Hybridoma cell pair and monoclonal antibody secreted by the same for detecting SMV Banxia strain

Abstract

The application discloses a hybridoma cell pair and application of a monoclonal antibody secreted by the hybridoma cell pair in detection of SMV Pinellia ternate strains, and belongs to the technical field of virus detection. The hybridoma cell pair disclosed by the application comprises two hybridoma cell strains, a first strain is named hybridoma cell strain SMVHBBX-1, the preservation number of which is CCTCC NO: C2026121, the preservation time is May 27, 2026, and the preservation unit is China Center for Type Culture Collection; and a second strain is named hybridoma cell strain SMVHBBX-2, the preservation number of which is CCTCC NO: C2026120, the preservation time is May 27, 2026, and the preservation unit is China Center for Type Culture Collection. The monoclonal antibody produced by the hybridoma cell pair has high specificity, high sensitivity and high titer, and is helpful to realize effective supervision on related plants and rapid screening and detection on diseased offspring.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to the application of a hybridoma cell pair and its secreted monoclonal antibody in the detection of SMV Banxia strain (soybean mosaic virus isolate BX, SMV-BX). Background Technology

[0002] Pinellia ternata ( Pinellia ternata Pinellia ternata (also known as Chinese pine) is a medicinal plant belonging to the Araceae family and a commonly used traditional Chinese medicine. Due to the medicinal properties of its tubers, such as drying dampness and resolving phlegm, relieving nausea and vomiting, and dissipating lumps and nodules, it is widely used in various traditional Chinese medicine formulas, resulting in a large market demand and significant economic value. However, under natural conditions, Pinellia ternata seeds have a low germination rate and slow seedling growth, while tuber propagation, due to its ease of operation and high survival rate, has become the most common method for Pinellia ternata production. However, the long-term reliance on tuber asexual reproduction in production has also revealed its drawbacks. Although tuber propagation is simple and has a high survival rate, the tubers are prone to carrying viruses. Infected Pinellia ternata plants can continuously transmit the virus to offspring through asexual reproduction, leading to the continuous accumulation and spread of the virus in the germplasm, resulting in germplasm degradation. Combined with field vector transmission, this further exacerbates the spread of viral diseases, ultimately causing serious negative impacts on the yield and quality of Pinellia ternata. With the expansion of Pinellia ternata cultivation, the occurrence and harm of Pinellia ternata viral diseases have become increasingly serious, becoming a key issue restricting the sustainable development of the Pinellia ternata industry.

[0003] There are several pathogens causing diseases in Pinellia ternata, including Soybean mosaic virus (SMV), Cucumber mosaic virus (CMV), Dasheen mosaic virus (DsMV), Konjac mosaic virus (KoMV), and Tobacco mosaic virus (TMV). Among them, SMV is the most prevalent, and SMV viruses infecting Pinellia ternata can evolve into separate strains, namely the Soybean mosaic virus isolate BX (SMV-BX). The amino acid homology among SMV Pinellia ternata strains is between 90% and 95%, with the CP protein homology between 96% and 98%. However, the protein homology between the SMV Pinellia ternata strain and the SMV soybean strain [there are many SMV strains infecting soybean, but here we uniformly refer to them as the SMV soybean strain (SMV-DD)] is only about 80% to 85%, with the CP protein homology being less than 82%. Under natural conditions, SMV (Symptomyces albuminosus) strains can infect both soybeans and Pinellia ternata, while SMV soybean strains can infect soybeans but are less likely to infect Pinellia ternata. When Pinellia ternata is infected with SMV, the plants exhibit symptoms such as mottling, mosaic, wrinkling, chlorosis, and stunting. Tubers develop poorly and are significantly smaller in size, sometimes even leading to plant death, resulting in a sharp decline in yield and varietal degeneration. Simultaneously, the content of effective components such as alkaloids decreases significantly, directly affecting the quality of the medicinal material.

[0004] Due to the lack of effective pesticides for control, early detection and diagnosis have become the core strategies for controlling the spread of the virus and screening for detoxified Pinellia ternata. Because the homology (<82%) of the coat protein between soybean strains and Pinellia ternata strains of Soybean Mosaic Virus (SMV) is low, the developed monoclonal antibodies against SMV soybean strains and their detection products cannot be used for the detection of SMV Pinellia ternata strains. Therefore, screening suitable cell lines and using them to prepare antibodies with high sensitivity and specificity against SMV Pinellia ternata strains, and processing them into rapid detection products such as immunogold test strips, virus cards, and enzyme-linked immunosorbent assay (ELISA) kits, has become an urgent problem to be solved in the sustainable development of the Pinellia ternata industry. Summary of the Invention

[0005] The purpose of this invention is to provide a hybridoma cell pair and use it to prepare antibodies with high sensitivity and specificity against SMV Pinellia ternata strains, so as to solve the technical bottleneck that existing SMV soybean strain antibodies and their detection products cannot be used for the detection of SMV Pinellia ternata strains, improve the detection and identification efficiency of soybean mosaic virus Pinellia ternata strains, and realize effective supervision of related plants and rapid screening and detection of infected offspring.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a hybridoma cell pair comprising two hybridoma cell lines. The first line is named hybridoma cell line SMVHBBX-1, with accession number CCTCC NO: C2026121, deposited on May 27, 2026, and deposited at the China Center for Type Culture Collection. The second line is named hybridoma cell line SMVHBBX-2, with accession number CCTCC NO: C2026120, deposited on May 27, 2026, and deposited at the China Center for Type Culture Collection.

[0007] Secondly, the present invention protects the use of the hybridoma cell pair in the preparation of monoclonal antibodies for detecting SMV Pinellia ternata strain.

[0008] Thirdly, the present invention protects a pair of monoclonal antibodies, the pair comprising two monoclonal antibodies, which are secreted by the first hybridoma cell line and the second hybridoma cell line described in the first aspect, respectively.

[0009] Furthermore, the preparation process of any one of the monoclonal antibodies in the monoclonal antibody pair is as follows: inject the first hybridoma cell line or the second hybridoma cell line into the peritoneal cavity of a mouse, collect the ascites fluid of the mouse 9-12 days later, and the supernatant obtained by centrifugation is the monoclonal antibody ascites fluid. The monoclonal antibody can be obtained by purifying the monoclonal antibody ascites fluid.

[0010] Fourthly, the present invention provides a detection kit for detecting SMV Pinellia ternata strains, the detection kit comprising the following components: (1) Pre-coated enzyme-labeled plate, prepared using any one of the monoclonal antibody pairs described in the third aspect; (2) The sample diluent is a phosphate buffer solution with a pH of 7.0 to 8.0; (3) Washing solution, the composition of which is phosphate buffer containing Tween-20; (4) The reaction termination solution is an H2SO4 solution; (5) Enzyme-labeled antibody; (6) The colorimetric reagent is TMB colorimetric reagent; (7) Standard of SMV Pinellia ternata strain.

[0011] More preferably, the preparation process of the pre-coated ELISA plate of component (1) includes the following steps: S1: Using 0.1 M Na2CO3-NaHCO3 buffer with a pH of 9.6, dilute the monoclonal antibody to 5 μg / mL and add 100 μL to each well of the microplate. Incubate at 37°C for 3 h. S2: Shake off the microplate solution in the microplate wells, add washing solution, soak for 5 minutes, then shake off the microplate solution in the microplate wells again, and then pat dry on absorbent paper. S3: Add the blocking solution to the microwells of the microplate, 150 μL per well, and react at 37°C for 3 h. The blocking solution is a carbonate buffer containing BSA. S4: Shake off the solution in the microplate wells, pat dry on absorbent paper, and then freeze dry. S5: Use a vacuum packaging machine to package the enzyme-labeled plate into an aluminum foil bag.

[0012] More preferably, the concentration of the H2SO4 solution in component (4) is 1.8 to 2.2 M.

[0013] More preferably, the SMV Pinellia ternata strain standard in component (7) is a freeze-dried powder of SMV Pinellia ternata strain or its CP protein.

[0014] Fifthly, the present invention provides a rapid test strip for detecting SMV Pinellia ternata strain. The rapid test strip includes a base plate, and a sample pad, a gold label pad, and an NC membrane that are partially overlapped and fixed on the base plate from top to bottom along the extension direction of the base plate. An absorbent filter paper is overlapped at the end of the NC membrane. The gold label pad is coated with one of the monoclonal antibodies in the monoclonal antibody pair described in the third aspect. The NC membrane is provided with a T line and a C line, wherein the T line is coated with the other monoclonal antibody in the monoclonal antibody pair described in the third aspect, and the C line is coated with goat anti-mouse IgG secondary antibody.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The two hybridoma cell lines provided by this invention were obtained by fusing spleen cells from mice infected with the CP protein of the SMV Pinellia ternata strain with tumor cells and then screening them. The monoclonal antibodies secreted by these cells have the advantages of good pairing effect with polyclonal antibodies, high specificity, and high titer in detecting the SMV Pinellia ternata strain. They have great application potential in double-antibody sandwich ELISA detection methods and colloidal gold test strips. They are expected to solve the technical bottleneck that existing SMV soybean strain antibodies and their detection products cannot be used for the detection of the SMV Pinellia ternata strain, and improve the detection and identification efficiency of soybean mosaic virus Pinellia ternata strain, so as to realize effective supervision of related plants and rapid screening and detection of infected offspring. Attached Figure Description

[0016] Figure 1This is an SDS-PAGE protein identification result of the CP protein small-scale induction expression reaction solution in Example 1. In the figure, lane M is the protein molecular weight standard, lane 1 is the blank control strain, lane 2 is the recombinant expression strain before IPTG induction, lane 3 is the blank control strain induced by IPTG, and lane 4 is the recombinant expression strain after IPTG induction. Figure 2 The image shows the SDS-PAGE protein identification results after lysis of the CP protein small-scale induction expression reaction solution in Example 1. In the image, lane M is the protein molecular weight standard, lane 1 is the precipitate of bacterial cell lysis after induction of the recombinant expression strain, and lane 2 is the supernatant of bacterial cell lysis after induction of the recombinant expression strain. Figure 3 The image shows the SDS-PAGE protein identification results of the eluent obtained from the large-scale expression and purification of CP protein in Example 1. In the image, lane M is the protein molecular weight standard, lane 1 is the unchromatographic sample, lane 2 is the unchromatographic sample after column chromatography, lane 3 is the 20 mM imidazole eluent, lane 4 is the 50 mM imidazole eluent, lane 5 is the 100 mM imidazole eluent, lane 6 is the 200 mM imidazole eluent, and lane 7 is the 500 mM imidazole eluent. Figure 4 The image shows the SDS-PAGE protein identification results after dialysis of the eluent obtained from the high-level expression and purification of CP protein in Example 1. In the image, lane M is the protein molecular weight standard, and the other lane is the purified sample. Figure 5 The image shows the Western Blot results of monoclonal antibodies prepared using FL624-42 against different viruses. Figure 6 The image shows the Western Blot results of monoclonal antibodies prepared using FL624-43 against different viruses. Figure 7 The image shows the Western Blot results of monoclonal antibodies prepared using FL624-44 against different viruses. Figure 8 The standard curve obtained by using the kit prepared in Example 2 to detect the soybean mosaic virus Pinellia ternata strain (SMV-BX); Figure 9 This is a schematic diagram of the rapid test strip provided in Example 3, where A is the front view and B is the top view. The labels in the figure are as follows: 1 is the sample pad; 2 is the gold label pad; 3 is the NC membrane (result observation area); 4 is the absorbent filter paper; 5 is the C line; 6 is the T line; and 7 is the base plate.

[0017] Figure 10 This is a picture of the actual test result of the rapid test strip provided in Example 3. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in this technical field, and specific reagents and equipment are shown in Tables 1-2.

[0019] Table 1 Summary of reagents involved in the embodiments of the present invention

[0020] Table 2 Summary of instruments involved in the embodiments of the present invention

[0021] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0022] Example 1 The main purpose of this embodiment is to screen hybridoma cell lines, and the process is as follows: 1. Expression and purification of SMV-BX viral CP protein (1) Sequence synthesis: Based on the SMV-BX CP protein gene sequence, the codons were optimized, the SMV-BX CP sequence was amplified and cloned into the vector pET30a to obtain the pET30a-SMV-BX-CP positive plasmid. The SMV-BX CP sequence was sequenced and identified by General Biotechnology (Chuzhou) Co., Ltd., and its sequence is shown in SEQ ID NO:1.

[0023] SEQ ID NO: .

[0024] (2) Activation of bacterial strain: BL21 (DE3) was transformed with the synthesized pET30a-SMV-BX-CP positive plasmid and spread on LB solid medium (kanamycin concentration 50 μg / mL). The next day, single colonies were picked and inoculated into 5 mL of LB liquid medium (kanamycin concentration 50 μg / mL) and cultured at 37℃ for 12 h-14 h.

[0025] (3) Small-scale expression: The next day, the bacterial strain was inoculated into 5 mL of LB liquid medium (kanamycin concentration 50 μg / mL) at a dilution of 1:50 and cultured at 37℃ until OD=0.4-0.6. 1 mL of the bacterial solution was centrifuged and used as a pre-induction control. 4 mL of the bacterial solution was added with 0.8 mM IPTG, and expression was induced at 25℃ for 6 h. The bacterial solution was then centrifuged at 8000 rpm and 4℃ for 1 min, and the bacterial cells were collected. SDS-PAGE was used to identify the protein form, and the results showed ( Figure 1 There was obvious expression of the target protein.

[0026] (4) Identification of protein expression form: The expressed bacterial cells were lysed by sonication in 1 mL of lysis buffer. Lysis conditions: ice bath, 40% power, sonication for 2 s, interval of 2 s, time 30 min. Centrifuge at 12000 rpm, 4℃ for 1 min, and collect the supernatant and precipitate. SDS-PAGE was used to identify the protein expression form, and the results showed that ( Figure 2 The target protein is mainly expressed in a soluble form.

[0027] (5) Large-scale expression and purification of protein: Single colonies of pET30a-SMV-BX-CP were picked from solid plates and inoculated into 5 mL of LB liquid medium (kanamycin concentration 50 μg / mL). The culture was incubated at 37℃ for 12-14 h. The next day, the bacterial strain was inoculated into 800 mL of LB liquid medium (kanamycin concentration 50 μg / mL) at a 1:50 ratio and incubated at 37℃ until OD=0.4-0.6. 0.8 mM IPTG was added, and expression was induced at 25℃ for 6 h. The bacterial culture was centrifuged at 8000 rpm and 4℃ for 15 min, and the bacterial cells were collected. 100 mL of lysis buffer was added for ultrasonic lysis. Lysis conditions: ice bath, 60% power, 2 s sonication, 2 s interval, 15 min time. Centrifugation was performed at 12000 rpm and 4℃ for 15 min, and the supernatant and precipitate were collected. The collected supernatant was purified using high-affinity NI resin, and the flow-through and eluent were collected. SDS-PAGE was used to detect the purification effect. The results showed that ( Figure 3 Protein purity was optimal when eluted with 200 mM imidazole. The 200 mM imidazole eluent was dialyzed to remove imidazole, and the dialysis efficiency was analyzed by SDS-PAGE. The results showed... Figure 4 The protein purity and concentration were acceptable after dialysis. Testing showed that the final target protein purity was greater than 90%, the concentration was 2 mg / mL, and the protein amount was 10 mg.

[0028] 2. Hybridoma cell lines were obtained by immunizing mice with SMV-BX virus CP protein. (1) Immunogen preparation: The CP protein of soybean mosaic virus Pinellia strain (SMV-BX) was mixed with an equal volume of Freund's adjuvant and emulsified evenly to form a water-in-oil state for immunizing mice.

[0029] (2) Immunization strategy: Four Balb / c mice were immunized with the CP protein of soybean mosaic virus Pinellia strain (SMV-BX) three times subcutaneously, with an interval of four weeks between each immunization. Finally, the mice's serum was collected and the antiserum titer was detected by indirect ELISA.

[0030] The steps of the indirect ELISA method are as follows: 1) Dilute the purified CP protein to 1 μg / mL with 0.1 M, pH 9.6 carbonate buffer, add 100 μL to each well of a 96-well microplate, and incubate at 37°C for 3 h or at 4°C overnight.

[0031] 2) Discard the liquid in the wells, add 250 μL of washing buffer, let stand for 30 seconds, discard the liquid in the plate, and repeat 3 times.

[0032] 3) Add the test sample, 100 μL per well, along with the positive control (positive mouse serum obtained in step (2), diluted in a gradient of 1K, 3K, 9K... before being added to the experiment), the negative control (pre-immunization mouse serum), and the blank control (no mouse serum added). Incubate at 37℃ for 45 min. 4) Repeat step 2). 5) Add 100 μL of HRP-labeled goat anti-mouse enzyme-labeled secondary antibody to each well and react at 37℃ for 45 min.

[0033] 6) Repeat step 2). 7) Add 100 μL of colorimetric reagent to each well and react at room temperature in the dark for 15 min.

[0034] 8) Add stop solution, 100 μL per well, and read the OD value at a wavelength of 450 nm using an ELISA reader. The resulting antiserum titers are shown in Table 3 (in this example, the highest dilution factor corresponding to a positive OD value / negative OD value > 2.5 is taken as the antiserum titer).

[0035] Table 3. Results of partial mouse antiserum titer determination

[0036] (3) Cell fusion: Two weeks after the last immunization, a booster immunization was performed by intraperitoneal injection of antigen, and cell fusion was performed three days later. Mice were euthanized by cervical dislocation, disinfected by soaking in 70% ethanol for 30 min, the abdominal cavity was cut open in a laminar flow hood, the spleen was removed, ground, and passed through an 80-mesh sieve to obtain spleen cells, which were then added to SP2 / 0 myeloma cells and fused under the action of PEG4000.

[0037] (4) Fusion screening: The fused cells were seeded into 96-well plates and cultured in HAT medium. After three days, the medium was changed to HT medium. After 10 days, the cell culture supernatant was collected for testing.

[0038] (5) Cloning and cell line establishment: The positive wells were cloned using the limiting dilution method. After 10 days, the clones were tested and the limiting dilution method was continued to clone the positive clones until all clones were positive, thus establishing positive cell lines. Finally, 44 positive cell lines were obtained and named FL624-1 to FL624-44.

[0039] (6) Expanded culture: The monoclonal cells of the established strain were expanded and cryopreserved for subsequent experiments.

[0040] 3. Monoclonal antibodies were prepared using the 44 screened positive cell lines (FL624-1 to FL624-44). (1) Ascites preparation: Mineral oil was injected into the peritoneal cavity of mice one week in advance. 44 positive cell lines (FL624-1~FL624-44) were injected into the peritoneal cavity of different mice. Ascites was collected after about 10 days. The supernatant was obtained by centrifugation at 4000 rpm.

[0041] (2) Monoclonal antibody purification: Centrifuge the ascites fluid for 15 min (4000 rpm, room temperature), collect the supernatant, slowly add saturated ammonium sulfate dropwise to half saturation while stirring at 4℃, continue stirring for 30 min, centrifuge for 30 min (13000 rpm, 4℃), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4); slowly add saturated ammonium sulfate dropwise to 33% while stirring at 4℃, continue stirring for 30 min, centrifuge for 30 min (13000 rpm, 4℃), discard the supernatant; dissolve the precipitate in an appropriate amount of PBS (0.01 M, pH 7.4), dialyze overnight at 4℃, determine the antibody content, and freeze at -20℃ for later use. After ammonium sulfate precipitation, purification was continued using Protein G columns. The new column was first passed through 5 mL of ultrapure water, then equilibrated with 5 mL of 0.4 M PB buffer (pH 7.0). Antibody was then passed through the column slowly to ensure better binding of the antibody protein to the binding site. The column was then equilibrated again with 10 mL of 0.4 MPB buffer (pH 7.0). The antibody at the binding site was eluted with 5 mL of 0.1 M glycine-hydrochloric acid buffer (pH 2.7), and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine, maintaining a neutral pH suitable for antibody preservation.

[0042] 4. Preparation of polyclonal antibodies by immunizing rabbits with SMV-BX virus CP protein. The prepared polyclonal antibody was used for subsequent screening and detection kit preparation. Its preparation process is as follows: (1) Immunogen preparation: The CP protein of soybean mosaic virus Pinellia strain (SMV-BX) was mixed with an equal volume of Freund's adjuvant and emulsified evenly to form a water-in-oil state for immunizing rabbits.

[0043] (2) Immunization strategy: Two New Zealand white rabbits were immunized with the CP protein of soybean mosaic virus strain Pinellia ternata (SMV-BX) three times subcutaneously, with an interval of 4 weeks between each immunization. Finally, rabbit serum was collected and its antiserum titer was detected by indirect ELISA.

[0044] The steps of the indirect ELISA method are as follows: 1) Dilute the purified CP protein to 1 μg / mL with 0.1 M, pH 9.6 carbonate buffer, add 100 μL to each well of a 96-well microplate, and incubate at 37°C for 3 h or at 4°C overnight.

[0045] 2) Discard the liquid in the wells, add 250 μL of washing buffer, let stand for 30 seconds, discard the liquid in the plate, and repeat 3 times.

[0046] 3) Add the test sample, 100 μL per well, along with the positive control (positive rabbit serum taken in step (2), diluted in a gradient of 1K, 3K, 9K... before being added to the experiment), the negative control (rabbit serum before immunization), and the blank control (no rabbit serum added). Incubate at 37℃ for 45 min. 4) Repeat step 2). 5) Add 100 μL of HRP-labeled goat anti-rabbit enzyme-labeled secondary antibody to each well and react at 37℃ for 45 min.

[0047] 6) Repeat step 2). 7) Add 100 μL of colorimetric reagent to each well and react at room temperature in the dark for 15 min.

[0048] 8) Add stop solution, 100 μL per well, and read the OD value at a wavelength of 450 nm using an ELISA reader. The antiserum titer is shown in Table 4 (in this example, the highest dilution factor corresponding to a positive OD value / negative OD value > 2.5 is taken as the antiserum titer).

[0049] Table 4 Results of rabbit antiserum titer determination

[0050] (3) Polyclonal antibody purification: Rabbit serum was centrifuged for 15 min (4000 rpm, room temperature), and the supernatant was collected. Saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4℃. Stirring was continued for 30 min, and centrifuged for 30 min (13000 rpm, 4℃). The supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4). Saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4℃. Stirring was continued for 30 min, and centrifuged for 30 min (13000 rpm, 4℃). The supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4), dialyzed overnight at 4℃, and the antibody content was determined. The antibody was stored at -20℃ for later use. After ammonium sulfate precipitation, purification was continued using Protein A columns. The new column was first passed through 5 mL of ultrapure water, then equilibrated with 5 mL of 0.4 M PB buffer (pH 7.0). Antibody was then passed through the column slowly to ensure better binding of the antibody protein to the binding site. The column was then equilibrated again with 10 mL of 0.4 MPB buffer (pH 7.0). The antibody at the binding site was eluted with 5 mL of 0.1 M glycine-hydrochloric acid buffer (pH 3.0), and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine, maintaining a neutral pH suitable for antibody preservation.

[0051] 5. Based on the performance results of 44 monoclonal antibodies, positive cell lines were further screened. The titers of antibodies obtained from purified positive cell lines were detected using an indirect ELISA method. The steps of the indirect ELISA method are as follows: 1) Dilute the purified CP protein to 1 μg / mL with 0.1 M, pH 9.6 carbonate buffer, add 100 μL to each well of a 96-well microplate, and incubate at 37°C for 3 h or at 4°C overnight.

[0052] 2) Discard the liquid in the wells, add 250 μL of washing buffer, let stand for 30 seconds, discard the liquid in the plate, and repeat 3 times.

[0053] 3) Add the test sample, 100 μL per well, along with positive controls (antibodies obtained from 44 different positive cell lines, each diluted in a serial gradient of 1K, 3K, 9K… before being added to the experiment), negative controls (pre-immunization mouse serum), and blank controls (without mouse serum). Incubate at 37℃ for 45 min. 4) Repeat step 2). 5) Add 100 μL of HRP-labeled goat anti-mouse enzyme-labeled secondary antibody to each well and react at 37℃ for 45 min.

[0054] 6) Repeat step 2). 7) Add 100 μL of colorimetric reagent to each well and react at room temperature in the dark for 15 min.

[0055] 8) Add stop solution, 100 μL per well, and read the OD value at a wavelength of 450 nm using an ELISA reader. The titers of each group of monoclonal antibodies are shown in Table 5 (in this example, the highest dilution factor corresponding to a positive OD value / negative OD value > 3 is taken as the titer).

[0056] Table 5 Summary of antibody titer test results obtained from different positive cell lines

[0057] 2) The antibody subtype was detected using a mouse antibody subtype detection kit, and the results are shown in Table 6.

[0058] Table 6 Subtype Detection Results

[0059] 3) The prepared antibodies with a titer ≥9 K were screened using a double-antibody sandwich ELISA method to identify antibody pairs that could be paired (FL624-18 and FL624-36 were excluded).

[0060] The double-antibody sandwich ELISA method is as follows: a. Dilute the coated antibody to 5 μg / mL with CB, 100 μL per well, and coat at 37℃ for 3 h; b. Wash the plate 3-5 times, pat dry, add 100 μL of positive standard / negative control / positive sample / negative sample to each well, and incubate at 25°C for 45 min. c. Wash the plate 3-5 times, pat dry, add enzyme-labeled antibody, 100 μL per well, and react at 25℃ for 45 min. The enzyme-labeled antibody includes 1 μg / mL of polyclonal antibody prepared by immunizing rabbits with SMV-BX virus CP protein and 0.2 μg / mL of horseradish peroxidase (HRP)-labeled goat anti-rabbit enzyme-labeled secondary antibody. d. Wash the plate 3-5 times, pat dry, add color developer, and react at 25℃ in the dark for 15 min; e. Add 100 μL of stop solution, OD 450 Read the OD values; the results are shown in Table 7.

[0061] Table 7 Summary of OD value detection results for different antibodies

[0062] As shown in Table 7, although most of the antibodies can be paired, it is easy to see that FL624-42, FL624-43, and FL624-44 are the three antibodies with the best pairing effect with polyclonal antibodies.

[0063] 4) Use Western blotting to perform further specificity analysis on the three antibodies selected in the previous step to determine whether they can be used for the development of virus detection products.

[0064] The method for Western blotting is as follows: a. Extraction of total plant protein: Plant tissues were quick-frozen in liquid nitrogen, ground into powder, and lysed with RIPA protein lysis buffer (containing PMSF and protease inhibitors) for 30 min on ice. Then, the tissues were centrifuged (12,000 rpm, 4℃, 15 min) and the supernatant was collected. The protein concentration was determined by the BCA method. The loading volume was standardized, and protein loading buffer was added. The tissues were denatured in a boiling water bath for 5-10 min and stored at -20℃ for later use.

[0065] b. SDS-PAGE electrophoresis: Prepare separating and stacking gels, insert combs to prepare the gels, and after solidification, load the gels into the electrophoresis tank, add electrophoresis buffer, and spot protein markers and samples. Then, maintain a constant voltage of 80 V on the stacking gel, and after the samples are transferred into the separating gel, adjust the voltage to 120 V. Stop electrophoresis by running bromophenol blue to the bottom of the gel.

[0066] c. Transfer: Cut the gel, NC / PVDF membrane (PVDF needs to be activated with methanol), and filter paper; clamp the membrane in the order of filter paper → gel → membrane → filter paper (without air bubbles), and place it in the transfer tank; transfer the membrane in a constant current of 200-300 mA and an ice bath for 60-90 min.

[0067] d. Sealing: Wash the membrane with TBST for 5 min x 3 times, then seal with 5% skim milk powder / BSA at room temperature for 1 h or overnight at 4°C.

[0068] e. Antibody incubation: Dilute the primary antibody with blocking buffer, incubate the NC membrane at room temperature for 2 h or at 4°C overnight, wash the membrane 3 times with TBST for 8 min each time. Incubate the NC membrane with HRP-labeled secondary antibody at room temperature for 1 h, and wash the membrane thoroughly with TBST.

[0069] f. Imaging: ECL luminescent solution is added to the membrane, and the strips are collected by exposure using a chemiluminescence analyzer. The gray values ​​are then analyzed.

[0070] Using five potato virus Y viruses (SMV-BX, SMV-DD, PVY, ChiVMV, TVBMV), one plant baculovirus (TMV), and one brome mosaic virus (CMV) as detection targets, the specificity of three monoclonal antibodies FL624-42, FL624-43, and FL624-44 was tested using the above method. The results are shown in [Figure number missing]. Figure 5 , Figure 6 , Figure 7FL624-42 and FL624-44 have good specificity, recognizing only its antigenic virus SMV-BX, while FL624-43 has weak specificity, recognizing not only its antigenic virus SMV-BX, but also SMV-DD, PVY and TVBMV of the same family. It is evident that the monoclonal antibodies secreted by FL624-42 and FL624-44 exhibit advantages such as good pairing with polyclonal antibodies, high specificity, and high titer, showing promising application prospects in SMV-BX detection. The hybridoma cell lines FL624-42 and FL624-44 demonstrate these advantages, and we have preserved them. The preservation information is as follows: FL624-42 is classified as hybridoma cell line SMVHBBX-1, with accession number CCTCC NO: C2026121, preservation date May 27, 2026, and depositary institution: China Center for Type Culture Collection, Wuhan University, Wuhan, China; FL624-44 is classified as hybridoma cell line SMVHBBX-2, with accession number CCTCC. NO: C2026120, deposited on May 27, 2026, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0071] Example 2 This embodiment provides a detection kit, which includes: (1) Pre-coated ELISA plates were prepared using one of the two monoclonal antibodies screened in Example 1. The preparation process was as follows: 0.1 M Na2CO3-NaHCO3 buffer with a pH of 9.6 was used as the coating solution; the monoclonal antibody was added to the coating solution to a concentration of approximately 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 reacted at 37°C for 3 h; a certain amount of BSA, sodium salicylate, and sucrose were added to the Na2CO3-NaHCO3 buffer (pH 9.6) to prepare the blocking working solution with concentrations of 1%, 0.05%, and 5%, respectively. The ELISA plate was blotted dry and washed twice with a plate washer, and the strips were patted dry on clean absorbent paper. Add 150 μL of the sealing working solution to each well of the microwell, bake at 37 ℃ for 3 hours, discard the sealing solution, pat the strip dry on clean absorbent paper, put it into a freeze vacuum dryer to dry for 3 hours, and vacuum heat seal. (2) The sample diluent is a phosphate buffer solution with a pH of 7.0 to 8.0; (3) Washing solution, the composition of which is phosphate buffer containing Tween-20; (4) The reaction termination solution is an H2SO4 solution with a concentration of 2 M; (5) Enzyme-labeled antibody, wherein the enzyme-labeled antibody includes 1 μg / mL of polyclonal antibody prepared by immunizing rabbits with SMV-BX virus CP protein in Example 1 and 0.2 μg / mL of horseradish peroxidase (HRP)-labeled goat anti-rabbit enzyme-labeled secondary antibody; (6) The colorimetric reagent is TMB colorimetric reagent; (7) SMV Pinellia ternata strain standard: The concentration of the SMV Pinellia ternata strain mother liquor was 1 mg / mL, diluted to 640 ng / mL, and 200 μL of 640 ng / mL standard was added to a 3 mL brown glass bottle. The standard was dried overnight in a freeze dryer to obtain freeze-dried powder.

[0072] The process of detecting SMV Pinellia ternata strains in plants using the above-mentioned detection kit includes the following steps: (1) Preparation of standard products and samples of unknown concentration 1) Preparation of standard products: The soybean mosaic virus Pinellia ternata strain (SMV-BX) standard was dissolved in 1 mL of sample extract, and the concentration of this solution was 128 ppb. Take 128 ppb of soybean mosaic virus Pinellia ternata strain (SMV-BX) standard, add 300 µL to 300 μL of sample extract, and the concentration of this solution is 64 ppb. Take 64 ppb of soybean mosaic virus Pinellia ternata strain (SMV-BX) standard, add 300 µL to 300 μL of sample extract, and the concentration of this solution is 32 ppb; Take 32 ppb of soybean mosaic virus Pinellia ternata strain (SMV-BX) standard and add 300 µL of sample extract to 300 µL of sample extract. The concentration of this solution is 16 ppb. Take 16 ppb of the soybean leaf virus Pinellia ternata strain (SMV-BX) standard and add 300 µL of the sample extract to 300 µL. The concentration of this solution is 8 ppb. After dilution, the standard solution should be protected from light and stored at -20°C for 14 days.

[0073] 2) Processing of samples with unknown concentrations: Blade pretreatment method: Take 5-10 mm 2 Leaf samples were placed in 1.5 mL centrifuge tubes, crushed, and 250 mL of diluent was added. The mixture was shaken for 5 minutes, centrifuged at 4000 rpm for 3 minutes, and 100 mL of the supernatant was used for analysis.

[0074] (2) Add standard or sample of unknown concentration: Add 100 mL of sample dilution (blank control) / standard / unknown concentration sample to the corresponding microwell, gently shake to mix, and react at 25℃ in the dark for 45 min.

[0075] (3) Washing the plate: Shake off the liquid in the well, wash thoroughly three times with 250 mL of washing solution per well, with a 1-minute interval between each wash, and pat dry with absorbent paper.

[0076] (4) Add enzyme-labeled antibody: Add 100 mL of enzyme-labeled antibody to each well, gently vortex to mix, and react at 25°C in the dark for 45 min. Remove the plate and repeat step 3 of washing.

[0077] (5) Color development: Add 100 mL of colorimetric reagent per well and react at 25°C in the dark for 15 min.

[0078] (6) Termination and Measurement: Add 100 mL of stop solution per well, gently vortex to mix, set the microplate reader to 450 nm, and measure the OD value of each well (preferably using dual wavelengths of 450 / 630 nm, reading the data within 5 minutes). If a microplate reader is unavailable, the stop solution can be omitted and the result can be determined visually.

[0079] (7) Calculate: Using statistical graphing software, a four-parameter fitting standard curve is plotted based on the measured OD values ​​of the standards. The concentration of SMV in the unknown sample can then be calculated using the standard curve and the OD values ​​of the sample with unknown concentration. The standard curve obtained in this embodiment is shown below. Figure 8 As shown, its R 2 The value of 0.9947 indicates that it has excellent linearity and can be used to calculate the concentration of soybean mosaic virus Banxia strain (SMV-BX) in the sample to be tested (sample with unknown concentration).

[0080] Example 3 This embodiment provides a quick test strip, such as Figure 9 As shown, it includes a base plate 7, and a sample pad 1, a gold-labeled pad 2, and an NC membrane 3, which are partially overlapped and fixed to the base plate from top to bottom along the extension direction of the base plate 7. An absorbent filter paper 4 is overlapped at the end of the NC membrane 3. The gold-labeled pad 2 is coated with one of the two monoclonal antibodies screened in Example 1. The NC membrane 3 has a C line 5 and a T line 6, where the T line 6 is coated with the other monoclonal antibody screened in Example 1, and the C line 5 is coated with goat anti-mouse IgG secondary antibody. The gold-labeled pad 2 is approximately 10 mm wide, with its upper edge overlapping the bottom of the NC membrane 3 by 1-2 mm; the sample pad 1 is approximately 20-30 mm wide, with its front edge overlapping the lower edge of the gold-labeled pad 2 by 1-2 mm; the absorbent filter paper 4 is approximately 20-30 mm wide, located at the top edge of the test strip, with its lower edge covering the upper part of the NC membrane 3 by 1-2 mm.

[0081] When using the test strip, insert it vertically into the sample solution. The sample solution will permeate along sample pad 1 to the colloidal gold-labeled antibody pad 2, forming a double-antibody sandwich structure with the specific antibody on the NC membrane 3. After 8-10 minutes, observe the color change in the result observation well and make a judgment. Result interpretation: Negative result (1 C line appears); Positive (+): C / T lines appear simultaneously.

[0082] The detailed method is as follows: 1) Wrapped A PALL170 nitrocellulose membrane (NC membrane) was selected and a gold-spraying membrane was used to streak a 2.0 mg / mL SMV-coated antibody solution (one of the monoclonal antibodies screened in Example 1) at a rate of 1.0 μL / cm to form a T line as the detection line; a 1 mg / mL goat anti-mouse secondary antibody (goat anti-mouse IgG) was streak a 0.7 μL / cm C line as the quality control line; the membrane was dried at 37°C for 24 hours and then ready for use; the antibody-coated nitrocellulose membrane was obtained.

[0083] 2) Preparation of colloidal gold a) Preparation: After washing the 500 mL beaker, 20 mL small beaker, rotor, brown bottle, glass rod, etc., place them in an acid tank (potassium dichromate: concentrated sulfuric acid: ultrapure water = 120 g: 200 mL: 1000 mL) and soak for 24 hours. Remove and rinse 3-4 times with tap water, then rinse 3-4 times with ultrapure water, and dry in a 37 ℃ oven for later use.

[0084] b) Preparation of chloroauric acid solution A. Weigh 1g of chloroauric acid powder (purchased from Sigma) into a brown bottle using a plastic weighing spoon, add 99 mL of ultrapure water to dissolve it completely, and store at 4°C protected from light.

[0085] c) Preparation of gold calcination solution B. Weigh 1g of trisodium citrate (purchased from Sigma) and dissolve it in 99 mL of ultrapure water, then mix well.

[0086] d) Preparation of colloidal gold: Measure 99 mL of ultrapure water into a beaker, add 1 mL of calcined gold solution A, place on a constant-temperature magnetic stirrer and stir until well mixed. Turn on the heat until the solution boils, then quickly add 2 mL of freshly prepared calcined gold solution B, continue stirring and heating. The solution gradually turns blue-black, then purple-black, then red upon further heating, and continues boiling until a transparent orange-red color appears. Continue boiling for 7-10 min, allow to cool naturally to room temperature, and add ultrapure water to bring the volume to 100 mL. Pour into a brown bottle and store at 4℃ protected from light; colloidal gold (particle size 40 nm) is obtained.

[0087] 3) Antibody labeling a) Antibody labeling: Take 1.5 mL of the colloidal gold prepared in step 1) above, adjust the pH value with 0.1 M K2CO3, add 10 μg of SMV-labeled antibody (i.e., the other monoclonal antibody screened in Example 1), mix well, and react at room temperature for 40 min. Add 10% BSA to stop the reaction, and let stand for 30 min.

[0088] b) Labeled antibody purification: First, centrifuge at low speed (1500 r / min) to discard the precipitate formed by the aggregated gold particles. Then, centrifuge at high speed (8500 r / min) for 30 minutes. Carefully aspirate the supernatant, and reconstitute the precipitate with 50 μL of 0.1M PBS (pH 7.4) containing 1% BSA, and store at 4°C to obtain the labeled colloidal gold antibody solution.

[0089] 4) Gold spraying The labeled colloidal gold antibody solution prepared in step 3) above was diluted to 0.1 mg / mL and sprayed onto the pretreated gold pad at a rate of 1 μL / cm. After drying, the gold pad with the specific monoclonal antibody immobilized with colloidal gold was obtained.

[0090] 5) Large panel assembly Arrange the components according to Figure 9 Assemble and fix it on the base plate (PVC adhesive base plate), and then place it in a vacuum dryer at 37 ℃ for 30 min.

[0091] 6) Cutting large boards Cut the assembled large board into 3mm wide strips, which become the SMV-BX speed test paper. See the attached diagram for details. Figure 9 .

[0092] The evaluation of the SMV-BX rapid test card (rapid test strip) for testing leaf samples includes the following steps: Leaf sample processing method: Place the leaf tissue between the cap and the body of a disposable tissue extraction tube, quickly close the cap, and repeat twice to obtain two circular leaf tissues; place the leaf at the bottom of the extraction tube using a pestle; insert the pestle into the tube, rotate the pestle or pipette tip to crush the leaf, and press continuously for 20-30 seconds. Add 0.5 mL (approximately 20 drops) of extraction buffer (10 mm PBS buffer); repeat the crushing step to ensure thorough mixing of the sample and buffer; allow to stand to precipitate or centrifuge to obtain the supernatant, which is the leaf sample extract. The detection results for 5× and 10× extractions are shown below. Figure 10 (Left: 5×, Right: 10×) It can be seen that the color of the detection line varies significantly with different sample concentrations.

[0093] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A hybridoma cell pair, characterized in that, The study includes two hybridoma cell lines. The first line is named SMVHBBX-1 Hybridoma cell line SMVHBBX-1, with accession number CCTCC NO: C2026121, deposited on May 27, 2026, at the China Center for Type Culture Collection. The second line is named SMVHBBX-2 Hybridoma cell line SMVHBBX-2, with accession number CCTCC NO: C2026120, deposited on May 27, 2026, at the China Center for Type Culture Collection.

2. The use of the hybridoma cell pair as described in claim 1 in the preparation of monoclonal antibodies for detecting SMV Pinellia ternata strain.

3. A monoclonal antibody pair, characterized in that, The monoclonal antibody pair comprises two monoclonal antibodies, which are secreted by the first hybridoma cell line and the second hybridoma cell line as described in claim 1, respectively.

4. The monoclonal antibody pair as described in claim 3, characterized in that, The preparation process of any one of the monoclonal antibodies in the monoclonal antibody pair is as follows: inject the first hybridoma cell line or the second hybridoma cell line into the peritoneal cavity of mice, collect the ascites fluid of mice after 9-12 days, and the supernatant obtained by centrifugation is the monoclonal antibody ascites fluid. The monoclonal antibody can be obtained by purifying the monoclonal antibody ascites fluid.

5. A detection kit for detecting SMV Pinellia ternata strains, characterized in that, The test kit comprises the following components: (1) Pre-coated enzyme-labeled plate, prepared using any one of the monoclonal antibody pairs as described in claim 3 or 4; (2) The sample diluent is a phosphate buffer solution with a pH of 7.0 to 8.0; (3) Washing solution, the composition of which is phosphate buffer containing Tween-20; (4) The reaction termination solution is an H2SO4 solution; (5) Enzyme-labeled antibody; (6) The colorimetric reagent is TMB colorimetric reagent; (7) Standard of SMV Pinellia ternata strain.

6. The detection kit as described in claim 5, characterized in that, The preparation process of the pre-coated ELISA plate of component (1) includes the following steps: S1: Using 0.1 M Na2CO3-NaHCO3 buffer with a pH of 9.6, dilute the monoclonal antibody to 5 μg / mL and add 100 μL to each well of the microplate. Incubate at 37°C for 3 h. S2: Shake off the microplate solution in the microplate wells, add washing solution, soak for 5 minutes, then shake off the microplate solution in the microplate wells again, and then pat dry on absorbent paper. S3: Add the blocking solution to the microwells of the microplate, 150 μL per well, and react at 37°C for 3 h. The blocking solution is a carbonate buffer containing BSA. S4: Shake off the solution in the microplate wells, pat dry on absorbent paper, and then freeze dry. S5: Use a vacuum packaging machine to package the enzyme-labeled plate into an aluminum foil bag.

7. The detection kit as described in claim 5, characterized in that, The concentration of the H2SO4 solution in component (4) is 1.8–2.2 M.

8. The detection kit as described in claim 5, characterized in that, The SMV Pinellia ternata standard in component (7) is a freeze-dried powder of SMV Pinellia ternata or its CP protein.

9. A rapid test strip for detecting SMV Pinellia ternata strains, characterized in that, The rapid test strip includes a base plate, and a sample pad, a gold label pad, and an NC membrane that are partially overlapped and fixed on the base plate from top to bottom along the extension direction of the base plate. An absorbent filter paper is overlapped at the end of the NC membrane. The gold label pad is coated with one of the monoclonal antibodies in the monoclonal antibody pair as described in claim 3 or 4. The NC membrane is provided with T lines and C lines, wherein the T lines are coated with the other monoclonal antibody in the monoclonal antibody pair as described in claim 3 or 4, and the C lines are coated with goat anti-mouse IgG secondary antibody.

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