Preparation method of bovine rotavirus monoclonal antibody and bovine rotavirus quantum dot immunochromatography test strip

Abstract

The invention discloses a bovine rotavirus monoclonal antibody and a preparation method of a bovine rotavirus quantum dot immunochromatography test strip, and belongs to the technical field of bovine rotavirus monoclonal antibodies. According to the invention, a hybridoma cell strain BRV 3G9 is utilized, and a monoclonal antibody is prepared by adopting an ascites method; the BRV 3G9 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46138. The BRV 3G9 is named as BRV 3G9. According to the present invention, the concentrated live virus immunized mouse is adopted to prepare the monoclonal antibody with strong immunoreaction, and the BRV quantum dot immunochromatography test strip is researched and developed on the basis of the monoclonal antibody, and has advantages of convenient use, high efficiency, high sensitivity, strong specificity and the like, and can achieve the rapid clinical differential diagnosis of calf diarrhea pathogens, the problem that grassroots veterinarians lack a clinical rapid detection technology is solved, the diagnosis and treatment level and the prevention and treatment effect are improved, and technical guarantee is provided for efficiently preventing and treating calf diarrhea and guaranteeing calf health.

Description

Technical Field

[0001] The invention relates to the field of bovine rotavirus monoclonal antibodies, in particular to a method for preparing a bovine rotavirus monoclonal antibody and a bovine rotavirus quantum dot immunochromatographic test strip. Background Art

[0002] Rotavirus (RV) belongs to the genus Rotavirus of the family Reoviridae and is one of the main pathogens of non-bacterial diarrhea in various young animals. Nearly 50% of diarrhea in newborn calves is related to rotavirus infection.

[0003] Bovine rotavirus (BRV) belongs to the family Reoviridae and the genus Rotavirus. The viral genome is a double-stranded RNA with 11 segments, encoding a total of six structural proteins (VP1-VP4, VP6, VP7). VP4 protein is the outer capsid spike protein of BRV, which can induce the body to produce neutralizing antibodies and is one of the important protective antigens of BRV. However, VP4 protein is relatively large, and it is difficult to express it completely and has poor solubility. VP4 protein is cleaved into VP8 and VP5 subunits under the action of trypsin. The core region of VP8 contains almost all the antigenic epitopes of VP4 protein. Like VP4 protein, it can stimulate the body to produce neutralizing antibodies and the immune response intensity is comparable. In addition, VP8 protein is highly conserved between the same serotypes and can stimulate homotypic protection.

[0004] Newborn calves infected with BRV are prone to mixed infection with other diseases, and the mortality rate of calves after the disease occurs is very high. Currently, no vaccine for BRV has been found to prevent infection. Therefore, monoclonal antibodies can be used to diagnose the virus early, and prevent and treat it as soon as possible. Isolating cattle with BRV can significantly reduce mortality and prevent further spread of the disease.

[0005] In 2018, Lu Yadong et al. developed a monoclonal antibody prepared with prokaryotically expressed VP6 protein as an antigen, and produced the corresponding colloidal gold immunochromatographic test strips. In 2020, Cao Lei et al. developed a monoclonal antibody prepared with the whole virus of BRV OSU strain as an antigen, and produced the corresponding colloidal gold immunochromatographic test strips. In 2019, ZhenXue Li et al. prepared and purified the monoclonal antibody of BRVVP6 protein and the rabbit polyclonal antibody of BRV whole virus, and produced the corresponding colloidal gold immunochromatographic test strips.

[0006] However, in the test strips prepared by the above three related research institutes, the antibodies coated with the C line and the T line have not been verified to see if the antigen epitope sites are different, and the antigens coated with the T line are mostly polyclonal antibodies, with poor specificity, long preparation cycle, and complex process, which will affect the accuracy and success rate of the test strips and are not conducive to the subsequent mass production of test strips. Compared with traditional colloidal gold immunochromatography test strips, fluorescent microsphere immunochromatography test strips have unique advantages. The colloidal gold immunochromatography method is that the sample can form a color visible to the naked eye after chromatography, and qualitative and semi-quantitative analysis is performed by color depth, but the content of the test object cannot be accurately analyzed. Moreover, any sample solution containing color will affect the test results of the colloidal gold method, such as serum sample testing. The marker of fluorescent immunochromatography is a substance that can emit fluorescence. By reading the intensity of fluorescence, the content of the test object can be accurately analyzed. Because the instrument only detects fluorescence, it is more suitable for detecting colored samples. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a bovine rotavirus monoclonal antibody and a bovine rotavirus quantum dot immunochromatographic test strip, and the monoclonal antibody is prepared by natural immune response, and a BRV quantum dot immunochromatographic test strip is developed based on this, which can realize the clinical rapid differential diagnosis of calf diarrhea pathogens, solve the problem of lack of clinical rapid detection technology for primary veterinarians, improve the diagnosis and treatment level and prevention and treatment effect, and provide technical guarantee for the efficient prevention and treatment of calf diarrhea and the protection of calf health.

[0008] To achieve the above object, the present invention provides a method for preparing a bovine rotavirus monoclonal antibody, using hybridoma cell line BRV 3G9 to prepare the monoclonal antibody by ascites method; BRV 3G9 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.46138.

[0009] Preferably, the ascites method for preparing monoclonal antibodies comprises:

[0010] The hybridoma cell line BRV 3G9 was injected into BALB / c mice by intraperitoneal injection. The state of the BALB / c mice was observed. When the BALB / c mice were slow in movement and had dry stool, ascites was collected. The collected ascites was centrifuged and purified to obtain monoclonal antibodies.

[0011] Preferably, BALB / c mice are pre-stimulated by intraperitoneal injection of Freund's incomplete adjuvant 10 to 15 days before injection of hybridoma cell line BRV 3G9.

[0012] A bovine rotavirus quantum dot immunochromatographic test strip prepared from the above monoclonal antibody;

[0013] The preparation method comprises the following steps:

[0014] S1, adjusting the buffer system of quantum dot fluorescent microspheres to activate the carboxyl groups of quantum dots;

[0015] S2, coupling the quantum dots activated in step S1 with the monoclonal antibody;

[0016] S3, pre-treating the sample pad and the conjugate pad, applying the monoclonal antibody solution labeled with quantum dots in step S1 on the conjugate pad and drying it, coating the monoclonal antibody solution with a concentration of 0.7-0.8 mg / mL on the detection line of the nitrocellulose membrane, and coating the goat anti-mouse IgG secondary antibody solution with a concentration of 0.7-0.8 mg / mL on the quality control line of the nitrocellulose membrane;

[0017] S4. Stack the processed sample pad, conjugate pad, nitrocellulose membrane, absorbent paper and carrier in sequence.

[0018] Preferably, step S1 comprises:

[0019] Use the same volume of 0.02M MES buffer with a pH value of 6.5-7.5 as the quantum dot fluorescent microspheres to dilute the quantum dot fluorescent microspheres, and then use a mixture of 15-25 mg / mL EDC-HCl and NHS to activate the quantum dot fluorescent microspheres. After mixing, react at 35-40°C for 10-20 minutes, centrifuge at 8000g for 10-25 minutes, and resuspend the quantum dot fluorescent microspheres with 0.02M MES buffer with a pH value of 6.5-7.5.

[0020] Preferably, in step S2, the volume ratio of the activated quantum dots to the monoclonal antibodies is 1:4-5.

[0021] Preferably, the interval between the detection line and the quality control line is 6.5-7.5 mm.

[0022] Therefore, the present invention discloses a method for preparing a bovine rotavirus monoclonal antibody and a bovine rotavirus quantum dot immunochromatographic test strip. The monoclonal antibody with strong immune response is prepared by immunizing mice with concentrated live virus, and a BRV quantum dot immunochromatographic test strip is developed based on the monoclonal antibody. The test strip has the advantages of being easy to use, highly efficient, highly sensitive, and highly specific. It can realize the clinical rapid differential diagnosis of the pathogen of calf diarrhea, solves the problem of lack of clinical rapid detection technology for primary veterinarians, improves the diagnosis and treatment level and the prevention and treatment effect, and provides technical guarantee for the efficient prevention and treatment of calf diarrhea and the protection of calf health.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1is a graph of the growth status of fused cells, wherein A is the growth status of cells on the 3rd day after cell fusion, B is the growth status of cells on the 5th day after cell fusion, and C is the growth status of cells on the 7th day after cell fusion;

[0025] Figure 2 This is a diagram of ascites purification results;

[0026] Figure 3 The IFA results of monoclonal antibodies 3G9, 7A1, and 7A2 are shown;

[0027] Figure 4 These are the subclass identification results for monoclonal antibodies 3G9, 7A1, and 7A2;

[0028] Figure 5 The results of the determination of the chromosome number of the monoclonal antibody, where A is 3G9, B is 7A1, and C is 7A2;

[0029] Figure 6 The Western Blot results of monoclonal antibodies and viral proteins, where A is 3G9, B is 7A1, and C is 7A2.

[0030] Figure 7 This is the IFA result diagram after the monoclonal antibody interacts with cells infected with different viruses;

[0031] Figure 8 The test strip test results of 6 paired combinations of 3 antibodies were assembled respectively;

[0032] Fig. 9 is a diagram of the specificity test results of the test strip after assembly;

[0033] Fig.10 This is a sensitivity test result diagram of the test strip after assembly; DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0036] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0037] Embodiment 1

[0038] Preparation of monoclonal antibodies against bovine rotavirus

[0039] Main materials: African green monkey embryonic kidney cells (MA104), myeloma cells (SP2 / 0), human ileocecal cancer cells (HRT-18), and bovine kidney cells (MDBK) are preserved by the Animal Infectious Disease Diagnosis and New Vaccine Laboratory of Heilongjiang Bayi Agricultural University.

[0040] Feeder cells were macrophages obtained from the peritoneal cavity of BALB / c mice. BRV G6 P1 strain, BRV positive and negative sera, bovine coronavirus (BCoV), bovine infectious rhinotracheitis virus (IBRV), and bovine viral diarrhea-mucosal disease (BVDV) virus strains were isolated and preserved in our laboratory.

[0041] BALB / c mice aged 4 to 6 weeks were purchased from Qingyuan Animal Farm in Lindian County. Quantum dot fluorescent microspheres were purchased from Beijing Nanogene Biotechnology Co., Ltd., and chromatography test strips and related materials were provided by Harbin Guosheng Biotechnology Co., Ltd.

[0042] Main reagents: PEG 1500, Freund's complete adjuvant and incomplete adjuvant were purchased from Sigma; plasmid mini-extraction kit and gel recovery kit were purchased from Quanshijin Company; HAT and HT culture media were purchased from Gibco, and mouse antibody subtype kit was purchased from Southern Bioetch; EDC, NHS, and MES were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0043] The preparation method comprises:

[0044] (1) Preparation of Antigen

[0045] The BRV strain was inoculated into MA104 cells, and the virus was collected after the pathological changes. When the total amount of virus was about 500 mL, it was concentrated by sucrose density gradient centrifugation. The concentrated virus protein was tested for protein concentration using the BCA protein concentration test kit, and the TCID50 of the concentrated virus and the unconcentrated virus was measured.

[0046] The TCID50 of BRV virus solution was 1×10 -5 / 0.1mL.

[0047] (2) Establishment of indirect ELISA method

[0048] The matrix method was used to determine that the optimal antigen protein coating amount for indirect ELISA was 2 μg / mL; the optimal antigen coating time was 2 hours; the optimal blocking solution was 1% BI serum; the optimal blocking time was 1 hour; and the optimal dilution concentration of goat anti-mouse secondary antibody was 1:4000.

[0049] (3) Mouse immunization

[0050] Five 6-week-old BALB / C female mice were immunized by footpad immunization. The BRV concentrated protein was diluted and mixed with Freund's complete adjuvant for emulsification, and then immunized in the mouse footpad. One week later, the same emulsification method and dose were mixed with Freund's incomplete adjuvant for emulsification for the second immunization. The third immunization method and interval were the same as the previous two times. At the same time, blood was collected from 4 mice, labeled as mouse 1, mouse 2, mouse 3, and mouse 4. The mouse serum titer was detected by indirect ELISA method with 4 immunized mouse sera. When the serum titer reached 1:8000 or above, the mice were boosted with immunization, and the cells were fused within 5 days.

[0051] (4) Cell fusion

[0052] Prepare feeder layer cells one day in advance, and prepare SP2 / 0 mouse myeloma cells. Blow down when fusion occurs, centrifuge at 800r / min for 5min, and count the cells for later use. Fix the immunized BALB / c mice in the cell operation table, take the spleen, and place it in a culture dish. Use a syringe needle to repeatedly inject the culture medium to rinse the spleen. Mix SP2 / 0 cells and spleen cells in proportion, centrifuge and discard the supernatant. Add culture medium and centrifuge again, aspirate and discard the supernatant, and break up and mix. Aspirate 750μL of PEG-1500 preheated at 37℃, add it slowly, and gently blow up the cells several times, and continue to stand in the water bath for 30s. Aspirate the culture medium and slowly add 30mL of culture medium, discard the supernatant. Then use 30mL of basal culture medium to slowly blow the cells and discard the supernatant. Move the fused cells to the culture dish, mix them, and slowly add them to the 96-well plate containing feeder layer cells. Culture them in an incubator at 37℃ and 5% CO2, and observe and record the cell fusion status every day.

[0053] Cell growth Figure 1 As shown, A is the growth state of cells on the 3rd day after cell fusion, and small cell colonies can be seen; B is the growth state of cells on the 5th day after cell fusion, and cells can be seen overlapping; C is the growth state of cells on the 7th day after cell fusion. When the fused cells grow to 1 / 10 of the well plate area, select the appropriate well to detect the supernatant. The fusion rate is calculated to be 75% (432 / 576).

[0054] (5) Positive hybridoma cell screening and subcloning

[0055] Mouse positive serum was used as a positive control, and SP2 / 0 culture supernatant was used as a negative control. Positive hybridoma cells were screened according to the method in step (2). Positive hybridoma cells were subcloned by multiple dilution method, and frozen for seed. Fusion cells in 12 cell wells were subcloned in total. Finally, three monoclonal antibody cells that could still secrete antibodies were obtained, namely 3G9, 7A1, and 7A2.

[0056] BRV 3G9 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.46138 and the deposit date is November 20, 2024.

[0057] (6) Preparation and purification of ascites

[0058] Twelve BALB / c female mice were intraperitoneally injected with 500 μL of Freund's incomplete adjuvant one week before the preparation of ascites. Ascites was prepared by intraperitoneal injection. When the abdomen of the mouse was obviously swollen, the ascites was collected and centrifuged to absorb the supernatant, and then stored at -80°C. The ascites titer of the monoclonal antibody was determined by indirect ELISA. The ascites was then purified according to the instructions of protein A / G agarose beads, and the purification was detected by PAGE gel, and stored at -80°C.

[0059] The titer of ascites was detected by indirect ELISA, and the results showed that the antibody titers of 3G9, 7A1, and 7A2 were 1:256000, 1:16000, and 1:8000, respectively. Among them, the ascites titer of 3G9 was the highest, so 3G9 ascites was purified.

[0060] The eluted antibody, flow-through and unpurified ascites were subjected to SDS-PAGE to observe the purification effect. Figure 2 shown. Figure 2 In the figure, M: 180 kDa protein marker; 1-5: elution 1-5 times; 6-7: blank wells; 8: flow-through; 9: unpurified ascites.

[0061] The results showed that compared with the unpurified ascites and flow-through fluid, the purified antibody had a light chain at about 25 kDa, a heavy chain at about 55 kDa, and no other miscellaneous bands.

[0062] Test Example 1

[0063] Indirect immunofluorescence (IFA) characterization of monoclonal antibodies

[0064] MA104 cells were infected with BRV, and IFA experiments were performed when MA104 cells showed pathological changes. 4% paraformaldehyde was used for fixation at 37°C, and the plates were washed with PBS. A mixture of 1% Triton X-100 and 1% BSA was added for permeabilization and blocking, and the plates were allowed to stand at 37°C for 30 minutes, and the plates were washed with PBS. The prepared mAb ascites was used as the primary antibody, incubated at 37°C for 1 hour, and the plates were washed with PBS. Goat anti-mouse IgG fluorescent antibody was used as the secondary antibody, incubated at 37°C in the dark for 1 hour, and the plates were washed with PBST. DAPI (1:1000 dilution) was used for staining for 5 minutes, and the plates were washed with PBST. Incubated at room temperature in the dark for 10 minutes, the plates were washed with PBST, and an appropriate amount of 90% glycerol was added to seal the plates, and the results were observed under a fluorescence microscope.

[0065] like Figure 3As shown, the results showed that 3G9, 7A1, and 7A2 could react with BRV to produce specific red fluorescence, while the negative serum control showed no fluorescence.

[0066] Test Example 2

[0067] Monoclonal antibody subclass identification

[0068] The subclasses of monoclonal antibodies 3G9, 7A1, and 7A2 were identified according to the instructions of the mouse monoclonal antibody subclass identification kit. The experimental results are shown in Table 2 and Figure 4 The results showed that the liquid was dark blue, the heavy chains were all IgG3, and the light chains were all Kappa chains.

[0069] Table 2 Antibody subclass identification table

[0070]

[0071] Test Example 3

[0072] Chromosome number test of hybridoma cells

[0073] Add 0.05 μg mL -1 Colchicine, culture at 37℃ for 4h to collect cells. Centrifuge and discard the supernatant. Slowly add 5mL of 0.4% KCl solution preheated at 37℃ to the cell pellet, mix well, and let stand at room temperature for 15min. Centrifuge under the same conditions to remove the supernatant, add fixative to 8mL, fix at room temperature for 25min, centrifuge and discard the supernatant. Secondary fixation: Centrifuge under the same conditions to remove the supernatant, add 8mL of fixative, and let stand at room temperature for half an hour. Centrifuge under the same conditions to remove the supernatant, resuspend the cell pellet with 500μL of fixative. Pipette 100μL of the cell suspension and drop it on a frozen slide. After drying, stain with Giemsa for 5min and observe the chromosome morphology and number under an oil microscope.

[0074] The results are as follows Figure 5 As shown, A is 3G9, B is 7A1, and C is 7A2. Through staining microscopy, it can be seen that the number of chromosomes is between 95 and 110, which is consistent with the expected result.

[0075] Test Example 4

[0076] Western blot identification of monoclonal antibodies

[0077] The cells were plated in a 12-well cell culture plate, and MA104 cells were infected with BRV, and uninfected MA104 cells were used as negative controls. After collecting sample proteins for quantification, they were electrophoresed by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk in TPBS at 37°C for 2h, and three strains of BRV monoclonal antibodies were used as primary antibodies at 4°C overnight. The membrane was washed with TBST. HRP-labeled goat anti-mouse IgG was used as secondary antibody at 37°C for 1h, washed as before, and the negative control was treated in the same way. The results were observed after color development.

[0078] The results are as follows Figure 6 As shown, A is 3G9, B is 7A1, and C is 7A2. The results showed that all three mAbs had a 50 kDa band, indicating that all three mAbs could effectively bind to the BRV viral structural protein, indicating that the prepared monoclonal antibodies were specific monoclonal antibodies against BRV.

[0079] Test Example 5

[0080] Neutralization ability test of monoclonal antibodies

[0081] The cells were plated in a 96-well cell culture plate, and the ascites of the three strains were filtered using a 0.22 μm filter membrane. The ascites were diluted in multiples using DMEM basal medium to 10 -6 The diluted ascites was mixed with the same volume of 200 TCID 50 After mixing the BRV virus dilutions, incubate them in a 37°C incubator for 1 hour. Add the mixed solution into the well plate, set up a virus-infected control group and a negative control group, and observe the cytopathic effect every day.

[0082] The experimental results showed that the neutralizing titer of 3G9 and 7A1 monoclonal antibodies in ascites was 1:100, and that of 7A2 was 1:1000.

[0083] Test Example 6

[0084] Monoclonal antibody specificity assay

[0085] The cells were plated in a 12-well cell culture plate, and MA104 cells were infected with BRV, HRT-18 cells were infected with BCoV, MDBK cells were infected with IBRV and BVDV, and uninfected cells were used as negative controls. When the cells were pathological, an IFA experiment was performed, and the method was as shown in Test Example 1.

[0086] The results are as follows Figure 7 As shown, 3G9, 7A1, and 7A2 can all react with BRV to produce red fluorescence, while cells infected with BCoV, IBRV, and BVDV and the control group showed no fluorescence, indicating that all three antibodies react specifically with BRV.

[0087] Test Example 7

[0088] Monoclonal antibody superimposed ELISA test

[0089] The BRV protein was diluted according to the optimal antigen coating concentration of the indirect ELISA method and coated on a blank ELISA plate. The ELISA plate was then coated according to the optimal blocking conditions. The three antibodies were diluted to a concentration of 1:8000, and then paired in pairs. They were used as primary antibodies. After incubation for 1 hour, the other corresponding paired strain was incubated for 1 hour. A control group and a negative control group were set up to incubate only one monoclonal antibody. After incubation, the goat anti-mouse secondary antibody was incubated. After HRP color development, the OD value was read with an enzyme reader, and the appreciation index (AI) was calculated:

[0090] AI=[(2A 1+2 / (A1+A2)-1]×100%;

[0091] Among them, A1 is the OD value of the incubation pair with the first antibody as the primary antibody; A2 is the OD value of the incubation pair with the second antibody as the primary antibody; 1+2 The two paired antibodies were incubated for 1 hour each to obtain the OD value of the primary antibody; when AI < 40%, it was determined that the two monoclonal antibodies were directed against the same antigen epitope. The larger the AI ​​value, the smaller the possibility of antigenic site overlap.

[0092] The test results are shown in Table 3:

[0093] Table 3 Monoclonal antibody superposition ELISA results

[0094] 3G9 7A1 7A2 7A1+7A2 3G9+7A1 3G9+7A2 OD value 1.251 1.095 0.468 1.866 1.390 1.854 AI Value 59.14％ 61.77％ 137.19％

[0095] The results showed that among the three monoclonal antibodies obtained, the AI ​​value of each combination of two antibodies was greater than 40%, so it was preliminarily determined that the three antibodies were directed against different antigenic epitopes of BRV.

[0096] Embodiment 2

[0097] The construction of the quantum dot detection test strip includes the following steps:

[0098] (1) Purification and concentration of monoclonal antibodies

[0099] The 3G9, 7A1 and 7A2 monoclonal antibodies with AI values ​​≥ 40% in the overlay experiment were purified using protein A / G agarose gel affinity chromatography medium, the concentration was determined, and then the concentration of each antibody was dialyzed and concentrated to 1 mg / mL.

[0100] (2) Preparation and optimization of quantum dot test strips

[0101] The buffer system of quantum dot fluorescent microspheres was adjusted. The microspheres were diluted with the same volume of 0.02 M MES buffer with a pH value of 6.5 and a pH value of 7, respectively. The microspheres were then activated with a mixture of 20 mg / mL EDC-HCl and NHS. After mixing, the mixture was reacted at 37°C for 15 min, centrifuged at 8000 g for 20 min, and the microspheres were resuspended with MES buffer. The results showed that when the pH of the MES buffer was 7, the microspheres were resuspended more thoroughly and the activation effect was better.

[0102] The antibody to be labeled and the activated microspheres were mixed at a volume ratio of 5:1, reacted at 37°C for 1 hour, and then the reaction system was blocked with a blocking agent, reacted at 37°C for 30 minutes, centrifuged at 8000g for 15 minutes, the supernatant was discarded, and the solution was resuspended in a quantum dot fluorescent microsphere preservation solution.

[0103] The sample pad and the conjugate pad were pretreated, the labeled antibody was coated on the conjugate pad and dried for later use, and different concentrations of primary antibody and goat anti-mouse secondary antibody were coated on the detection line (T line) and quality control line (C line) of the nitrocellulose membrane respectively.

[0104] The three antibodies were combined and paired, respectively, sprayed on the T line and prepared on the conjugate pad, dried, and assembled.

[0105] The experimental results showed that the optimal coating concentration of the monoclonal antibody on the test line (T line) was 0.75 mg / mL; the optimal coating concentration of the goat anti-mouse IgG secondary antibody on the quality control line (C line) was 0.75 mg / mL, and the interval between the T line and the C line was 7 mm.

[0106] The three antibodies were paired in pairs and coated on the T line and the binding pad respectively, for a total of 6 paired combinations. BRV positive virus liquid was added to each pair. The test results were determined a few minutes after the sample was added. The experiment was repeated 3 times. The results are as follows Figure 8 As shown, the assembly order from left to right is 1: 7A1 is T line, 7A2 is marker; 2: 7A1 is T line, 3G9 is marker; 3: 7A2 is T line, 7A1 is marker; 4: 7A2 is T line, 3G9 is marker; 5: 3G9 is T line, 7A2 is marker; 6: 3G9 is T line, 7A1 is marker. The test results show that when 3G9 is the marker antibody placed on the conjugate pad, the test strip has the best effect, such as samples 2 and 4 in the figure.

[0107] (3) Performance measurement of quantum dot test strips

[0108] The test strips assembled after the optimized conditions were used for specific experiments: the test strips were used to detect BCoV, IBRV, BVDV and BRV virus solutions respectively. The test results were determined a few minutes after the samples were added. The experiment was repeated 3 times. The test strips only showed positive bands for BRV virus, and other samples were negative. The experimental results are shown in Fig. 9 .

[0109] Sensitivity test: TCID50 is 1×10 -5 / 0.1mL of BRV virus solution was diluted at 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, and the test results were determined a few minutes after the sample was added. The experiment was repeated 3 times. It can be observed that the fluorescence intensity at the T line gradually fades with the increase of the sample dilution multiple. When the dilution multiple is 1:128, there is still a faint band at the T line, and when the dilution multiple reaches 1:256, the antigen is completely undetectable. The experimental results are shown in Fig.10 .

[0110] Stability test: After the prepared test strips were placed in a 37°C incubator for 30 days, 10 samples that were positive for BRV by PCR were tested for stability of the test strips. The test results were determined a few minutes after the samples were added. The experiment was repeated 3 times. The results showed that the positive samples all showed two bands, and the negative samples had no bands on the T line, indicating that the prepared test strips were still stable after being stored at 37°C for 30 days.

[0111] Therefore, this experiment invented a method for preparing a bovine rotavirus monoclonal antibody and a bovine rotavirus quantum dot immunochromatographic test strip, prepared and screened three hybridoma cell lines 3G9, 7A1, and 7A2 that can stably secrete antibodies, and identified that they have good biological activity; 3G9 with the highest ascites titer was selected for purification and preparation of quantum dot immunochromatographic test strips, and the prepared quantum dot immunochromatographic test strips have the advantages of being easy to use, cheap, efficient, highly sensitive, and highly specific, and can achieve clinical rapid differential diagnosis of calf diarrhea pathogens, solve the problem of lack of clinical rapid detection technology for primary veterinarians, improve the diagnosis and treatment level and prevention and treatment effect, and provide technical guarantee for the efficient prevention and treatment of calf diarrhea and the protection of calf health.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a bovine rotavirus monoclonal antibody, characterized in that: The monoclonal antibody was prepared by the ascites method using the hybridoma cell line BRV 3G9; BRV 3G9 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number: CGMCC No.46138.

2. The method for preparing a bovine rotavirus monoclonal antibody according to claim 1, characterized in that: The ascites method for preparing monoclonal antibodies comprises: The hybridoma cell line BRV 3G9 was injected into BALB / c mice by intraperitoneal injection. The state of the BALB / c mice was observed. When the BALB / c mice were slow in movement and had dry stool, ascites was collected. The collected ascites was centrifuged and purified to obtain monoclonal antibodies.

3. The method for preparing a bovine rotavirus monoclonal antibody according to claim 2, characterized in that: BALB / c mice were pre-stimulated by intraperitoneal injection of Freund's incomplete adjuvant 10 to 15 days before injection of hybridoma cell line BRV 3G9.

4. A bovine rotavirus quantum dot immunochromatographic test strip, characterized in that: Prepared by the monoclonal antibody according to any one of claims 1 to 3; The preparation method comprises the following steps: S1, adjusting the buffer system of quantum dot fluorescent microspheres to activate the carboxyl groups of quantum dots; S2, coupling the quantum dots activated in step S1 with the monoclonal antibody; S3, pre-treating the sample pad and the conjugate pad, applying the monoclonal antibody solution labeled with quantum dots in step S1 on the conjugate pad and drying it, coating the monoclonal antibody solution with a concentration of 0.7-0.8 mg / mL on the detection line of the nitrocellulose membrane, and coating the goat anti-mouse IgG secondary antibody solution with a concentration of 0.7-0.8 mg / mL on the quality control line of the nitrocellulose membrane; S4. Stack the processed sample pad, conjugate pad, nitrocellulose membrane, absorbent paper and carrier in sequence.

5. A bovine rotavirus quantum dot immunochromatographic test strip according to claim 4, characterized in that: Step S1 includes: Use the same volume of 0.02M MES buffer with a pH value of 6.5-7.5 as the quantum dot fluorescent microspheres to dilute the quantum dot fluorescent microspheres, and then use a mixture of 15-25 mg / mL EDC-HCl and NHS to activate the quantum dot fluorescent microspheres. After mixing, react at 35-40°C for 10-20 minutes, centrifuge at 8000g for 10-25 minutes, and resuspend the quantum dot fluorescent microspheres with 0.02M MES buffer with a pH value of 6.5-7.

5.

6. A bovine rotavirus quantum dot immunochromatographic test strip according to claim 4, characterized in that: In step S2, the volume ratio of the activated quantum dots and the monoclonal antibodies coupled is 1:4-5.

7. A bovine rotavirus quantum dot immunochromatographic test strip according to claim 4, characterized in that: The interval between the test line and the quality control line is 6.5-7.5mm.

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