Colloidal gold test strip for rapidly detecting SFTSV antigen and preparation method of colloidal gold test strip

Abstract

According to the rapid detection colloidal gold test strip for the SFTSV antigen and the preparation method of the rapid detection colloidal gold test strip, the SFTSVNP antigen is detected by adopting a double-antibody sandwich method, an N protein monoclonal antibody 1G, prepared in a laboratory, of a human-derived Dualtada virus is used as a detection antibody, and an N protein monoclonal antibody 1E, labeled by colloidal gold, of the human-derived Dualtada virus is used as a labeled antibody; the test strip is formed by sequentially adhering a sample pad, a colloidal gold pad, a cellulose acetate membrane (NC membrane) and absorbent paper to a PVC (polyvinyl chloride) bottom plate along the chromatography direction; the NC membrane is sequentially coated with a detection line and a quality control line along the chromatography direction; the prepared rapid detection colloidal gold test strip for the SFTSV antigen can qualitatively detect the Databean virus in peripheral blood, serum and plasma samples, and a rapid and reliable virus detection technology suitable for grassroots units and field application is established.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a colloidal gold test strip for rapid detection of SFTSV antigen and a preparation method thereof. Background Art

[0002] Fever with thrombocytopenia syndrome virus (SFTSV), which was named Dabebanda virus by the International Committee on Taxonomy of Viruses (ICTV) in recent years, is also known as new Bunyavirus. It is an emerging tick-borne pathogen. Infection with this virus can cause fever with thrombocytopenia syndrome (SFTS), which is clinically manifested by fever, thrombocytopenia and leukocytopenia, gastrointestinal symptoms and multiple organ dysfunction, with a mortality rate of up to 12% to 50%. In 2017, SFTS was listed as one of the top ten infectious diseases that require priority research by the World Health Organization (WHO). Antibodies against SFTSV NP protein are usually detected early after SFTSV infection, indicating that NP protein can be used as a target antigen for early diagnosis of SFTSV infection. At present, the diagnostic method for SFTSV is mainly based on nucleic acid detection, and the viral RNA in SFTSV-infected samples is amplified and detected by a fully automatic fluorescent PCR instrument to achieve the detection of new SFTSV. However, this method is complicated to operate, requires specific laboratories and professional personnel, has a long determination cycle and high cost. Lateral flow immunochromatography (LFIA) is simple, fast and intuitive. Currently, there are more than 1,000 immunochromatographic test systems for testing various analytes. The colloidal gold immunochromatography method is simple to operate, has a short reaction time, and is easy to store reagents. It is of great significance for the rapid detection and prevention of the virus. Summary of the invention

[0003] The object of the present invention is to provide a colloidal gold test strip for rapid detection of SFTSV antigen and a preparation method thereof. The present invention uses colloidal gold as a tracer to prepare a test strip, which can qualitatively detect Dabiebanda virus in peripheral blood, serum, and plasma samples. At present, colloidal gold has been widely used in tracing, imaging, and labeling due to its excellent properties, and immunochromatographic test strip detection is in a particularly important position in the detection field because of its advantages such as simplicity, rapidity, low price, and ability to be used anytime and anywhere. The present invention establishes a fast, reliable virus detection technology suitable for grassroots units and field applications. The detection method is simple and fast to operate, and can be used for clinical auxiliary diagnosis.

[0004] To achieve the above-mentioned purpose and other related purposes, the technical scheme provided by the present invention is: a colloidal gold test paper for rapid detection of SFTSV antigen, characterized in that: the colloidal gold test paper for rapid detection is composed of a sample pad, a colloidal gold binding pad, a cellulose acetate membrane and a water-absorbing paper sequentially pasted on a PVC bottom plate along the chromatography direction; the cellulose acetate membrane is sequentially coated with a detection line and a quality control line along the chromatography direction; the colloidal gold binding pad is coated with a colloidal gold-labeled human N protein monoclonal antibody 1E of the big bebebanda virus, and the coating amount is 10 μg / cm 2 The detection line is coated with 1G monoclonal antibody of the N protein of human Dabiebandha virus, and the coating amount is 20 μg / cm; the quality control line is coated with the N protein of Dabiebandha virus, and the coating amount is 5 μg / cm.

[0005] The preferred technical solution is: the colloidal gold binding pad is pressed on the cellulose acetate membrane by 1-2 mm; the sample pad is pressed on the colloidal gold binding pad by 2-3 mm; and the absorbent paper is pressed on the cellulose acetate membrane by 1-2 mm.

[0006] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for preparing the colloidal gold test paper for rapid detection of SFTSV antigen, characterized in that it comprises the following steps: Step 1: Coating of cellulose acetate membrane The cellulose acetate membrane was pasted onto the PVC base plate, coated with the quality control line coating solution and the detection line coating solution, and dried; the SFTSV-NP protein was diluted to 0.5 mg / mL with 10 mM PBS buffer to prepare the quality control line coating solution; the human N protein monoclonal antibody 1G of the Dabiebandha virus was diluted to 2 mg / mL with 10 mM PBS buffer to prepare the detection line coating solution; Step 2: Preparation of colloidal gold conjugate pad The colloidal gold binding pad treatment solution was dripped onto the glass fiber to make it soaked, and then dried to obtain the binding pad; the colloidal gold binding pad treatment solution was a 50 mM phosphate buffer solution containing 5% by mass of sucrose, 1% by mass of trehalose, and 0.3% by mass of Tween 20; the colloidal gold solution was added to 0.1 M K 2 CO 3 Adjust the pH to 7.5-8.5, mix well, add human N protein monoclonal antibody 1E of Dabebanda virus, mix well, react; add 10% bovine serum albumin to block the reaction; centrifuge to remove the supernatant, re-dissolve with a resuspension solution, dilute, and spread on the binding pad, and dry to obtain a colloidal gold binding pad; the resuspension solution is a 50mM phosphate buffer containing 5% by mass of sucrose and 10% by mass of bovine serum albumin; Step 3: Preparation of sample pad The sample pad treatment solution is added dropwise onto the glass fiber to completely soak it, and then dried to obtain a sample pad; the sample pad treatment solution is a 50 mM Tris-HCl buffer solution with a pH of 8.0 containing 0.15 M sodium chloride and 0.25% Triton-X 100; Step 4: Test strip assembly and cutting Cut the absorbent paper and stick it to the side of the PVC base plate close to the cellulose acetate membrane quality control line, overlapping the cellulose acetate membrane by 1-2mm; stick the colloidal gold binding pad to the side of the PVC base plate close to the cellulose acetate membrane detection line, overlapping the cellulose acetate membrane by 1-2mm; stick the sample pad to the side of the PVC base plate close to the colloidal gold binding pad, overlapping the colloidal gold binding pad by 2-3mm; cut into test strips with a width of about 3.8mm.

[0007] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The present invention adopts the double antibody sandwich method of immunochromatography technology to qualitatively detect Dabiebanda virus in peripheral blood, serum and plasma samples, and interprets the results by directly observing the quality control line (C) and the detection line (T). During the test, the sample treatment liquid is mixed with the peripheral blood, serum and plasma samples for lysis and dilution, and the Dabiebanda virus in the sample is treated by the sample treatment liquid to release N protein, and the N protein is combined with the labeled human Dabiebanda virus N protein monoclonal antibody 1E pre-coated on the colloidal gold pad. The conjugate moves forward under the action of chromatography, and then it will be combined and captured by the human Dabiebanda virus N protein monoclonal antibody 1G fixed on the detection line (T), so that a red strip appears on the detection line (T); the uncaptured conjugate and the labeled human Dabiebanda virus N protein monoclonal antibody 1E continue to chromatograph to the quality control line (C) and combine with the SFTSV-NP protein fixed on the quality control line (C), and a red strip appears. Regardless of whether there is N protein in the sample, the quality control line (C) will have a red strip. This device is a rapid detection device with high detection sensitivity. It can be used for the rapid and accurate detection of Dabiebandha virus antigens. It can be used both clinically and for personal use, and can achieve early detection and early control of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 It is a structural diagram of the test strip of the present invention.

[0009] Figure 2 This is the SFTSV-NP protein detection result of Example 4 of the present invention.

[0010] Figure 3 This is the serum sample test result of Example 5 of the present invention. DETAILED DESCRIPTION

[0011] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0012] See also Figure 1-3 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0013] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0014] Reagents and materials: Reagent: Chloroauric acid (HAuCl 4 · 4H 2 O), trisodium citrate (Na 3 C 6 H 5 O 7 · 2H 2 O), SFTSV nucleoprotein antigen (NP), human Dabiebandha virus N protein monoclonal antibodies 1E and 1G, K 2 CO 3 , BSA, sucrose, trehalose, Na 2 HPO 4 12H 2 O、NaH 2 PO 4 ·2H 2 O, Tween-20, Triton X-100, NaCl, distilled water; Consumables: nitrocellulose membrane (NC membrane), sample pad, colloidal gold binding pad, absorbent pad, PVC base plate, centrifuge tube, pipette and tip, ruler, signature pen and cotton fiber thick pen tip.

[0015] Example 1: Preparation of colloidal gold solution 1) Preparation before preparation Impurities on the surface of the glass container have a great interference effect on the synthesis of colloidal gold particles. Therefore, during the experiment, all containers should be washed with aqua regia, soaked overnight, washed three times with distilled water, and dried in an oven for later use.

[0016] 2) Preparation of colloidal gold solution Take 5 mL of 0.2% chloroauric acid aqueous solution and add it to 95 mL of ultrapure water in a round-bottom flask. The water in the condenser tube is refluxed and heated to 120°C in an oil bath to boil. After boiling, use a pipette to quickly and accurately add freshly prepared 1% trisodium citrate (Na 3 C 6 H 5 O 7 ·2H 2 O) 2mL of aqueous solution. After a few minutes, the color of the solution changes from black to purple to dark blue to wine red. When the color of the solution completely changes to transparent wine red, continue boiling for 15 minutes to obtain the required colloidal gold. Take out the flask from the oil bath, continue stirring at room temperature and cool to room temperature. After cooling, place it in a brown bottle and store it in a refrigerator at 4°C for later use.

[0017] Example 2: Preparation of colloidal gold-labeled human Dabiebandha virus N protein monoclonal antibody 1E Place 1 mL of the 30-40 nm colloidal gold solution prepared in Example 1 in a centrifuge tube and 2 CO 3 Adjust the pH, mix well, add 10μg human N protein monoclonal antibody 1E of Bepbanda virus, mix the solution with an oscillator, react for 1 hour, add 10% BSA, and continue to react for 0.5 hours. Then centrifuge at 10000 rpm for 10 minutes, remove the supernatant, and resuspend the precipitate with a resuspension solution (5% sucrose, 10% bovine serum albumin in 50mM phosphate buffer) to a volume of 100μL and store at 4℃ for later use.

[0018] Example 3: Preparation of colloidal gold rapid test strips for SFTSV nucleoprotein antigen 1. Preparation of sample pad Prepare sample pad pretreatment solution: Prepare 0.25% (v / v) Triton-X100 and 0.15M sodium chloride solution with Tris-HCl buffer at pH 8.0. Cut the glass cellulose membrane sample pad into strips with a width of 4 mm and a length of 10 mm, soak them in the above solution at room temperature, and store them in a drying dish for later use.

[0019] 2. Preparation of colloidal gold binding pad Preparation of immunogold colloid: Prepare an antibody solution labeled with colloidal gold according to Example 2.

[0020] Preparation of colloidal gold binding pad pretreatment solution: Prepare 0.3% (v / v) Tween-20, 1% (w / v) trehalose and 5% (w / v) sucrose solution with PBS buffer at pH = 7.4.

[0021] Cut the glass cellulose membrane sample pad into strips of 4 mm wide and 4 mm long, soak them in the above solution at room temperature, and store them in a drying dish for later use. Take 10 μL of the above colloidal gold-labeled antibody solution and drop it onto the treated conjugate pad, dry it, and prepare a colloidal gold conjugate pad for later use.

[0022] 3. Preparation of nitrocellulose membrane (NC membrane) Prepare a nitrocellulose membrane coated with a specific antibody detection T line and a control C line coated with goat anti-mouse antibody, dry at 27°C, and seal the bag for later use. Dilute the human Dabiebanda virus N protein monoclonal antibody 1G to 2 mg / mL, and dilute the SFTSV-NP protein to 0.5 mg / mL; evenly draw the diluted human Dabiebanda virus N protein monoclonal antibody on the cellulose membrane to obtain a detection T line with an antibody content of 0.02 mg / cm; evenly draw the diluted goat anti-mouse antibody on the cellulose membrane to obtain a control C line with an antibody content of 0.005 mg / cm, and coat at 37°C for later use.

[0023] Preparation of NC membrane treatment solution: Use PBS buffer with pH=7.4 to prepare BSA solution to non-specifically block the prepared NC membrane. After drying, cut the NC membrane into strips with a width of 4 mm and a length of 25 mm for later use.

[0024] 4. Assembly of immunocolloidal gold rapid test strips The bottom plate, nitrocellulose membrane, sample pad, colloidal gold binding pad and water absorbent pad are combined, and the water absorbent pad is pasted on the top of the nitrocellulose membrane on the bottom plate. The nitrocellulose membrane and the water absorbent pad overlap each other by 1 to 2 mm, and the colloidal gold binding pad and the sample pad are pasted in sequence below the nitrocellulose membrane, and the nitrocellulose membrane and the colloidal gold binding pad and the colloidal gold binding pad and the sample pad overlap each other by 1 to 2 mm, so as to obtain the SFTSV immune colloidal gold rapid detection test paper strip.

[0025] Example 4: Validation of NP protein detection using colloidal gold rapid test strips for SFTSV nucleoprotein antigen 1. Test strip test The test strip prepared in the above example was placed flat on the table, 50 μL of 250 / 100 / 50 / 10 / 1 ng of SFTSV nucleoprotein antigen to be tested was added to the sample pad, and the fluid began to flow forward under capillary action, and the result was read after 10 to 15 minutes.

[0026] 2. Result judgment During the detection process, the SFTSV nucleoprotein antigen to be tested first forms a conjugate with the immunocolloidal gold, and then reacts with the corresponding T-line antibody on the nitrocellulose membrane to form a specific double antibody sandwich complex, which is fixed and accumulated to form a red line visible to the naked eye. The unreacted immunogold colloid continues to chromatographically move forward. When it reaches the SFTSV NP pre-coated on the control C line, the immunocolloidal gold is bound by it, so that a red line visible to the naked eye also appears on the C line, which is fixed and accumulated by the colloidal gold particles. Result: The red T line indicates that the SFTSV virus test is positive, and the T line does not show red, indicating that the SFTSV virus test is negative, that is, the test sample does not contain SFTSV virus antigen or the SFTSV virus antigen content is extremely low, and the C line shows red, indicating that the test paper is valid. If the C line does not show red, it means that the test paper is invalid regardless of the T line result, and the test result is invalid.

[0027] 3. Verification results: See the results for details. Figure 2 , 250 / 100 / 50 / 10 / 1ng of SFTSV nucleoprotein antigen can be detected by the colloidal gold rapid test strip of SFTSV nucleoprotein antigen with obvious C line and T line, and the color depth of T line gradually weakens with the decrease of the content of SFTSV nucleoprotein antigen added. The colloidal gold rapid test strip of SFTSV nucleoprotein antigen can effectively detect SFTSV nucleoprotein antigen, and can still effectively detect it when the protein amount is as low as 1ng, indicating that the test strip has high sensitivity.

[0028] Example 5: Application of colloidal gold rapid detection test strips for SFTSV nucleoprotein antigen Take 10 nucleic acid-positive and 10 nucleic acid-negative case sera for testing, place the test strip prepared in the above example flat on the table, add 15 μL of the test serum on the sample pad and add 2 drops of sample lysate, under the action of capillary, the fluid begins to chromatograph forward, and read the result after 10 to 15 minutes. The result judgment method is as in Example 4.

[0029] Verification results: The results are shown in Figure 3 , 10 sera from nucleic acid-positive cases and 10 sera from nucleic acid-negative cases were tested, and it was found that 7 of the 10 nucleic acid-positive samples had obvious C lines and T lines, and 3 had only C lines detected; 9 of the 10 sera from nucleic acid-negative cases had only C lines detected, and 1 had both C and T lines detected.

[0030] Example 6: A colloidal gold test strip for rapid detection of SFTSV antigen and its preparation method A colloidal gold test paper for rapid detection of SFTSV antigen, wherein the colloidal gold test paper is formed by sequentially pasting a sample pad, a colloidal gold binding pad, a cellulose acetate membrane and a water-absorbing paper onto a PVC bottom plate along a chromatography direction; the cellulose acetate membrane is sequentially coated with a detection line and a quality control line along the chromatography direction; the colloidal gold pad is coated with a colloidal gold-labeled human N protein monoclonal antibody 1E of Dabebanda virus, and the coating amount is 10 μg / cm 2 The detection line is coated with 1G monoclonal antibody of the N protein of human Dabiebandha virus, and the coating amount is 20 μg / cm; the quality control line is coated with the N protein of Dabiebandha virus, and the coating amount is 5 μg / cm.

[0031] The preferred technical solution is: the colloidal gold pad is pressed 1 mm onto the cellulose acetate membrane; the sample pad is pressed 2 mm onto the colloidal gold pad; and the absorbent paper is pressed 1 mm onto the cellulose acetate membrane.

[0032] The method for preparing the colloidal gold test paper for rapid detection of SFTSV antigen is characterized by comprising the following steps: Step 1: Coating of cellulose acetate membrane The cellulose acetate membrane was pasted onto the PVC base plate, coated with the quality control line coating solution and the detection line coating solution, and dried; the SFTSV-NP protein was diluted to 0.5 mg / mL with 10mM PBS buffer to prepare the quality control line coating solution; the human Dabiebandha virus N protein monoclonal antibody 1G was diluted to 2 mg / mL with 10mM PBS buffer to prepare the detection line coating solution; Step 2: Preparation of colloidal gold conjugate pad The colloidal gold binding pad treatment solution was dripped onto the glass fiber to make it soaked, and then dried to obtain the binding pad; the colloidal gold binding pad treatment solution was a 50 mM phosphate buffer solution containing 5% sucrose, 1% trehalose, and 0.3% Tween 20; the colloidal gold solution was added to 0.1 M K 2 CO 3 Adjust the pH to 8, mix well, add human N protein monoclonal antibody 1E of Dabebanda virus, mix well, and react; add 10% bovine serum albumin to block the reaction; centrifuge to remove the supernatant, re-dissolve with a resuspending solution, dilute, and spread on the binding pad, and dry to obtain a colloidal gold binding pad; the resuspending solution is a 50mM phosphate buffer containing 5% sucrose and 10% bovine serum albumin; Step 3: Preparation of sample pad The sample pad treatment solution is added dropwise onto the glass fiber to completely soak it, and then dried to obtain a sample pad; the sample pad treatment solution is a 50 mM Tris-HCl buffer solution with a pH of 8.0 containing 0.15 M sodium chloride and 0.25% Triton-X 100; Step 4: Test strip assembly and cutting Cut the absorbent paper and stick it to the side of the PVC base plate close to the cellulose acetate membrane quality control line, overlapping the cellulose acetate membrane by 1mm; stick the colloidal gold binding pad to the side of the PVC base plate close to the cellulose acetate membrane detection line, overlapping the cellulose acetate membrane by 1mm; stick the sample pad to the side of the PVC base plate close to the colloidal gold binding pad, overlapping the colloidal gold binding pad by 2mm; cut into test strips with a width of about 3.8mm.

[0033] Example 7: A colloidal gold test strip for rapid detection of SFTSV antigen and its preparation method The present invention provides a rapid detection test strip for Dabiebandha virus antigen; The test strip is composed of a sample pad, a colloidal gold pad, a cellulose acetate membrane (NC membrane) and a blotting paper adhered to a PVC bottom plate in sequence along the chromatography direction; the NC membrane is coated with a detection line and a quality control line in sequence along the chromatography direction; the colloidal gold pad is coated with a colloidal gold-labeled human large benda virus N protein monoclonal antibody 1E, and the coating amount is 10 μg / cm 2 ; The detection line is coated with 1G monoclonal antibody against the N protein of human Dabiebandha virus, with a coating amount of 20 μg / cm; the quality control line is coated with the N protein of Dabiebandha virus, with a coating amount of 5 μg / cm;.

[0034] The test strip is about 3 mm wide; the NC membrane is pasted on the PVC base plate and is about 2.5 cm long; the microsphere pad is about 0.7 cm long, and the microsphere pad is pressed on the NC membrane by 1-2 mm; the sample pad is about 1.7 cm long, and the sample pad is pressed on the colloidal gold pad by 2-3 mm; the absorbent paper is about 1.7 cm long, and the absorbent paper is pressed on the NC membrane by 1-2 mm.

[0035] The present invention also provides a rapid detection kit for Dabiebandha virus antigen, which contains the rapid detection test strip for Dabiebandha virus antigen of the present invention and a sample processing solution.

[0036] Preferably, the sample processing solution is a 20-200 mM phosphate buffer containing 0.9%-1% sodium chloride, 0.1-0.2% Tween 20, 0.1-0.5% bovine serum albumin, and 0.05-0.1% proclin300, and its pH value is 8.0-8.2.

[0037] The present invention also provides a method for preparing the above-mentioned rapid detection reagent strip for Dabiebandha virus antigen, comprising the following steps: (1) NC membrane coating Cut the NC membrane into a width of about 2.5 cm and paste it on the PVC base plate, coat it with the quality control line coating solution and the test line coating solution, and dry it; dilute the SFTSV-NP protein to 0.5 mg / mL with 10mM PBS buffer as the quality control line coating solution; dilute the human Bigbebanda virus N protein monoclonal antibody 1G to 2 mg / mL with 10mM PBS buffer as the test line coating solution; (2) Preparation of colloidal gold pad Add the binding pad treatment solution dropwise onto the glass fiber to completely soak it, and dry it to obtain the binding pad; the colloidal gold pad treatment solution is a 50 mM phosphate buffer solution containing 5% sucrose, 1% trehalose, and 0.3% Tween 20; Add 0.1M K colloidal gold solution 2 CO 3 Adjust the pH, mix well, add human N protein monoclonal antibody 1E of Dabebanda virus, mix well, react; add 10% bovine serum albumin to block the reaction; centrifuge to remove the supernatant, re-dissolve with a resuspension solution, dilute, spread on the binding pad, and dry; the resuspension solution is a 50mM phosphate buffer containing 5% sucrose and 10% bovine serum albumin; (3) Preparation of sample pad The sample pad treatment solution is added dropwise onto the glass fiber to completely soak it, and then dried to obtain a sample pad; the sample pad treatment solution is a 50 mM Tris-HCl buffer solution with a pH of 8.0 containing 0.15 M sodium chloride and 0.25% Triton-X 100; (4) Test strip assembly and cutting Cut the absorbent paper into a width of about 1.7 cm and stick it to the PVC base plate coated with NC membrane on the side close to the NC membrane quality control line, overlapping the NC membrane by 1-2 mm; cut the colloidal gold pad into a width of about 0.7 cm and stick it to the PVC base plate on the side close to the NC membrane detection line, overlapping the NC membrane by 1-2 mm; cut the sample pad into a width of about 1.7 cm and stick it to the PVC base plate on the side close to the microsphere pad, overlapping the microsphere pad by 2-3 mm; cut the test strips into a width of about 3.8 mm.

[0038] The present invention also provides a method for rapid detection of Dabiebandha virus antigen, wherein the rapid detection test strip of the present invention is used. According to the present invention, the detection method is not for the purpose of disease diagnosis. Specifically, the peripheral blood, serum, and plasma samples are lysed and diluted, transferred to the sample pad of the test strip, and the test strip begins chromatography, and it takes about 15 minutes to obtain the test result.

[0039] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A colloidal gold test paper for rapid detection of SFTSV antigen, characterized in that: The rapid detection colloidal gold test paper is composed of a sample pad, a colloidal gold binding pad, a cellulose acetate membrane and a water-absorbing paper adhered to a PVC bottom plate in sequence along the chromatography direction; the cellulose acetate membrane is coated with a detection line and a quality control line in sequence along the chromatography direction; the colloidal gold binding pad is coated with a colloidal gold-labeled human large benda virus N protein monoclonal antibody 1E, and the coating amount is 10 μg / cm 2 The detection line is coated with 1G monoclonal antibody of the N protein of human Dabiebandha virus, and the coating amount is 20 μg / cm; the quality control line is coated with the N protein of Dabiebandha virus, and the coating amount is 5 μg / cm.

2. The rapid detection colloidal gold test paper for SFTSV antigen according to claim 1, characterized in that: The colloidal gold binding pad is pressed on the cellulose acetate membrane by 1-2 mm; the sample pad is pressed on the colloidal gold binding pad by 2-3 mm; and the absorbent paper is pressed on the cellulose acetate membrane by 1-2 mm.

3. A method for preparing a colloidal gold test paper for rapid detection of SFTSV antigen according to claim 1 or 2, characterized in that: The steps include: Step 1: Coating of cellulose acetate membrane The cellulose acetate membrane was pasted onto the PVC base plate, coated with the quality control line coating solution and the detection line coating solution, and dried; the SFTSV-NP protein was diluted to 0.5 mg / mL with 10 mM PBS buffer to prepare the quality control line coating solution; the human N protein monoclonal antibody 1G of the Dabiebandha virus was diluted to 2 mg / mL with 10 mM PBS buffer to prepare the detection line coating solution; Step 2: Preparation of colloidal gold conjugate pad Add colloidal gold binding pad treatment solution dropwise onto the glass fiber to make it soaked, and dry to obtain the binding pad; the colloidal gold binding pad treatment solution is a 50mM phosphate buffer containing 5% by mass of sucrose, 1% by mass of trehalose, and 0.3% by mass of Tween 20; adjust the pH value of the colloidal gold solution to 7.5-8.5, mix well, add human N protein monoclonal antibody 1E of big band virus, mix well, and react; add 10% bovine serum albumin to block the reaction; centrifuge to remove the supernatant, re-dissolve with a resuspension solution, dilute, and spread on the binding pad, and dry to obtain the colloidal gold binding pad; the resuspension solution is a 50mM phosphate buffer containing 5% by mass of sucrose and 10% by mass of bovine serum albumin; Step 3: Preparation of sample pad The sample pad treatment solution is added dropwise onto the glass fiber to completely soak it, and then dried to obtain a sample pad; the sample pad treatment solution is a 50 mM Tris-HCl buffer solution with a pH of 8.0 containing 0.15 M sodium chloride and 0.25% Triton-X 100; Step 4: Test strip assembly and cutting Cut the absorbent paper and stick it to the side of the PVC base plate close to the cellulose acetate membrane quality control line, overlapping the cellulose acetate membrane by 1-2mm; stick the colloidal gold binding pad to the side of the PVC base plate close to the cellulose acetate membrane detection line, overlapping the cellulose acetate membrane by 1-2mm; stick the sample pad to the side of the PVC base plate close to the colloidal gold binding pad, overlapping the colloidal gold binding pad by 2-3mm; cut into test strips with a width of about 3.8mm.

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