Monoclonal antibody and application thereof in detection of pepper vein mottle virus

Abstract

The invention belongs to the technical field of agricultural biology, and relates to a monoclonal antibody and application thereof in detection of pepper vein mottle virus, the monoclonal antibody for detection of pepper vein mottle virus comprises a first monoclonal antibody and / or a second monoclonal antibody; wherein the first monoclonal antibody is generated by a first hybridoma cell strain, the second monoclonal antibody is generated by a second hybridoma cell strain, the preservation number of the first hybridoma cell strain is CCTCC (China Center For Type Culture Collection) NO.C2024333, and the preservation number of the second hybridoma cell strain is CCTCC NO.C2024334. The monoclonal antibody has the advantages of strong specificity, high sensitivity, high titer, accurate and quantitative detection of the capsicum vein mottle virus in different organs of crops, accurate and reliable identification result, and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and relates to a monoclonal antibody and an application thereof in detecting pepper vein mottle virus. Background Art

[0002] Chilli veinal mottle virus (ChiVMV) is a species of Potyvirus in the family Potyviridae. In recent years, the virus has spread rapidly in my country, causing serious economic losses to Solanaceae plants. It is one of the important viruses for port quarantine in my country.

[0003] The natural hosts of ChiVMV are mainly peppers, tomatoes and other Solanaceae crops. When it harms in the field, it can cause symptoms such as mottled leaves, mosaics, shrinkage, and dwarfed plants on the host plants. Under natural conditions, it mainly relies on aphids for non-persistent transmission. Mechanical transmission and sap transmission can also spread the virus, but the virus cannot be transmitted through seeds. In my country, the virus was first found on peppers in Shaanxi in 2003. Subsequently, there were reports of the virus harming Hainan, Yunnan, Fujian, Hunan, Guangdong, Guangxi and other regions. ChiVMV has become the dominant virus species of pepper virus disease in southern my country. The World Vegetable Center (formerly the Asian Vegetable Research and Development Center) conducted a survey in 16 countries or regions in Asia, and the results showed that 30% of peppers were affected by CHiVMV. Not only that, the virus has also spread to other crops, among which tobacco is the most seriously damaged. Reports of ChiVMV causing epidemics of tobacco virus disease have been found in tobacco-growing areas in Yunnan, Anhui, Sichuan, Guizhou and other provinces. In addition, ChiVMV often co-infects with other viruses, causing more serious economic losses.

[0004] Therefore, pepper vein mottle virus (ChiVMV) is currently an important virus that harms Solanaceae plants, but its rapid detection method is lacking, and there are few research reports on related antibodies, hybridoma cells, etc., which urgently need breakthroughs. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides monoclonal antibodies and their use in detecting pepper vein mottle virus. In order to improve the detection and identification of pepper vein mottle virus (ChiVMV), effectively supervise related plants and quickly screen and detect infected offspring, the present invention provides a hybridoma cell pair and a monoclonal antibody pair secreted by the hybridoma cell pair and their use in detecting pepper vein mottle virus (ChiVMV). The main purpose is to provide a rapid and sensitive detection antibody and detection method for pepper vein mottle virus (ChiVMV) in plants.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] The present invention provides a monoclonal antibody, wherein the monoclonal antibody comprises a first monoclonal antibody and / or a second monoclonal antibody;

[0008] The first monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO.C2024333, and the second monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO.C2024334.

[0009] Furthermore, when the monoclonal antibodies are the first monoclonal antibody and the second monoclonal antibody, the first monoclonal antibody and the second monoclonal antibody are stored independently.

[0010] The present invention also provides a hybridoma cell strain secreting the above monoclonal antibody, wherein the deposit number of the hybridoma cell strain secreting the first monoclonal antibody is CCTCC NO.C2024333, and the deposit number of the hybridoma cell strain secreting the second monoclonal antibody is CCTCC NO.C2024334.

[0011] The present invention also provides the use of the above monoclonal antibody in preparing a product for detecting pepper vein mottle virus, wherein the product is a kit or a test paper.

[0012] The present invention also provides a test kit for detecting pepper vein mottle virus, which comprises: the first monoclonal antibody or the second monoclonal antibody pre-coated ELISA plate, sample diluent, washing solution, reaction stop solution, enzyme-labeled antibody, and color developer.

[0013] Furthermore, the kit also includes a pepper vein mottle virus standard. Further preferably, the pepper vein mottle virus standard is extracted from a plant that becomes diseased after being inoculated with ChiVMV, the extraction method is sucrose density gradient centrifugation, and the inoculated virus strain is ChiVMV Guizhou tobacco strain (sequence accession number: OP378160.1).

[0014] Further, the sample diluent is a phosphate buffer;

[0015] Further, the washing solution is a phosphate buffer containing Tween-20;

[0016] Furthermore, the reaction termination solution is a sulfuric acid solution; preferably, the sulfuric acid solution is 1.8-2.2M, more preferably 1.9-2.1M, and more preferably 2M.

[0017] Furthermore, the developer is TMB developer.

[0018] Furthermore, the enzyme-labeled antibody is an enzyme-labeled polyclonal antibody, and further, the enzyme is horseradish peroxidase.

[0019] Preferably, the concentration of the enzyme-labeled antibody is 8 to 12 μg / mL, preferably 9 to 11 μg / mL, more preferably 10 μg / mL;

[0020] Preferably, the incubation temperature after adding the antibody is 22-26° C., preferably 25° C., and the incubation time is 40-50 minutes, preferably 45 minutes.

[0021] The present invention also provides a method for detecting pepper vein mottle virus (ChiVMV), using the above-mentioned kit, comprising the following steps:

[0022] S100, extracting from the sample to be tested to obtain a sample extract;

[0023] S110, using the above kit to detect the sample extract, the main process of which includes adding sample, incubation, washing, adding enzyme, incubation, washing, color development, termination and reading;

[0024] S120. Calculate the concentration of pepper vein mottle virus (ChiVMV) in the sample to be tested using a concentration-absorbance standard curve prepared using a pepper vein mottle virus (ChiVMV) standard substance.

[0025] The present invention also provides a pepper vein mottle virus rapid test paper, comprising the above-mentioned monoclonal antibody pair.

[0026] The present invention also provides a method for preparing the above-mentioned pepper vein mottle virus rapid test paper:

[0027] S200, assembly of a large plate of rapid test paper: a NC membrane labeled with a specific monoclonal antibody (one of the above-mentioned monoclonal antibodies) and a goat anti-mouse secondary antibody is fixed in the middle of the large plate, and a gold-labeled pad labeled with a specific monoclonal antibody (another of the above-mentioned monoclonal antibodies) is covered under the NC membrane, with a width of about 10 mm and a front edge thereof overlapping 1-2 mm from the bottom of the NC membrane; a sample pad is about 20-30 mm wide and covers the gold-labeled pad, with a front edge thereof overlapping 1-2 mm from the bottom of the gold-labeled pad; an absorption pad is about 20-30 mm wide and is located at the top of the test strip, with a bottom thereof covering 1-2 mm from the top of the NC membrane.

[0028] S210, cutting of rapid test paper: Use a strip cutter to cut the large board into finished rapid test paper with a width of about 3-4 mm.

[0029] The present invention also provides a detection method for the above-mentioned pepper vein mottle virus rapid test paper, wherein the above-mentioned rapid test paper is vertically inserted into the sample solution, and the sample solution will penetrate along the sample pad to the label pad of the colloidal gold labeled antibody, and form a double antibody sandwich structure with the specific antibody on the NC membrane, and after 5-8 minutes, observe the color change in the result observation hole to make a judgment. Result judgment: negative result (1 C line appears); positive (+): C / T lines are colored at the same time.

[0030] The beneficial effects of the present invention are as follows: the monoclonal antibody provided by the present invention has strong specificity, high sensitivity, and high titer, and can specifically and accurately perform quantitative detection of pepper vein mottle virus (ChiVMV) in different organs of crops, and the identification results are accurate, reliable, and highly sensitive.

[0031] The present invention immunizes rabbits with ChiVMV virus to obtain polyclonal antibodies, immunizes mice with ChiVMV virus, and obtains specific monoclonal antibodies through screening, and finally develops an enzyme-linked immunosorbent assay quantitative detection kit for quantitatively detecting ChiVMV content, which comprises: immunizing 2-month-old New Zealand white rabbits with pepper vein mottle virus (ChiVMV) to obtain polyclonal antibodies against ChiVMV virus, immunizing 6-week-old female BALB / c mice with pepper vein mottle virus (ChiVMV), fusing spleen cells of immune mice with myeloma cells SP2 / 0, obtaining specific monoclonal antibody cell strains through screening, preparing ascites, and obtaining specific monoclonal antibodies through purification; using a double antibody sandwich ELISA method to screen paired antibodies; establishing a double antibody sandwich ELISA method, and obtaining an ELISA kit system through optimization, which can quantitatively detect pepper vein mottle disease (ChiVMV). The present invention can perform qualitative and quantitative detection of pepper vein mottle virus (ChiVMV) in plants through ELISA experiments, and has the advantages of strong specificity, high sensitivity, simplicity and rapidity, etc.

[0032] Deposit of biological materials: The hybridoma cell line secreting the first monoclonal antibody of the present invention was screened by the inventor of the present invention, and its preservation number is CCTCC NO.C2024333, the cell line preservation name is hybridoma cell line (Hybridoma cell line) ChiVMV-2, the preservation date is October 22, 2024, the preservation unit is the China Center for Type Culture Collection, the preservation institution is abbreviated as CCTCC, and the address is the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, China.

[0033] The hybridoma cell line secreting the second monoclonal antibody of the present invention is obtained by fusion screening by the inventor of the present invention, and its preservation number is CCTCC NO.C2024334, the cell line preservation name is hybridoma cell line (Hybridoma cell line) ChiVMV-5, the preservation date is October 22, 2024, the preservation unit is China Center for Type Culture Collection, the preservation institution is abbreviated as CCTCC, and the address is Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a standard curve diagram of the kit of the present invention;

[0036] Figure 2 It is a schematic diagram of the side structure of the rapid test paper of the present invention;

[0037] Figure 3 It is a schematic diagram of the top view of the rapid test paper of the present invention;

[0038] Figure 4 It is a structural schematic diagram of the rapid test paper of the present invention;

[0039] Figure 5 It is the detection effect of the rapid test strip of the present invention at different dilution multiples of the sample.

[0040] Reference numerals: 1 - sample pad, 2 - colloidal gold binding pad, 3 - chromatographic membrane, 4 - water-absorbing material, 5 - detection line T, 6 - quality control line C, 7 - PVC rubber plate. DETAILED DESCRIPTION

[0041] The principles and features of the present invention are described below (in conjunction with the accompanying drawings). The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0042] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0043] The sources of some experimental materials and experimental instruments of the present invention are shown in Table 1 below:

[0044] Table 1 Experimental materials and experimental instruments

[0045]

[0046]

[0047]

[0048] The present invention provides a monoclonal antibody for detecting pepper vein mottle virus, wherein the monoclonal antibody comprises a first monoclonal antibody and / or a second monoclonal antibody;

[0049] The first monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO.C2024333, and the second monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO.C2024334.

[0050] Furthermore, when the monoclonal antibodies are the first monoclonal antibody and the second monoclonal antibody, the first monoclonal antibody and the second monoclonal antibody are stored independently.

[0051] The present invention also provides a hybridoma cell strain secreting the above monoclonal antibody, wherein the deposit number of the hybridoma cell strain secreting the first monoclonal antibody is CCTCC NO.C2024333, and the deposit number of the hybridoma cell strain secreting the second monoclonal antibody is CCTCC NO.C2024334.

[0052] The present invention also provides the use of the above monoclonal antibody in preparing a product for detecting pepper vein mottle virus, wherein the product is a kit or a test paper.

[0053] The present invention also provides a test kit for detecting pepper vein mottle virus, which comprises: the first monoclonal antibody or the second monoclonal antibody pre-coated ELISA plate, sample diluent, washing solution, reaction stop solution, enzyme-labeled antibody, and color developer.

[0054] The present invention immunizes rabbits with ChiVMV virus to obtain polyclonal antibodies, immunizes mice with ChiVMV virus, and obtains specific monoclonal antibodies through screening, and finally develops an enzyme-linked immunosorbent assay quantitative detection kit for quantitatively detecting the content of pepper vein mottle virus (ChiVMV), which includes: immunizing 2-month-old New Zealand white rabbits with pepper vein mottle virus (ChiVMV) to obtain polyclonal antibodies against ChiVMV virus, immunizing 6-week-old female BALB / c mice with pepper vein mottle virus (ChiVMV), fusing spleen cells of immune mice with myeloma cells SP2 / 0, obtaining specific monoclonal antibody cell strains through screening, preparing ascites, and obtaining specific monoclonal antibodies through purification; using a double antibody sandwich ELISA method to screen paired antibodies; establishing a double antibody sandwich ELISA method, and obtaining an ELISA kit system through optimization, which can quantitatively detect pepper vein mottle virus (ChiVMV). The present invention can perform qualitative and quantitative detection of pepper vein mottle virus (ChiVMV) in plants through ELISA experiments, and has the advantages of strong specificity, high sensitivity, simplicity and rapidity.

[0055] Furthermore, the kit also includes a pepper vein mottle virus standard. Further preferably, the pepper vein mottle virus standard is extracted from a plant infected with the virus.

[0056] It is understood that the kit includes: a first monoclonal antibody pre-coated ELISA plate, a sample diluent, a washing solution, a reaction stop solution, an enzyme-labeled antibody, a color developer and a ChiVMV standard. Or the kit includes: a second monoclonal antibody pre-coated ELISA plate, a sample diluent, a washing solution, a reaction stop solution, an enzyme-labeled antibody, a color developer and a ChiVMV standard.

[0057] The steps of pre-coating the ELISA plate with monoclonal antibodies are as follows: diluting the first monoclonal antibody or the second monoclonal antibody to a certain concentration, adding to a 96-well ELISA plate, 100 μL per well, and reacting at 37° C. for 3 hours; drying the well solution, adding a washing solution, soaking for 5 minutes, drying the well solution, and patting dry on absorbent paper; adding a blocking solution to the 96-well ELISA plate, 150 μL per well, and reacting at 37° C. for 2 hours; drying the well solution, patting dry on absorbent paper, and drying with a freeze dryer; and packaging in an aluminum foil bag with a vacuum packaging machine.

[0058] The coating buffer is a carbonate buffer, and more preferably, the carbonate buffer is a Na2CO3-NaHCO3 buffer, with a concentration of 0.1 M and a pH value of 9.6. Preferably, the blocking solution is a carbonate buffer containing BSA.

[0059] As an optional embodiment, the sample diluent may be a phosphate buffer;

[0060] As an optional embodiment, the washing solution is a phosphate buffer containing Tween-20;

[0061] As an optional embodiment, the reaction termination solution is a sulfuric acid solution; preferably, the sulfuric acid solution is 1.8-2.2M, more preferably 1.9-2.1M, and more preferably 2M.

[0062] As an optional embodiment, the color developer is TMB color developer.

[0063] As an optional embodiment, the enzyme-labeled antibody is an enzyme-labeled polyclonal antibody. Further, the enzyme is horseradish peroxidase. Preferably, the concentration of the monoclonal antibody labeled with horseradish peroxidase is 8-12 μg / mL, preferably 9-11 μg / mL, and more preferably 10 μg / mL. The method for labeling the polyclonal antibody with horseradish peroxidase can be a conventional enzyme labeling scheme in the art.

[0064] Preferably, the incubation temperature after adding the antibody is 22-26° C., preferably 25° C., and the incubation time is 40-50 minutes, preferably 45 minutes.

[0065] The present invention also provides a method for detecting pepper vein mottle virus (ChiVMV), using the above-mentioned kit, comprising the following steps:

[0066] S100, extracting from the sample to be tested to obtain a sample extract;

[0067] S110, using the above kit to detect the sample extract, the main process of which includes adding sample, incubation, washing, adding enzyme, incubation, washing, color development, termination and reading;

[0068] S120. Calculate the concentration of pepper vein mottle virus (ChiVMV) in the sample to be tested using a concentration-absorbance standard curve prepared using a pepper vein mottle virus (ChiVMV) standard substance.

[0069] The present invention also provides a pepper vein mottle virus rapid test paper, comprising the above-mentioned monoclonal antibody pair.

[0070] The present invention also provides a method for preparing the above-mentioned pepper vein mottle virus rapid test paper:

[0071] S200, assembly of the large plate of rapid test paper: the NC membrane marked with specific monoclonal antibody (one of the antibodies of the above-mentioned monoclonal antibody pair) and goat anti-mouse secondary antibody is fixed in the middle of the large plate, and the gold label pad marked with specific monoclonal antibody (the other antibody of the above-mentioned monoclonal antibody pair) is covered under the NC membrane, the width of the label pad is about 10mm, and its front edge is overlapped 1-2mm from the bottom of the NC membrane; the width of the sample pad is about 20-30mm, covering the gold label pad, and its front edge is overlapped 1-2mm from the bottom of the gold label pad; the width of the absorption pad is about 20-30mm, located at the top of the test strip, and its bottom covers 1-2mm from the top of the NC membrane.

[0072] S210, cutting of rapid test paper: Use a strip cutter to cut the large board into finished rapid test paper with a width of about 3-4 mm.

[0073] It can be understood that in step S200, the NC membrane marked with the first monoclonal antibody and the goat anti-mouse secondary antibody can be fixed in the middle of the large plate, and the gold label pad marked with the second monoclonal antibody can be covered under the NC membrane. The width of the label pad is about 10 mm, and its front edge is 1-2 mm above the bottom of the NC membrane; the sample pad is about 20-30 mm wide and covers the gold label pad, and its front edge is 1-2 mm below the gold label pad; the absorption pad is about 20-30 mm wide and is located at the top of the test strip, and its lower part covers 1-2 mm above the upper part of the NC membrane.

[0074] Alternatively, step S200 is: the NC membrane labeled with the second monoclonal antibody and the goat anti-mouse secondary antibody is fixed in the middle of the large plate, and the gold label pad labeled with the first monoclonal antibody is covered under the NC membrane, the width of the label pad is about 10 mm, and its front edge overlaps 1-2 mm from the bottom of the NC membrane; the sample pad is about 20-30 mm wide, covering the gold label pad, and its front edge overlaps 1-2 mm from the bottom of the gold label pad; the absorption pad is about 20-30 mm wide, located at the top of the test strip, and its lower part covers 1-2 mm from the upper part of the NC membrane.

[0075] The present invention also provides a detection method for the above-mentioned pepper vein mottle virus rapid test paper, wherein the above-mentioned rapid test paper is vertically inserted into the sample solution, and the sample solution will penetrate along the sample pad to the label pad of the colloidal gold labeled antibody, and form a double antibody sandwich structure with the specific antibody on the NC membrane, and after 5-8 minutes, observe the color change in the result observation hole to make a judgment. Result judgment: negative result (1 C line appears); positive (+): C / T lines are colored at the same time.

[0076] The present invention is described in detail below in conjunction with embodiments and experimental data.

[0077] Example 1 Polyclonal Antibody Preparation

[0078] 1. Preparation of immunogen: Mix ChiVMV virus with an equal volume of Freund's adjuvant and emulsify evenly to form an oil-in-water state for immunization of rabbits.

[0079] 2. Immunization strategy: Two New Zealand white rabbits were immunized with ChiVMV virus, subcutaneously for three times with an interval of 4 weeks, and finally detected by indirect ELISA.

[0080] The steps of the indirect ELISA method are:

[0081] 1) Dilute the expressed and purified protein to 1 μg / mL with 0.1 mol / L, pH=9.6 carbonate buffer, add 100 μL to each well of a 96-well ELISA plate, react at 37°C for 3 h or stand at 4°C overnight;

[0082] 2) Shake off the liquid in the plate wells, add 250 μL of washing buffer, let stand for 30 seconds, shake off the liquid in the plate, and repeat 3 times;

[0083] 3) Add the test sample, 100 μL per well, and add the positive control (positive rabbit serum obtained in step (2)), negative control (pre-immune rabbit serum) and blank control (no rabbit serum) at 37°C for 45 min;

[0084] 4) Repeat step 2);

[0085] 5) Add HRP-labeled goat anti-rabbit enzyme-labeled secondary antibody, 100 μL per well, and react at 37°C for 45 min;

[0086] 6) Repeat step 2);

[0087] 7) Add chromogen, 100 μL per well, and react at room temperature in the dark for 15 min;

[0088] 8) Add stop solution, 100 μL per well, and read the OD value at a wavelength of 450 using a microplate reader. The antiserum titer is as shown in Table 2:

[0089] Table 2 Antiserum titer

[0090]

[0091] 3. Polyclonal antibody purification: Rabbit serum was centrifuged for 15 min (4000 rpm, room temperature), the supernatant was taken, saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded; the precipitate was dissolved in an appropriate amount of PBS (0.01M, pH7.4); saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded; the precipitate was dissolved in an appropriate amount of PBS (0.01M, pH7.4), dialyzed overnight at 4°C, the antibody content was determined, and frozen at -20°C for later use; after ammonium sulfate precipitation, Protein A column was continued for purification, and the new column was first passed with 5 mL of ultrapure water, and then 5 mL of 0.4 M PB buffer (pH7.0) was used to balance the purification column; the antibody was passed through the column, and the column was required to pass slowly during the process so that the antibody protein could better bind to the binding site; continue 10 mL The purification column was equilibrated with 0.4 M PB buffer (pH 7.0); the antibody on the binding site was eluted with 5 mL 0.1 M glycine-HCl buffer (pH 3.0), and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine to keep the pH at a neutral value suitable for antibody storage.

[0092] Example 2 Monoclonal Antibody Preparation

[0093] 1. Monoclonal cell lines

[0094] 1. Preparation of immunogen: Mix ChiVMV virus with an equal volume of Freund's adjuvant and emulsify evenly to form an oil-in-water state for immunization of mice;

[0095] 2. Immunization strategy: ChiVMV virus was used to immunize four Balb / c mice, subcutaneously for three times, with an interval of 4 weeks, and finally detected by indirect ELISA;

[0096] The steps of the indirect ELISA method are:

[0097] 1) Dilute the expressed and purified protein to 1 μg / mL with 0.1 mol / L, pH 9.6 carbonate buffer, add 100 μL to each well of a 96-well ELISA plate, react at 37°C for 3 h or stand at 4°C overnight.

[0098] 2) Shake off the liquid in the plate wells, add 250 μL of washing buffer, let stand for 30 seconds, shake off the liquid in the plate, and repeat 3 times.

[0099] 3) Add the test sample, 100 μL per well, and add the positive control (positive mouse serum obtained in step (2)), negative control (pre-immune mouse serum) and blank control (no mouse serum) at 37°C for 45 min.

[0100] 4) Repeat step 2);

[0101] 5) Add HRP-labeled goat anti-mouse enzyme-labeled secondary antibody, 100 μL per well, and react at 37°C for 45 min.

[0102] 6) Repeat step 2);

[0103] 7) Add 100 μL of developer to each well and react at room temperature in the dark for 15 min.

[0104] 8) Add stop solution, 100 μL per well, and read the OD value at a wavelength of 450 using a microplate reader. The antiserum titer is as shown in Table 3:

[0105] Table 3 Antiserum titer table

[0106]

[0107]

[0108] 3. Cell fusion: Two weeks after the last immunization, the antigen was injected intraperitoneally for booster immunization. Three days later, cell fusion was performed. The mice were killed by cervical dislocation and disinfected by soaking in 70% ethanol for 30 minutes. The abdominal cavity was cut open on a clean bench, the spleen was taken out, ground, and passed through an 80-mesh sieve to obtain spleen cells. SP2 / 0 myeloma cells were added and cell fusion was performed under the action of PEG4000.

[0109] 4. Fusion screening: The fused cells were plated into a 96-well plate and cultured with HAT culture medium. After three days, the medium was changed to HT culture medium. After 10 days, the cell culture supernatant was taken for detection.

[0110] 5. Cloning and strain establishment: Use the limiting dilution method to clone the positive wells, test after 10 days, and continue to clone the positive clones by limiting dilution until all the clones are positive. Positive cell lines can be established, and finally 6 positive cell lines are obtained;

[0111] 6. Expand the culture: Expand the culture of the established monoclonal cells and freeze them.

[0112] 2. Ascites Preparation and Purification

[0113] 1. Preparation of ascites: One week in advance, mineral oil was injected into the peritoneal cavity of mice, and a certain number of cells were injected into the peritoneal cavity of mice. The ascites was collected about 10 days later and centrifuged at 4000 rpm. The supernatant was the monoclonal antibody ascites.

[0114] 2. Monoclonal antibody purification: centrifuge the ascites for 15 min (4000 rpm, room temperature), take the supernatant, add saturated ammonium sulfate drop by drop slowly to half saturation under stirring at 4°C, continue stirring for 30 min, centrifuge for 30 min (13000 rpm, 4°C), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4); add saturated ammonium sulfate drop by drop slowly to 33% under stirring at 4°C, continue stirring for 30 min, centrifuge for 30 min (13000 rpm, 4°C), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4), dialyze overnight at 4°C, determine the antibody content, and freeze at -20°C for later use. After ammonium sulfate precipitation, the Protein G column was used for purification. The new column was first passed through with 5 mL of ultrapure water, and then the purification column was equilibrated with 5 mL of 0.4 M PB buffer (pH 7.0); the antibody was passed through the column slowly during the process so that the antibody protein could better bind to the binding site; 10 mL of 0.4 M PB buffer (pH 7.0) was used to equilibrate the purification column; 5 mL of 0.1 M glycine-hydrochloric acid buffer (pH 2.7) was used to elute the antibody on the binding site, and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine to keep the pH at a neutral value suitable for antibody storage, thereby obtaining the purified monoclonal antibody.

[0115] 3. Antibody Screening

[0116] 1. Potency test

[0117] The purified antibodies were tested for potency using the indirect ELISA method. The data are shown in Table 4:

[0118] Table 4

[0119] Test date 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 Cell number FL596-1 FL596-2 FL596-3 FL596-4 FL596-5 FL596-6 Detection source V V V V V V V V V V V V 1K 1.9113 1.8526 1.8639 2.1626 2.3603 1.9048 3K 1.1142 1.9769 1.698 1.2847 2.38 1.4654 9K 0.9049 1.9366 1.5242 1.3813 2.2872 0.9354 27K 0.5947 1.6377 1.3017 0.9976 2.1671 1.09 81K 0.3018 1.0304 0.8368 0.4744 1.7132 0.4699 243K 0.2882 0.4967 0.3707 0.1581 0.7958 0.259 729K 0.1684 0.2559 0.1967 0.1094 0.4334 0.1982 Negative 0.0732 0.0772 0.1564 0.1415 0.1505 0.082 Potency 27K 81K 81K 81K 729k 81K

[0120] 2. Subtype detection

[0121] The antibody was tested for subtype using a mouse antibody subtype detection kit. The results are shown in Tables 5 and 6:

[0122] Table 5

[0123] Antibody ID Clone number Subtype FL596-1 6C64F13F1 IgG1, kappa FL596-2 6E95E64C5 IgG2b, kappa FL596-3 7F46A115D6 IgG1, kappa FL596-4 7C107B26C1 IgG1, kappa FL596-5 6C516F712G10 IgG1, kappa FL596-6 8B718D813C6 IgG1, kappa

[0124] Table 6

[0125] Test date 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 2024 / 3 / 12 Cell number FL596-1 FL596-2 FL596-3 FL596-4 FL596-5 FL596-6 Detection V V V V V V V V V V V V 1K 1.9113 1.8526 1.8639 2.1626 2.3603 1.9048 3K 1.1142 1.9769 1.698 1.2847 2.38 1.4654 9K 0.9049 1.9366 1.5242 1.3813 2.2872 0.9354 27K 0.5947 1.6377 1.3017 0.9976 2.1671 1.09 81K 0.3018 1.0304 0.8368 0.4744 1.7132 0.4699 243K 0.2882 0.4967 0.3707 0.1581 0.7958 0.259 729K 0.1684 0.2559 0.1967 0.1094 0.4334 0.1982 Negative 0.0732 0.0772 0.1564 0.1415 0.1505 0.082 Potency 27K 81K 81K 81K 729k 81K

[0126] 3. Antibody pairing

[0127] The prepared antibodies were screened using the double antibody sandwich ELISA method to screen out the antibody pairs that can be paired.

[0128] The double antibody sandwich ELISA method is as follows:

[0129] a. Dilute the coating antibody to 10 μg / mL with CB, add 100 μL to each well, and coat at 37°C for 3 h.

[0130] b. Wash the plate 3-5 times, pat dry, add positive standard / negative control / positive sample / negative sample, 100 μL per well, react at 25°C for 45 min; the positive standard is chiVMV virus, the negative control is the standard diluent, the positive sample is the virus-infected sample, and the negative sample is the healthy sample;

[0131] c. Wash the plate 3-5 times, pat dry, add enzyme-labeled antibody, 100 μL per well, react at 25°C for 45 min;

[0132] d. Wash the plate 3-5 times, pat dry, add color developer, and react at 25℃ in the dark for 15 minutes;

[0133] e. Add 100 μL of stop solution and read the OD value at OD450. The results are shown in Table 7:

[0134] Table 7

[0135] Serial number 1 2 3 4 5 6 Antibody ID FL596-1 FL596-2 FL596-3 FL596-4 FL596-5 FL596-6 Standard product 1000ppb 2.6735 3.2927 1.6686 2.6443 3.2718 3.2286 Standard product 100ppb 0.9974 0.9488 0.8491 1.2317 2.6347 2.7592 Standard product 10ppb 0.7464 0.453 0.6777 0.9727 1.233 1.4217 Standard product 0 0.7055 0.4031 0.6636 0.9314 0.1937 0.3849 Sample 100X 0.9288 0.9163 1.5636 2.4872 1.9031 2.0661 Sample 1000X 0.8027 0.6794 0.8871 0.9223 0.4224 0.5954 Sample 10000X 0.7951 0.6512 0.8038 0.5408 0.2256 0.4349 Negative control: Pothos scindapsus 0.8181 0.6767 0.8136 0.5036 0.1968 0.4206

[0136] It can be seen that the pairing effect of FL596-05, FL596-02 and polyclonal antibodies is the best.

[0137] The present invention preserves the cell line producing FL596-05 antibody, the preservation number is CCTCCNO.C2024334, the cell line preservation name is hybridoma cell line ChiVMV-5, the preservation date is October 22, 2024, the preservation unit is China Center for Type Culture Collection, the preservation institution is abbreviated as CCTCC, and the address is Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, China (opposite the First Affiliated Primary School of Wuhan University).

[0138] The present invention preserves the cell line producing FL596-02 antibody, the preservation number is CCTCCNO.C2024333, the cell line preservation name is hybridoma cell line ChiVMV-2, the preservation date is October 22, 2024, the preservation unit is China Center for Type Culture Collection, the preservation institution is abbreviated as CCTCC, and the address is Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, China (opposite the First Affiliated Primary School of Wuhan University).

[0139] Example 3 Assembly of the kit of the present invention:

[0140] 1. Preparation of buffer

[0141] (1) Coating buffer, 0.1 M carbonate buffer CB, pH 9.6;

[0142] (2) Sample diluent, 0.01 mM phosphate buffer (PBS), pH 7.4;

[0143] (3) washing solution, PBS containing 0.2% Tween-20;

[0144] (4) blocking solution, CB containing 1% BSA;

[0145] (5) Stop solution: 2M H2SO4 solution.

[0146] 2. Preparation of ELISA Plate

[0147] (1) Coating

[0148] A certain amount of ChiVMV antibody (i.e., one of FL596-05 and FL596-06 obtained in Example 2) was transferred to the required volume of coating solution to make the concentration about 5 μg / mL to prepare the coating working solution. 100 μL of the coating working solution was added to each well of the ELISA plate and allowed to stand overnight at 4°C (be careful to prevent water evaporation).

[0149] (2) Closed

[0150] Add a certain amount of BSA, sodium salicylate, and sucrose to CB buffer (pH 9.6) to make the concentrations of BSA, sodium salicylate, and sucrose 1%, 0.05%, and 5%, respectively, to prepare a blocking working solution. Use a plate washer to dry and wash twice, and pat the plate dry on clean absorbent paper. Add 150 μL of the blocking working solution to each well of the microwell and bake at 37°C for 3 hours.

[0151] (3) Dry and seal

[0152] The blocking solution was discarded, the strips were patted dry on clean absorbent paper, placed in a freeze vacuum dryer to dry for 3 hours, and vacuum heat-sealed.

[0153] 3. Preparation of ChiVMV standard lyophilized powder

[0154] The concentration of ChiVMV mother solution was 1 mg / mL, which was diluted to 225 ng / mL. 200 μL of 225 ng / mL standard was added to a 3 mL brown glass bottle and dried overnight in a freeze dryer to obtain freeze-dried powder of the standard.

[0155] 4. Preparation of enzyme-labeled antibody working solution

[0156] Preparation of antibody working solution: The purified ChiVMV polyclonal antibody (ie, the polyclonal antibody obtained in Example 1) was diluted to 1 μg / mL, and the concentration of horseradish peroxidase (HRP) labeled goat anti-rabbit was 0.2 μg / mL.

[0157] 5. Assembly of the kit, see Table 8:

[0158] Table 8

[0159] name quantity Pre-coated ELISA plate 1 block Standard lyophilized powder 2 bottles Antibody working solution 1 bottle ELISA working solution 1 bottle Sample diluent 2 bottles Color developer 1 bottle Terminator 1 bottle Washing liquid 1 bottle manual 1 serving

[0160] Example 4 Detection of pepper vein mottle virus (ChiVMV) in plants using the kit of the present invention

[0161] (1) Prepare standards and samples of unknown concentration

[0162] 1) Preparation of standard products:

[0163] Dissolve the pepper vein mottle virus (ChiVMV) standard in 1 mL of sample extract solution. The concentration of this solution is 32 ppb.

[0164] Take 32 ppb of pepper vein mottle virus (ChiVMV) standard, 300 μL and add 300 μL of sample extract, the concentration of this solution is 16 ppb;

[0165] Take 16 ppb pepper vein mottle virus (ChiVMV) standard, 300 μL and add 300 μL of sample extract, the concentration of this solution is 8 ppb;

[0166] Take 8 ppb of pepper vein mottle virus (ChiVMV) standard, 300 μL and add 300 μL of sample extract, the concentration of this solution is 4 ppb;

[0167] Take 4 ppb of pepper vein mottle virus (ChiVMV) standard, 300 μL and add 300 μL of sample extract, the concentration of this solution is 2 ppb;

[0168] After dilution, keep the standard solution away from light and store at -20℃ for 14 days.

[0169] 2) Processing of samples with unknown concentration:

[0170] Leaf pre-treatment method: Take 5-10mm 2 The leaf samples were placed in a 1.5 mL centrifuge tube and crushed. 250 μL of diluent was added and the mixture was shaken for 5 minutes. The samples were centrifuged at 4000 rpm for 3 minutes and 100 μL of the supernatant was taken for analysis.

[0171] (2) Add standard or unknown concentration sample:

[0172] Add 100 μL of sample diluent (blank control) / standard sample / sample of unknown concentration into the corresponding microwells, shake gently to mix, and react at 25°C in a dark environment for 45 minutes.

[0173] (3) Washing plate:

[0174] The liquid in the wells was shaken dry, and the wells were thoroughly washed three times with 250 μL of washing solution / well, each time with an interval of 1 min, and then patted dry with absorbent paper.

[0175] (4) Add enzyme-labeled antibody:

[0176] Add 100 μL / well of enzyme-labeled antibody, gently shake to mix, react at 25°C in a dark environment for 45 min, take out and repeat step 3 of plate washing.

[0177] (5) Color rendering:

[0178] Add 100 μL / well of color developer and place in a dark environment at 25°C for 15 min.

[0179] (6) Termination and determination:

[0180] Add 100 μL stop solution / well, shake gently to mix, set the microplate reader to 450nm, and measure the OD value of each well (preferably using dual wavelength 450 / 630nm for detection, and reading the data within 5 minutes). If there is no microplate reader, the determination can be made by visual inspection without adding stop solution.

[0181] (7) Calculation:

[0182] Using statistical drawing software, a four-parameter fitting standard curve was drawn according to the OD value of the measured standard, such as Figure 1 As shown; the concentration of ChiVMV in the unknown sample can be calculated by the standard curve and the OD value of the unknown concentration sample.

[0183] The test results are shown in Table 9:

[0184] Table 9

[0185] ppb OD1 32 2.5382 16 1.377 8 0.8436 4 0.4818 2 0.312 Sample 100X 1.9031 0 0.1446

[0186] Example 5 Preparation of ChiVMV rapid test paper and evaluation of sample detection effect

[0187] 1. Preparation of ChiVMV rapid test paper

[0188] S200, assembly of a large plate of rapid test paper: a NC membrane labeled with a specific monoclonal antibody (one of the monoclonal antibodies obtained in Example 2) and a goat anti-mouse secondary antibody is fixed in the middle of the large plate, and a gold label pad labeled with a specific monoclonal antibody (another monoclonal antibody obtained in Example 2) is covered under the NC membrane, the width of the label pad is about 10 mm, and its front edge is 1-2 mm above the bottom of the NC membrane; the sample pad is about 20-30 mm wide, covering the gold label pad, and its front edge is 1-2 mm below the gold label pad; the absorption pad is about 20-30 mm wide, located at the top of the test strip, and its lower part covers 1-2 mm above the upper part of the NC membrane.

[0189] S210, cutting of rapid test paper: Use a strip cutter to cut the large board into finished rapid test paper with a width of about 3-4 mm.

[0190] When using, insert the rapid test paper vertically into the sample solution. The sample solution will penetrate along the sample pad to the label pad of the colloidal gold labeled antibody and form a double antibody sandwich structure with the specific antibody on the NC membrane. After 5-8 minutes, observe the color change in the result observation hole and make a judgment. Result judgment: negative result (1 C line appears); positive (+): C / T lines are colored at the same time.

[0191] The detailed method is as follows:

[0192] 1) Coating

[0193] Select the nitrocellulose membrane (NC membrane) of PALL170 and use a film spray gold machine to draw a T line at 1.0 μL / cm with a ChiVMV coated antibody solution (i.e., one of the monoclonal antibodies prepared in Example 2) with a concentration of 2.0 mg / mL as the detection line; draw a C line at 0.7 μL / cm with a goat anti-mouse secondary antibody (goat anti-mouse IgG) with a concentration of 1 mg / mL as the quality control line; dry at 37°C for 24 hours for standby use; and obtain a nitrocellulose membrane coated with an antibody.

[0194] 2) Preparation of colloidal gold

[0195] a) Preparation: Wash the 500mL beaker, 20mL small beaker, rotor, brown bottle, glass rod, etc. and place them in an acid tank (potassium dichromate: concentrated sulfuric acid: ultrapure water = 120g: 200mL: 1000mL) and soak for 24 hours. Take them out and rinse them with tap water for 3-4 times, then rinse them with ultrapure water for 3-4 times, and place them in a 37℃ oven to dry for later use.

[0196] b) Preparation of Au solution A. Use a plastic weighing spoon to weigh 1 g of chloroauric acid powder (purchased from Sigma) into a brown bottle, add 99 mL of ultrapure water to fully dissolve, and store at 4°C away from light.

[0197] c) Preparation of calcined gold solution B. Weigh 1 g of trisodium citrate (purchased from Sigma) and dissolve it in 99 mL of ultrapure water and mix well.

[0198] d) Preparation of colloidal gold: 99 mL of ultrapure water was measured in a beaker, 1 mL of burnt gold solution A was added, and the mixture was stirred on a constant temperature magnetic stirrer. The solution was heated until the solution boiled, and 2 mL of the newly prepared burnt gold solution B was quickly added. The solution gradually turned blue-black, then purple-black, and then red after heating. The solution turned transparent orange-red after boiling for 7-10 minutes, and then cooled to room temperature naturally. The ultrapure water was added to make the volume 100 mL. The solution was poured into a brown bottle and stored at 4°C away from light to obtain colloidal gold (particle size 40 nm).

[0199] 3) Antibody labeling

[0200] a) Antibody labeling: Take 1.5 mL of the colloidal gold prepared in 1) above, adjust the pH value with 0.1 M K2CO3, add 10 μg of ChiVMV labeled antibody (i.e., another monoclonal antibody prepared in Example 2), mix well, react at room temperature for 40 minutes, add 10% BSA to terminate, and let stand for 30 minutes.

[0201] b) Purification of labeled antibody: First centrifuge at low speed (1500r / min) to discard the precipitate formed by the condensed gold colloid particles. Then centrifuge at high speed (8500r / min) for 30 minutes. Carefully remove the supernatant, and re-dissolve the precipitate with 50μL of 0.1M PBS (PH7.4) containing 1% BSA and store at 4°C to obtain the labeled colloidal gold antibody solution.

[0202] 4) Gold spraying

[0203] The labeled colloidal gold antibody solution prepared in 3) above was diluted to 0.1 mg / mL, sprayed on the pretreated gold label pad at 1 uL / cm, and dried for standby use to obtain a label pad fixed with colloidal gold-labeled specific monoclonal antibody.

[0204] 5) Large panel assembly

[0205] The components were assembled and fixed on a base plate (PVC base plate with adhesive), and then placed in a vacuum dryer at 37°C for 30 min.

[0206] 6) Large board cutting

[0207] Cut the assembled large board into 3mm wide strips, which are ChiVMV rapid test paper. The specific structure is as follows: Figure 2-4 shown.

[0208] 2. Evaluation of the effect of ChiVMV rapid test card (rapid test paper) on leaf samples

[0209] Leaf sample processing method: Place the leaf tissue between the lid and the tube body of a disposable tissue extraction tube, quickly cover the lid, repeat twice to obtain two circular leaf tissues; place the leaf at the bottom of the extraction tube with a pestle; insert the pestle into the tube, rotate the pestle or the gun head to crush the leaf, and continue pressing for 20 to 30 seconds. Add 0.5mL (about 20 drops) of extraction buffer (10mm PBS buffer); repeat the crushing steps to fully contact and mix the sample and buffer; let it settle or centrifuge to obtain the supernatant, which is the leaf sample extract. The test results are shown in Figure 5 , the rapid test strip detection buffer is negative, and the positive sample 10X, 50X, and 100X extraction can all detect positive and show a gradient change.

[0210] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody comprises a first monoclonal antibody and / or a second monoclonal antibody; wherein the first monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO.C2024333, and the second monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO.C2024334.

2. Use of the monoclonal antibody according to claim 1 in preparing a product for detecting pepper vein mottle virus, characterized in that: The product is a test kit or a test paper.

3. A test kit for detecting pepper vein mottle virus, characterized in that: The kit comprises: an ELISA plate pre-coated with the first monoclonal antibody or the second monoclonal antibody as claimed in claim 1, a sample diluent, a washing solution, a reaction stop solution, an enzyme-labeled antibody, and a color developer.

4. A test kit for detecting pepper vein mottle virus according to claim 3, characterized in that: The kit also includes a pepper vein mottle virus standard.

5. A test kit for detecting pepper vein mottle virus according to claim 3, characterized in that: The sample diluent is phosphate buffer.

6. A test kit for detecting pepper vein mottle virus according to claim 3, characterized in that: The washing solution is a phosphate buffer containing Tween-20.

7. A test kit for detecting pepper vein mottle virus according to claim 4, characterized in that: The reaction termination solution is a sulfuric acid solution.

8. A test kit for detecting pepper vein mottle virus according to claim 4, characterized in that: The color developer is TMB color developer.

9. A test kit for detecting pepper vein mottle virus according to claim 4, characterized in that: The enzyme-labeled antibody is an enzyme-labeled polyclonal antibody.

10. A pepper vein mottle virus rapid test paper, characterized in that: The method comprises the first monoclonal antibody and the second monoclonal antibody according to claim 1.

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