A monkeypox virus antigen detection kit and preparation method

Abstract

The present invention belongs to the technical field of in vitro diagnosis, that is, a monkeypox virus antigen detection kit also relates to its preparation method. The present invention includes a test card and a sample diluent, the test card includes an upper cover, a lower cover and a monkeypox virus antigen detection test strip, the monkeypox virus antigen detection test strip includes a sample pad, a latex binding pad, a solid phase antibody reaction membrane, a wicking paper and a PVC bottom plate, and the upper cover is provided with a sample addition hole and an observation window and an upper self-adhesive window. The monkeypox antigen rapid detection kit developed by the present invention is based on the immunochromatography principle of latex immunochromatography and antigen-antibody specific binding reaction, which is one of the methods for rapid screening of monkeypox patients. The present invention is to process biological raw materials through coating, labeling, drying, assembly and other processes, and pre-fix them on the corresponding carrier. The user only needs to mix the collected sample into the diluent, then add the diluted sample to the product, and wait for the time to read the result.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis, that is, a monkeypox virus antigen detection kit and also relates to a preparation method thereof. Background Art

[0002] Monkeypox occurs in monkeys in the rainforests of central and western Africa. It can also infect other animals and occasionally humans. The clinical manifestations are similar to smallpox, but the condition is milder. The disease is caused by the monkeypox virus. It belongs to a group of viruses that includes smallpox virus, the virus used in smallpox vaccines, and cowpox virus. It needs to be distinguished from smallpox and chickenpox. The virus can be transmitted from animals to humans through direct close contact, and can also be transmitted from person to person, mainly through blood and body fluids.

[0003] Monkeypox is a viral zoonosis caused by infection with the monkeypox virus, an enveloped double-stranded DNA virus that, like smallpox virus and cowpox virus, belongs to the genus Orthopoxvirus of the Poxviridae family. Its main hosts are African rodents (such as African squirrels, tree squirrels, Gambian kangaroos, dormice, etc.).

[0004] Monkeypox virus can be transmitted through direct contact with infected ulcers, body fluids, respiratory droplets, and contaminated clothing. The vast majority of monkeypox cases occur in men, most of which occur in gay, bisexual, and men who have sex with men. There is currently no specific treatment for monkeypox virus, and smallpox can be used for localized outbreaks. Summary of the invention

[0005] The purpose of the present invention is to provide a monkeypox virus antigen detection kit and a preparation method thereof, which can be applicable to the detection of oropharyngeal swabs, body fluids, blood and skin lesion tissue (including blister fluid, pustular fluid, and scab) samples, and has high sensitivity, can effectively shorten the window period for virus diagnosis, thereby reducing the risk of invisible transmission caused by the incubation period and effectively controlling the large-scale spread of the epidemic.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A monkeypox virus antigen detection kit comprises a detection card and a sample diluent. The detection card comprises an upper cover, a lower cover and a monkeypox virus antigen detection test strip. The monkeypox virus antigen detection test strip comprises a sample pad, a latex binding pad, a solid phase antibody reaction membrane, absorbent paper and a PVC bottom plate. The upper cover is provided with a sample addition hole, an observation window and an upper self-adhesive window.

[0008] The solid-phase antibody reaction membrane is pasted on the PVC board, and two ends of the solid-phase antibody reaction membrane are respectively overlapped with one end of a latex binding pad and one end of an absorbent paper, and the other end of the latex binding pad is pasted on the PVC board, and the other end of the latex binding pad is overlapped with one end of a sample pad, and the other end of the sample pad is pasted on the PVC board, and the other end of the absorbent paper is pasted on the PVC board.

[0009] The sample diluent includes 1.2% by mass of tris(hydroxymethyl)aminomethane, 0.1% by mass of Casein-Na, 0.7% by mass of TritonX-100, 2% by mass of KCl, 1% by mass of glycine, 0.04% by mass of CMC, 1% by mass of sodium carbonate, 0.5% by mass of fatty alcohol polyoxyethylene ether, 0.2% by mass of BND-10 and 0.05% by volume of Proclin-300.

[0010] The manufacturing method of the monkeypox virus antigen detection kit includes a preparation method of a latex conjugate pad, a preparation method of a sample pad and a preparation method of a solid phase antibody reaction membrane, wherein the preparation method of the latex conjugate pad is as follows:

[0011] (1) vortexing and ultrasonicating the latex, wherein the latex is particles with a particle size of 300 nm;

[0012] (2) Resuspend the latex and washing solution in a ratio of 1:4;

[0013] (3) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend and sonicate again with the same volume of washing solution;

[0014] (4) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend with the same volume of reaction buffer and perform sonication;

[0015] (5) Add EDAC to the latex reaction solution at 20% of the original latex volume and incubate under vortex for 30 min at room temperature;

[0016] (6) Add NHS to the latex reaction solution at 10% of the original latex volume and continue vortexing and incubating for 30 min at room temperature;

[0017] (7) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend with an equal volume of conjugate diluent, and perform sonication;

[0018] (8) Add labeled monkeypox virus monoclonal antibody at a ratio of 1.0 mg / mL and incubate with vortexing at room temperature for 1.5 h;

[0019] (9) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend with the same volume of blocking solution and sonicate; then block for another 30 min;

[0020] (10) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, and use the reconstitution solution to make the precipitate eight times the volume of the original latex, then resuspend and sonicate to obtain the latex labeling working solution;

[0021] (11) The latex marker working stock solution was sputtered onto the glass fiber at 1.5 ul / cm and dried at 45°C overnight to prepare a latex binding pad.

[0022] The cleaning solution in step (2) comprises 3.8% by mass of borax.

[0023] The reaction buffer of step (4) includes 50 mM MES buffer with a pH value of 5.5.

[0024] The conjugate diluent in step (7) is 1.2% by mass of Na2HPO4·12H2O and 0.148% by mass of NaH2PO4·2H2O.

[0025] The sealing solution of step (9) comprises 0.09% by mass of Na2OH with a pH value of 8.0, 1% by mass of sodium caseinate and 0.618% by mass of borax.

[0026] The reconstituted solution of step (10) comprises 0.125% by mass of tris(hydroxymethyl)aminomethane, 0.25% by mass of Casein, 20% by mass of sucrose and 5% by mass of trehalose.

[0027] The preparation method of the sample pad is as follows: soak the K8964 glass cellulose membrane with the sample pad treatment solution, 3.3 mL / strip, and dry at 45° C. overnight to obtain the sample pad.

[0028] The sample pad treatment solution includes 0.6% by mass of tris(hydroxymethyl)aminomethane with a pH value of 8.0, 1% by mass of PVP-30, 0.5% by mass of S9, 0.5% by mass of Casein, 0.5% by mass of TritonX-100, 1% by mass of S-17, 0.3% by mass of EDTA, 1% by mass of BSA and 0.5% by mass of fatty alcohol polyoxyethylene ether.

[0029] The preparation method of the solid phase antibody reaction membrane is as follows: the solid phase antibody reaction membrane is coated with a detection T line and a quality control C line in sequence, the detection T line is coated with monkeypox monoclonal antibody, and the quality control C line is coated with goat anti-mouse IgG antibody and DNP-BSA.

[0030] The preparation method of the solid phase antibody reaction membrane is as follows: the concentration of the monkeypox monoclonal antibody 1 is 1.0 mg / mL, the concentration of the goat anti-mouse IgG antibody is 0.5 mg / mL, and the concentration of DNP-BSA is 0.5 mg / mL.

[0031] The present invention adopts the above technical solution and has the following advantages:

[0032] 1. The present invention adopts 300nm latex labeling. Compared with traditional colloidal gold labeling, latex labeling adopts covalently coupled gold particle conjugates, which are more stable and avoid the influence of gold stability and uniformity caused by sample addition method, heating time, stirring speed, reaction temperature and container; in addition, latex is 300nm large particles, and the sensitivity can be increased by up to 20 times, which is more suitable for the detection of multiple sample types. It can detect low-level viruses in oropharyngeal swabs, body fluids and blood before the patient's rash appears, shorten the window period of virus diagnosis, effectively reduce the risk of invisible transmission caused by the incubation period, and control the large-scale spread of the epidemic.

[0033] 2. Currently, there is no monkeypox virus antigen diagnostic reagent at home and abroad. The present invention can maximize the sample compatibility of the reagent by optimizing the sample pad and sample diluent. The components in the sample diluent and sample pad of the monkeypox virus antigen detection kit of the present invention eventually form a composite system with strong anti-interference and strong buffering capacity through cooperation with each other, which can effectively buffer electrolytes between different samples of oropharyngeal swabs, body fluids, blood and skin lesion tissues (including blister fluid, pustular fluid, and scab), and is compatible with a variety of sample types.

[0034] 3. The addition of surfactants, hydrophilic polymers, anti-RBC monoclonal antibodies, etc. in the sample diluent and sample pad can effectively prevent the non-specific binding of fat, bacteria, cell debris, heterophilic antibodies, red blood cells and other factors in the sample with antigen-antibody. On the basis of protecting the antigen to be tested, it reduces the occurrence of false positives and improves the detection accuracy.

[0035] In short, the monkeypox antigen rapid detection kit developed by the present invention is based on the immunochromatography principle of latex immunochromatography and antigen-antibody specific binding reaction, which is one of the methods for rapid screening of monkeypox patients. The present invention processes biological raw materials through coating, labeling, drying, assembly and other processes, and pre-fixes them on the corresponding carrier. The user only needs to mix the collected sample into the diluent, then add the diluted sample to the product, and wait for the time to read the result. Compared with the conventional monkeypox PCR detection method, this monkeypox antigen rapid detection kit has lower requirements for the detection environment, and the detection process is simple and easy to understand. It is generally considered to be suitable for personal and family self-testing, as well as large-scale rapid screening applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the structure of a detection card in a monkeypox virus antigen detection kit of the present invention;

[0037] Figure 2 It is a schematic structural diagram of a monkeypox virus antigen detection test strip in a monkeypox virus antigen detection kit of the present invention;

[0038] Figure 3 This is a schematic structural diagram of an upper cover in a monkeypox virus antigen detection kit of the present invention;

[0039] Figure 4 The figure is a schematic flow chart of a method for preparing a monkeypox virus antigen detection kit according to the present invention (the gray steps are the key points).

[0040] The labels include: 3-monkeypox virus antigen detection test strip, 4-PVC plate, 5-sample pad, 6-latex binding pad, 7-solid phase antibody reaction membrane, 8-absorbent paper, 9-detection T line, 10-quality control C line, 11-sample addition hole, 12-observation window, 13-upper cover, 14-lower cover, 15-upper self-adhesive window. DETAILED DESCRIPTION

[0041] The embodiments of the present invention are described in detail below with reference to the examples. In the examples, the specific conditions are not specified, and the experiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased on the market.

[0042] The described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] Embodiment 1:

[0044] like Figure 1-4As shown, a monkeypox virus antigen detection kit includes a detection card and a sample diluent, the detection card includes an upper cover 13, a lower cover 14 and a monkeypox virus antigen detection test strip 3, the upper cover 13 and the lower cover 14 are snap-connected to each other, a receiving cavity is formed between the upper cover 13 and the lower cover 14, the monkeypox virus antigen detection test strip 3 is located in the receiving cavity, the monkeypox virus antigen detection test strip 3 includes a sample pad 5, a latex binding pad 6, a solid phase antibody reaction membrane 7, absorbent paper 8 and a PVC board 4, the solid phase antibody reaction membrane 7 is adhered to the PVC board 4, and both ends of the solid phase antibody reaction membrane 7 are One end of a latex binding pad 6 and one end of an absorbent paper 8 are overlapped respectively, the other end of the latex binding pad 6 is pasted on the PVC board 4, the other end of the latex binding pad 6 is overlapped with one end of a sample pad 5, the other end of the sample pad 5 is pasted on the PVC board 4, and the other end of the absorbent paper 8 is pasted on the PVC board 4. The width of the monkeypox virus antigen detection test strip 3 is 3.5 mm, and a sample addition hole 11, an observation window 12 and an upper adhesive window 15 are provided on the upper cover 13. The position of the sample addition hole 11 corresponds to the position of the sample pad 5, so that the sample to be tested can be dripped onto the sample pad 5.

[0045] A method for preparing a monkeypox virus antigen detection kit comprises a method for preparing a latex binding pad, a method for preparing a sample pad, and a method for preparing a solid-phase antibody reaction membrane.

[0046] The preparation method of the latex bonding pad is as follows:

[0047] (1) Vortex and sonicate the latex (particles with a particle size of 300 nm);

[0048] (2) Resuspend the latex and cleaning solution at a ratio of 1:4 (volume ratio 1:4, small sample mark 200ul latex: 800ul cleaning solution);

[0049] (3) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend and sonicate again with the same volume of washing solution;

[0050] (4) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend with the same volume of reaction buffer and perform sonication;

[0051] (5) Add EDAC to the latex reaction solution at 20% of the original latex volume and incubate under vortex for 30 min at room temperature;

[0052] (6) Add NHS to the latex reaction solution at 10% of the original latex volume and continue vortexing and incubating for 30 min at room temperature;

[0053] (7) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend with an equal volume of conjugate diluent, and perform sonication;

[0054] (8) Add labeled monkeypox virus monoclonal antibody at a ratio of 1.0 mg / mL and incubate with vortexing at room temperature for 1.5 h;

[0055] (9) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, resuspend with the same volume of blocking solution and sonicate; then block for another 30 min;

[0056] (10) Centrifuge at 4°C and 12,000 rpm for 15 min, remove the supernatant, and use the reconstitution solution to make the precipitate eight times the volume of the original latex, then resuspend and sonicate to obtain the latex labeling working solution;

[0057] (11) The latex marker working stock solution was sputtered onto the glass fiber at 1.5 ul / cm and dried at 45°C overnight to prepare a latex binding pad.

[0058] In step (1), the latex is particles with a particle size of 300 nm.

[0059] The cleaning solution in step (2) includes 3.8% by mass of borax.

[0060] The reaction buffer in step (4) includes 50 mM MES buffer with a pH value of 5.5.

[0061] In step (7), the conjugate diluent has a mass percentage of 1.2% Na2HPO4·12H2O and a mass percentage of 0.148% NaH2PO4·2H2O.

[0062] The sealing solution in step (9) includes 0.09% by mass of Na2OH with a pH value of 8.0, 1% by mass of sodium caseinate and 0.618% by mass of borax.

[0063] The reconstitution solution in step (10) includes 0.125% by mass of tris(hydroxymethyl)aminomethane, 0.25% by mass of Casein, 20% by mass of sucrose and 5% by mass of trehalose.

[0064] The sample pad was prepared as follows:

[0065] The sample pad treatment solution was soaked in K8964 glass cellulose membrane, 3.3 mL / strip, and dried at 45°C overnight to prepare a sample pad.

[0066] The sample pad treatment liquid includes 0.6% tris(hydroxymethyl)aminomethane with a pH value of 8.0, 1% PVP-30, 0.5% S9, 0.5% Casein, 0.5% TritonX-100, 1% S-17, 0.3% EDTA, 1% BSA, and 0.5% fatty alcohol polyoxyethylene ether.

[0067] The preparation method of the solid phase antibody reaction membrane is as follows:

[0068] The solid phase antibody reaction membrane is coated with a detection T line 9 and a quality control C line 10 in sequence. The detection T line 9 is coated with monkeypox monoclonal antibody, and the quality control C line 10 is coated with goat anti-mouse IgG antibody and DNP-BSA.

[0069] The concentration of monkeypox monoclonal antibody was 1.0 mg / mL, the concentration of goat anti-mouse IgG antibody was 0.5 mg / mL, and the concentration of DNP-BSA was 0.5 mg / mL.

[0070] The position of the observation window 12 corresponds to the position of the detection T line 9 and the quality control C line 10.

[0071] The sample diluent includes 1.2% by mass of tris(hydroxymethyl)aminomethane, 0.1% by mass of Casein-Na, 0.7% by mass of TritonX-100, 2% by mass of KCl, 1% by mass of glycine, 0.04% by mass of CMC, 1% by mass of sodium carbonate, 0.5% by mass of fatty alcohol polyoxyethylene ether, 0.2% by mass of BND-10 and 0.05% by volume of Proclin-300.

[0072] Results evaluation:

[0073] A detection method of a monkeypox virus antigen detection kit comprises the following steps:

[0074] (1) Detection:

[0075] a. Test samples include oropharyngeal swabs, blood and skin lesion tissues;

[0076] b. Add 0.4 mL of sample diluent into the attached reagent tube;

[0077] c. Oropharyngeal swab: Wipe the tongue root and tonsils on both sides at least three times with a cotton swab. Collect the scab with sterilized tweezers. Soak the collected samples in the sample diluent in the reagent tube and stir them thoroughly to obtain the sample solution to be tested.

[0078] d. Add 80 μL of whole blood or the sample solution prepared in step c to the sample well of the test card. The sample solution reaches the test strip through the sample well. Observe the result for 15 minutes. The result will be invalid after 20 minutes.

[0079] (2) Result judgment:

[0080] a. Negative: Only one red band appears on the quality control C line 10, and no red band appears on the detection T line 9. b. Positive: Two red bands appear. One red band is located on the detection T line 9, and the other red band is located on the quality control C line 10.

[0081] c. Invalid: No red stripe appears on the quality control C line 10, indicating that the operation process is incorrect or the test card is damaged.

[0082] Sensitivity and specificity verification of the kit of the present invention:

[0083] (1) Sensitivity

[0084] The monkeypox virus recombinant antigen was purchased and graded diluted to 1000, 500, 200, 20, 2, and 0.2 pg / mL for sensitivity study. The results are shown in Table 1 (Sensitivity study):

[0085] Table 1 Sensitivity study

[0086]

[0087] Note: "+++" represents strong positive, "++" represents positive, "+" represents weak positive, and "-" represents negative.

[0088] The minimum detection limit of the present invention for detecting the monkeypox virus recombinant antigen is 2 pg / mL.

[0089] (2) Analytical specificity

[0090] The cross-reactive pathogenic microorganisms in the table below that are likely to cause the same or similar symptoms were verified. The results showed that the specificity for cross-reactive substances was good under the following concentration conditions:

[0091] Table 2 Analytical specificity studies

[0092]

[0093] In order to better demonstrate that the kit of the present invention has good sensitivity and specificity, taking Example 1 as a reference, through 2 comparative examples, the kit in Example 1 of the present invention and the kit in Comparative Examples 1-2 were used to simultaneously detect negative samples, and the specific detection results are shown in Table 3; different concentrations of recombinant proteins were detected, and the specific detection results are shown in Table 4.

[0094] Comparative Example 1:

[0095] The difference from Example 1 is that the preparation of the gold binding pad is different. In Example 1, the latex binding pad is used and latex labeling is adopted. In Comparative Example 1, the colloidal gold binding pad is used and colloidal gold labeling is adopted by conventional process. The rest of the operations are the same, as follows:

[0096] The preparation method of the colloidal gold binding pad is as follows: adjust the pH value of a 40 nm colloidal gold solution to 9.0 with 0.2 mol / L K2CO3, add 10 μg / mL monkeypox virus monoclonal antibody 2, and after sufficient reaction, centrifuge at 4°C and 10,000 rpm for 30 min, remove the supernatant, and concentrate 25 times to obtain a colloidal gold labeled antibody. Use a gold sprayer to sputter on a glass cellulose membrane at 1.5 μL / cm, and dry at 45°C overnight to obtain a colloidal gold binding pad.

[0097] Comparative Example 2:

[0098] The difference from Example 1 is the preparation of the sample pad. In Comparative Example 2, the sample pad is not added with TritonX-100 and S-17, but with RBC. The other operations are the same, as follows:

[0099] The sample pad was prepared as follows:

[0100] A K8964 glass cellulose membrane was soaked with a sample pad treatment solution, 5 mL / strip, and dried overnight at 45°C to prepare a sample pad. The sample pad treatment solution included 0.6% tris(hydroxymethyl)aminomethane with a pH value of 8.0, 1% PVP-30, 0.5% S9, 0.5% Casein, 0.3% EDTA, 1% BSA, 0.5% fatty alcohol polyoxyethylene ether, and 1% RBC.

[0101] Table 3

[0102] Example Oropharyngeal swabs 100 cases 100 cases of body fluids 100 cases of blood 100 cases of scab Example 1 100％ 100％ 100％ 100％ Comparative Example 1 86.2％ 87.7％ 95.2％ 93.3％ Comparative Example 2 100％ 100％ 100％ 100％

[0103] Table 4

[0104]

[0105] It is found from Table 3 and Table 4 that, compared with Comparative Examples 1 and 2, the kit in Example 1 of the present invention has higher sensitivity and stronger specificity in actual detection applications.

[0106] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. The examples of the implementation methods and the parts not described in detail are common knowledge of ordinary technicians in the field. For those skilled in the art, they can still modify the technical solutions recorded in the above embodiments, or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A monkeypox virus antigen detection kit, characterized in that: It comprises a test card and a sample diluent, wherein the test card comprises an upper cover, a lower cover and a monkeypox virus antigen test strip, wherein the monkeypox virus antigen test strip comprises a sample pad, a latex binding pad, a solid phase antibody reaction membrane, absorbent paper and a PVC bottom plate, and the upper cover is provided with a sample addition hole, an observation window and an upper self-adhesive window; The sample diluent includes 1.2% by mass of tris(hydroxymethyl)aminomethane, 0.1% by mass of Casein-Na, 0.7% by mass of TritonX-100, 2% by mass of KCl, 1% by mass of glycine, 0.04% by mass of CMC, 1% by mass of sodium carbonate, 0.5% by mass of fatty alcohol polyoxyethylene ether, 0.2% by mass of BND-10 and 0.05% by volume of Proclin-300; The manufacturing method of the monkeypox virus antigen detection kit includes a preparation method of a latex conjugate pad, a preparation method of a sample pad and a preparation method of a solid phase antibody reaction membrane, wherein the preparation method of the latex conjugate pad is as follows: (1) vortexing and ultrasonicating the latex, wherein the latex is particles with a particle size of 300 nm; (2) Resuspend the latex and washing solution in a ratio of 1:4; (3) Centrifuge, remove the supernatant, resuspend with the same volume of washing solution and sonicate again; (4) centrifugation, removal of supernatant, resuspending with an equal volume of reaction buffer and sonication; (5) Add EDAC to the latex reaction solution at 20% of the original latex volume and incubate under vortexing at room temperature; (6) Add NHS to the latex reaction solution at 10% of the original latex volume and continue vortexing and incubating at room temperature; (7) Centrifuge, remove the supernatant, resuspend with the same volume of conjugate diluent and perform ultrasonic treatment; (8) adding labeled monkeypox virus monoclonal antibody at a ratio of 1.0 mg / mL and incubating under vortexing at room temperature; (9) Centrifuge, remove the supernatant, resuspend with the same volume of blocking buffer and perform ultrasonic treatment; The cells were then sealed for another 30 min. (10) centrifuging, removing the supernatant, and using a reconstitution solution to adjust the precipitate to eight times the volume of the original latex, and then resuspending and ultrasonicating to obtain a latex labeling working stock solution; (11) Sputtering the latex marker working stock solution onto the glass fiber and drying it at 45° C. overnight to prepare a latex binding pad; The preparation method of the sample pad is as follows: soaking a K8964 glass cellulose membrane with a sample pad treatment solution, 3.3 mL / strip, and drying at 45° C. overnight to obtain a sample pad; the sample pad treatment solution includes 0.6% by mass of tris(hydroxymethyl)aminomethane with a pH value of 8.0, 1% by mass of PVP-30, 0.5% by mass of S9, 0.5% by mass of Casein, 0.5% by mass of TritonX-100, 1% by mass of S-17, 0.3% by mass of EDTA, 1% by mass of BSA, and 0.5% by mass of fatty alcohol polyoxyethylene ether.

2. A monkeypox virus antigen detection kit according to claim 1, characterized in that: The solid-phase antibody reaction membrane is pasted on the PVC board, and two ends of the solid-phase antibody reaction membrane are respectively overlapped with one end of a latex binding pad and one end of an absorbent paper, and the other end of the latex binding pad is pasted on the PVC board, and the other end of the latex binding pad is overlapped with one end of a sample pad, and the other end of the sample pad is pasted on the PVC board, and the other end of the absorbent paper is pasted on the PVC board.

3. A monkeypox virus antigen detection kit according to claim 1, characterized in that: The cleaning solution in step (2) comprises 3.8% by mass of borax.

4. A monkeypox virus antigen detection kit according to claim 1, characterized in that: The reaction buffer of step (4) includes 50 mM MES buffer with a pH value of 5.

5.

5. A monkeypox virus antigen detection kit according to claim 1, characterized in that: The conjugate diluent in step (7) is 1.2% by mass of Na2HPO4·12H2O and 0.148% by mass of NaH2PO4·2H2O.

6. A monkeypox virus antigen detection kit according to claim 1, characterized in that: The reconstituted solution of step (10) comprises 0.125% by mass of tris(hydroxymethyl)aminomethane, 0.25% by mass of Casein, 20% by mass of sucrose and 5% by mass of trehalose.

7. A monkeypox virus antigen detection kit according to claim 1, characterized in that: The preparation method of the solid phase antibody reaction membrane is as follows: a detection T line and a quality control C line are coated on the solid phase antibody reaction membrane in sequence, the detection T line is coated with a monkeypox monoclonal antibody, and the quality control C line is coated with a goat anti-mouse IgG antibody and DNP-BSA; the concentration of the monkeypox monoclonal antibody is 1.0 mg / mL, the concentration of the goat anti-mouse IgG antibody is 0.5 mg / mL, and the concentration of the DNP-BSA is 0.5 mg / mL.

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