gold solution and mixing them evenly to obtain a first mixed solution; Gradually add 100 μL of 5% sodium casein to the first mixed solution and mix well to obtain a first blocking mixed solution; The first blocking mixed solution was centrifuged at a speed of 3000-5000 rpm for 5-8 minutes and at a speed of 10000-12000 rpm for 10-15 minutes, respectively. The supernatant was discarded and the precipitate was fixed to 100 μL with a preservation solution to obtain a conventional colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody. The large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody is prepared by the following steps: Take 50 μL of large-particle colloidal gold, add 50-75 μL of 0.01M pH 7.0 phosphate buffer or 0.01M pH 8.5 boric acid borax solution, and vortex to mix to obtain a large-particle colloidal gold solution; adding 3-6 μg of anti-monkeypox virus E8L protein monoclonal antibody to the large particle colloidal gold solution, and incubating at room temperature to obtain a second mixed solution; Gradually add 50 μL of 5% casein sodium salt to the second mixed solution and mix well to obtain a second blocking mixed solution; The second blocking mixed solution was centrifuged at a speed of 8000-10000 rpm for 10-15 min, the supernatant was discarded, and 500-1000 μL of preservation solution was added to the precipitate and vortexed to obtain large-particle colloidal gold-labeled anti-monkeypox virus E8L protein monoclonal antibody.

5. The monkeypox virus antigen detection test strip according to claim 1, characterized in that: When the test line is coated with a monoclonal antibody against the monkeypox virus E8L protein, the coating concentration of the monoclonal antibody against the monkeypox virus E8L protein is 1.0-1.5 mg / mL, and the coating volume is 1.0 μL / cm; When the quality control line was coated with Super C+, the coating concentration of Super C+ was 0.5 mg / mL and the coating volume was 1.0 μL / cm.

6. A monkeypox virus antigen detection kit, characterized in that: Comprising the monkeypox virus antigen detection test strip and sample extract according to any one of claims 1 to 5; Each 1000 mL of the sample extract comprises the following components in percentage by mass: 0.3% disodium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 0.5-1% sodium chloride, 1% potassium chloride, 0.1-0.5% casein sodium salt, and 0.1-0.5% Tritonx-100.

7. The monkeypox virus antigen detection kit according to claim 6, characterized in that The test kit also includes a cartridge; the monkeypox virus antigen detection test strip is installed in the cartridge, the cartridge is provided with a sample addition hole at a location corresponding to the sample pad of the monkeypox virus antigen detection test strip, and the cartridge is provided with a display area in an area corresponding to the detection line and the quality control line.

8. Use of the monkeypox virus antigen detection kit according to claim 6 or 7 in detecting monkeypox virus for purposes other than disease diagnosis and treatment.

9. A method for detecting monkeypox virus using the monkeypox virus antigen detection kit according to claim 6 or 7, wherein the method is for the purpose of non-disease diagnosis and treatment, comprising the steps of: placing the sample to be tested into a sample extracting solution to obtain a sample solution; The sample solution was added to the sample pad, and the test results were as follows: 1) When the test line shows color and the quality control line also shows color, the test result of the sample to be tested is positive; 2) If the test line does not show color and the quality control line shows color, the test result of the sample to be tested is negative; 3) If the quality control line does not show color, the kit is invalid and needs to be re-measured with a new kit.

10. The method according to claim 9, characterized in that The sample to be tested is a scab fluid or pustular fluid sample.

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