Hybridoma cell strain 3D7 producing monoclonal antibody of canine parvovirus and application thereof

Abstract

The application discloses a hybridoma cell strain 3D7 for producing canine parvovirus monoclonal antibody and an application thereof, and belongs to the technical field of biological detection. The preservation number of the hybridoma cell strain 3D7 is CCTCC NO: C2024216. The monoclonal antibody produced by the hybridoma cell strain 3D7 provided in the application is used as a capture antibody to prepare a colloidal gold detection test strip for canine parvovirus disease, which can accurately detect the canine parvovirus, has good specificity and high sensitivity, can recognize CPV-2, CPV-2a, CPV-2b and CPV-2c various subtypes of CPV strains, has good broad-spectrum property, and is superior to commercially available colloidal gold test strips in terms of minimum detection amount. The test strip has high coincidence rate with PCR. The application provides a technical means for rapid on-site diagnosis of CPV.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a hybridoma cell strain 3D7 producing canine parvovirus monoclonal antibody and application thereof. BACKGROUND

[0002] Canine parvovirus disease (CPVD) is an acute and severe infectious disease widely spread in dogs caused by canine parvovirus (CPV). The host of CPV is very wide, which can infect raccoon, cat, wolf and almost all mammals, and can be directly transmitted through the fecal-oral route or indirectly transmitted through fecal contamination of parasites. The virus recognizes mesenteric lymph nodes and thymic lymphoid tissue as target tissues for replication, and is transmitted to intestinal crypts of small intestine through blood, but the transmission of the virus is not limited to the intestinal tract, but also includes lung, spleen, liver, heart, bone marrow, kidney and brain, etc., which is a systemic disease. The virus completes mutation by changing the amino acid of VP2 protein combined with the transferrin receptor of host cells. So far, the antigenic subtypes of CPV circulating in the world include CPV-2, CPV-2a, CPV-2b, CPV-2c, new CPV-2a and new CPV-2b.

[0003] CPVD has rapid onset, fast transmission and high mortality, and the sick dogs die rapidly after the onset. Rapid diagnosis is very important for the prevention and control of CPVD. At present, many kinds of laboratory detection methods have been applied to the diagnosis of canine parvovirus disease, such as hemagglutination test (HA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence test, PCR, etc. Although the above methods have good sensitivity and specificity, the above methods are time-consuming and need professional instruments, and can only be detected by professional personnel in the laboratory, which is not suitable for rapid diagnosis of canine parvovirus disease on site.

[0004] Colloidal gold immunochromatography test strip has the advantages of rapidity, simplicity, low price, intuitive and simple result determination, and does not need additional instruments and equipment. At present, there are colloidal gold immunochromatography test strips for rapid detection of canine parvovirus on the market. However, the test strips on the market have the problems of incomplete coverage of detected virus strains, low sensitivity of detection and low coincidence rate with PCR detection results.

[0005] Due to the long-term evolution and mutation of CPV, currently there are CPV-2, CPV-2a, CPV-2b and CPV-2c different subtype strains in China. However, there is no effective detection test strip that can recognize the above-mentioned multiple subtype strains in China. Therefore, based on the current CPV epidemic trend in China, it is of great significance to screen a monoclonal antibody that can effectively recognize CPV-2, CPV-2a, CPV-2b and CPV-2c and other multiple subtypes, and then to recognize the four strains of CPV in a broad spectrum, to quickly diagnose suspected CPV infected animals and to break the technical monopoly of imported test strips. SUMMARY

[0006] The purpose of the present application is to provide a hybridoma cell strain 3D7 producing canine parvovirus monoclonal antibody and its application, in order to solve the problems existing in the prior art. The monoclonal antibody produced by the hybridoma cell strain 3D7 provided by the present application is used as a capture antibody to prepare a colloidal gold detection test strip for canine parvovirus disease, which can accurately detect canine parvovirus, has good specificity and high sensitivity, and can recognize CPV-2, CPV-2a, CPV-2b and CPV-2c multiple subtype CPV strains, has good broad spectrum, and is superior to commercially available colloidal gold test strips in terms of minimum detection amount.

[0007] To achieve the above-mentioned purpose, the present application provides the following scheme:

[0008] The present application provides a hybridoma cell strain 3D7 producing canine parvovirus monoclonal antibody, which is preserved in China Center for Type Culture Collection on October 18, 2024, and the preservation number is CCTCC NO: C2024216.

[0009] The present application also provides the application of the hybridoma cell strain 3D7 in preparing canine parvovirus monoclonal antibody.

[0010] The present application also provides a canine parvovirus monoclonal antibody produced by the hybridoma cell strain 3D7.

[0011] The present application also provides the application of the canine parvovirus monoclonal antibody in preparing a colloidal gold detection kit for canine parvovirus disease.

[0012] The present application also provides a colloidal gold detection test strip for canine parvovirus disease, which is composed of the following components: sample pad, gold-labeled antibody binding pad, absorbent paper and nitrocellulose membrane; the nitrocellulose membrane has a quality control line and a detection line, the detection line is coated with the canine parvovirus monoclonal antibody, and the quality control line is coated with a goat anti-mouse antibody.

[0013] Further, the gold-labeled antibody binding pad is coated with colloidal gold-labeled antibody 2A10.

[0014] Further, the coating concentration of the goat anti-mouse antibody is 4.5 mg / mL.

[0015] Further, the coating concentration of the canine parvovirus monoclonal antibody is 2.5 mg / mL.

[0016] Further, the gold spraying amount of the colloidal gold labeled antibody 2A10 is 5 μL / cm.

[0017] The present application discloses the following technical effects:

[0018] The present application applies hybridoma cell technology to screen 8 hybridoma cell strains capable of secreting anti-CPV monoclonal antibodies, and antibody subclass identification results show that 3D7, 1B3, 6C3, 5D4 and 1D3 are IgG subtypes, and the obtained 5 IgG subtype monoclonal antibodies are used for antibody cross pairing test with commercial antibody 2A10, 3D7 is selected as the best capture antibody, and 2A10 is selected as the best gold labeled antibody, and the colloidal gold immunochromatography test strip prepared therefrom can accurately detect canine parvovirus, has good specificity and high sensitivity, and can identify CPV strains of CPV-2, CPV-2a, CPV-2b and CPV-2c subtypes, has good broad spectrum, and is superior to commercially available colloidal gold test strips in terms of minimum detection amount. 70 clinical samples are detected by using the test strip and PCR respectively. The results show that the positive coincidence rate between the two is 91.8% (45 / 49), the negative coincidence rate is 100% (21 / 21), the total coincidence rate is 94.3%, and the coincidence rate with PCR is higher. The present application provides a technical means for rapid on-site diagnosis of CPV. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0020] Figure 1 SDS-PAGE analysis (A) and Western blot detection verification immunogenicity (B) of CPV virus sucrose density gradient centrifugation;

[0021] Figure 2 Indirect ELISA determination of serum titer after immunization of 6 mice with CPV;

[0022] Figure 3 Antibody titer (A) and ascites antibody titer (B) of hybridoma cell culture supernatant;

[0023] Figure 4The results of indirect immunofluorescence detection of CPV monoclonal antibodies secreted by different hybridoma cells (A: 3D7; B: 1B3; C: 6C3; D: 5D4; E: 1D3; F: 3A9; G: 1D9; H: 1H2);

[0024] Figure 5 The results of SDS-PAGE verification after purification of 5 IgG subtype monoclonal antibodies;

[0025] Figure 6 The results of Western blot identification of anti-CPV monoclonal antibodies;

[0026] Figure 7 The results of preliminary screening of antibody cross-pairing;

[0027] Figure 8 The results of optimization of the best gold spraying amount;

[0028] Figure 9 The results of optimization of the best coating amount of 3D7 capture antibody T line;

[0029] Figure 10 The results of optimization of the best coating amount of goat anti-mouse antibody C line;

[0030] Figure 11 The results of specific detection of test strips;

[0031] Figure 12 The results of sensitivity determination of test strips against different strains of CPV, A is the sensitivity determination result of the test strip against CPV-2; B is the sensitivity determination result of the test strip against CPV-2a; C is the sensitivity determination result of the test strip against CPV-2b; D is the sensitivity determination result of the test strip against CPV-2c;

[0032] Figure 13 The results of stability detection of test strips;

[0033] Figure 14 The comparison of the minimum detection amount of self-made colloidal gold detection test strips and commercial test strips for detecting CPV-2 (A), CPV-2a (B), CPV-2b (C), and CPV-2c (D), wherein the first row of each figure is the self-made test strip, and the second row is the commercial test strip.

[0034] Biological preservation

[0035] Hybridoma cell line 3D7.

[0036] Accession number: CCTCC NO: C2024216;

[0037] Preservation time: October 18, 2024;

[0038] Depository: China General Microbiological Culture Collection Center

[0039] Address of Deposit: Wuhan, China, Wuhan University. DETAILED DESCRIPTION

[0040] Various illustrative embodiments of the present application are now described in detail. Such description, however, should not be taken to limit the scope of the present application, but rather, should be taken as merely illustrative of some aspects, features and embodiments thereof.

[0041] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range is inclusive of its end points.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0043] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples and embodiments described herein are exemplary only and are not intended to be limiting.

[0044] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0045] The CPV virus, different subtypes of CPV-2, CPV-2a, CPV-2b, CPV-2c virus and specific detection of canine coronavirus (CCV), canine distemper virus (CDV), canine parainfluenza virus (CPIV) and canine adenovirus (CAV) used as antigens in the embodiments of the present application are preserved by the National Key Laboratory of Agricultural Microbial Resources Exploration and Utilization of Huazhong Agricultural University, and a commitment is made to the public for 20 years from the filing date. The canine nasopharyngeal swabs are provided by the experimental dog base of Wuhan Keqian Biological Co., Ltd. in Hubei Province, a total of 70 clinical samples, PCR experiments are carried out according to the primer sequence provided in standard GB / T 34746-2017 to detect CPV, of which 49 are positive and 21 are negative. The primers are synthesized by Wuhan Qikai Biological Company. Other experimental materials, if not specified, can be obtained by conventional purchase.

[0046] Example 1 Preparation of CPV monoclonal antibody

[0047] 1. Preparation and purification of CPV antigen

[0048] The CPV was inoculated into the single F81 cell in good growth state for amplification culture, and cultured for 3-4 days. When the cell pathological change (CPE) reached 80%, the virus was collected by repeated freeze-thawing three times. The collected virus liquid was placed in a centrifuge tube, the centrifuge tube was placed in a sleeve, and centrifuged at 28000 rpm, 4℃ for 2h. At the end of centrifugation, the supernatant was discarded, and the lower precipitate was dissolved with a small amount of PBS.

[0049] The concentrated virus was purified by sucrose density gradient, 2mL of 20%, 30%, 40%, 50% and 60% mass concentration sucrose solution was added in sequence in the ultracentrifuge tube, and the uppermost layer was added with ultracentrifuged concentrated virus liquid. The centrifuge tube was placed in the sleeve, centrifuged at 28000 rpm, 4℃ for 2h. After centrifugation, the virus was distributed in each sucrose concentration layer. After the virus liquid distributed in each layer was sucked out, it was added into the ultracentrifuge tube, and PBS solution was used to make up, and the sugar was removed again by ultracentrifugation. After centrifugation, the supernatant was discarded, and the lower precipitate was dissolved with a small amount of PBS.

[0050] The virus liquid of each sucrose layer obtained was verified by SDS-PAGE to check the distribution of virus protein in each sucrose layer. The results are shown in Figure 1 A, which shows that the virus protein is mainly gathered in the 20-30% sucrose layer, and the virus purification effect is good. Then Western blot was used to verify the recognition of virus protein in each layer, and the immunogenicity was verified. The results are shown in Figure 1 B, which shows that the main immunogenic protein VP2 exists in the purified CPV antigen. The virus layer with the most virus protein and recognizing the VP2 protein of the virus was selected as the immunogen for immunizing mice.

[0051] 2. Animal immunization

[0052] Six healthy 6-8 week old BALB / c mice were immunized with purified concentrated CPV virus solution. The first immunization was emulsified with an equal amount of CPV and Freund's complete adjuvant, and injected intraperitoneally at a dose of 0.2 mL per mouse. The second and third immunizations were emulsified with Freund's incomplete adjuvant, and the dose was the same as the first immunization, with a two-week interval. The mice were eye bled at the sixth week after the third CPV immunization. The mouse serum dilution factors were 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 , 1:10 9 , and 1:10 10 , and their titers were determined by indirect ELISA. The results are shown in Table 1 and Figure 2 , which shows that the serum titers of the six mice after immunization were all greater than 10 5 , and the in vivo antibody levels reached the fusion standard. The mice were then subjected to a booster immunization followed by cell fusion.

[0053] Table 16 Determination of cell serum titers of six mice

[0054]

[0055] The mice with higher indirect ELISA detection values were subjected to a booster immunization with a dose of 0.2 mL per mouse of CPV virus solution without adjuvant, and cell fusion was performed three days after the booster immunization.

[0056] 3. Cell fusion

[0057] Two days before cell fusion, feeder layer cells were prepared from non-immunized mice. The mice were eye bled to death, and negative serum was collected. The mice were soaked in 75% anhydrous ethanol for 10 min, and then their limbs were fixed on a sterilized foam board. A small incision was carefully made in the mouse's abdominal skin to avoid damaging the peritoneum. After the incision, the skin was pulled towards both sides to fully expose the peritoneum. A 5 mL injection of HAT medium (containing 2% HAT, 20% FBS, and 1% ampicillin and streptomycin double-antibiotic RPMI-1640) containing 20% serum was injected into the mouse's abdominal cavity, and the abdomen was massaged to mix the medium with the contents of the abdominal cavity. The peritoneum was lifted with tweezers, and the mouse's abdominal cavity fluid was extracted and placed in a 50 mL centrifuge tube. This step was repeated three times, and the cell suspension was plated in a 96-well culture plate and cultured in a 37°C, 5% CO2 incubator.

[0058] The mice with high serum titer were taken blood from eyeball, and executed by breaking the neck after disinfecting with 75% anhydrous alcohol for 10 minutes. Then the mice were fixed on the disinfected fixing plate, and the spleen was taken out and grinded on the cell screen with the push rod of the syringe. After the spleen was grinded completely, the cells on the cell screen were washed down carefully, and the prepared spleen cell suspension was centrifuged at 1000 r / min for 10 minutes. The supernatant was discarded, and the spleen cells were resuspended and counted.

[0059] Fusion process: the spleen cell suspension and the SP2 / 0 myeloma cells in logarithmic growth phase were mixed uniformly at a ratio of 5:1, centrifuged at 1000 r / min for 10 minutes, and the supernatant was discarded (the supernatant must be discarded to avoid affecting the PEG concentration and causing low cell fusion efficiency). The centrifuge tube was repeatedly scraped several times on the edge of the clean bench to keep the centrifuged cells loose and improve the contact efficiency with PEG. After the cells were loosened, 1 mL of preheated 50% PEG was slowly added to the mixed cells in a 37°C water bath, and the process was continuously stirred for 30 seconds and then static for 30 seconds. After complete addition, preheated RPMI-1640 base medium was added for termination, and the cells were centrifuged at 1000 r / min for 10 minutes. The cells were suspended in HAT medium, and then uniformly plated in the feeder layer cells at about 200 μL / well, and placed in the cell culture box for culture.

[0060] 4. Screening of hybridoma cells

[0061] The ELISA plate was coated with the optimized antigen concentration (6.8 μg / mL), and the hybridoma cells were screened by indirect ELISA method when the hybridoma cells grew into single clones and the culture solution turned slightly yellow. The hybridoma cells with positive antibody detection were expanded in culture, and the other part was subcloned (limiting dilution method) to prevent loss of hybridoma cell lines due to cell contamination.

[0062] After 3-4 times of subcloning, 8 positive hybridoma cell lines were obtained, which were named 3D7, 1B3, 6C3, 5D4, 1D3, 3A9, 1D9 and 1H2, respectively.

[0063] 5. Identification of hybridoma cell antibody subtype

[0064] According to the immunoglobulin typing detection kit, the CPV antigen was coated on the enzyme-labeled plate, and the hybridoma cell culture supernatant was used as the primary antibody, and different types of goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM and IgA were used as the secondary antibody for indirect ELISA experiment, and the subclasses of the obtained 8 monoclonal antibodies were determined. The results are shown in Table 2. It can be seen that 5 of the 8 hybridomas are IgG subtype, and 3 are IgM subtype. The heavy chains of 3D7, 6C3 and 5D4 are IgG2a subtype, the heavy chains of 1B3 and 1D3 are IgG1 subtype, and the heavy chains of 3A9, 1D9 and 1H2 are IgM subtype; among them, the light chains of 3D7, 1D3, 3A9, 1D9 and 1H2 are κ chains, and the light chains of 1B3, 6C3 and 5D4 are λ chains.

[0065] Table 2: Subtype identification of 8 hybridoma cell strains

[0066]

[0067] 6. Hybridoma supernatant titer and ascites titer determination

[0068] Preparation of hybridoma ascites: 8-week-old female Balb / c mice were selected and injected with 500 μL of Freund's incomplete adjuvant in the abdominal cavity. After 1 week, the well-grown hybridoma cells were collected and resuspended in RPMI-1640 medium. The cell counting plate was used for counting, and each mouse was injected with 10 6 hybridoma cells in the abdominal cavity. Continue to feed for 1 week, observe the state of the mouse abdomen, and when the ascites is produced, the ascites can be sucked with a syringe, and stored at -20℃ after sub-packaging.

[0069] The cell supernatant of 8 hybridomas was diluted by 2 times, and the ascites was diluted by 10 times, which was used as the primary antibody. The indirect ELISA method was used to determine the titer of the ascites and the cell culture supernatant of the monoclonal antibodies.

[0070] Through the indirect ELISA method, the dilution multiples of the hybridoma cell culture supernatant were 1:2, 1:2 2 , 1:2 3 , 1:2 4 , 1:2 5 , 1:2 6 , 1:2 7 , 1:2 8 , 1:2 9 , 1:2 10 , 1:2 11 , 1:2 12 , and the dilution multiples of the hybridoma cell mouse ascites were 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:105 1:10 6 1:10 7 1:10 8 1:10 9 1:10 10 Potency was determined by dilution. The results are shown in Table 3 and... Figure 3 As shown, the titers of the supernatant from the 8 hybridoma strains ranged from 2. 4 ~2 12 The potency of ascites ranges from 10. 3 ~10 8 .

[0071] Table 3 Antibody titers in hybridoma cell culture supernatant and ascites fluid

[0072]

[0073] 7. Indirect immunofluorescence identification of monoclonal antibodies

[0074] CPV-2, CPV-2a, CPV-2b, and CPV-2c were seeded into well-grown monolayers of F81 cells and cultured at 37°C for 72 h. The culture supernatant was discarded, and the cells were fixed with methanol for 15 min. The cells were then carefully and slowly washed three times with PBS for 5 min each time (taking care not to wash away the cells). The supernatant of the hybridoma cells to be tested was added, and the cells were incubated at 37°C for 1 h. Simultaneously, the supernatant of SP2 / 0 cells was used as a negative control well; after washing, goat anti-mouse FITC-IgG and FITC-IgM (1:3000) were added, and the cells were incubated at 37°C in the dark for 1 h. The experimental results were observed under an inverted fluorescence microscope and photographed. The results are as follows: Figure 4 As shown, 3D7, 6C3, 1D3, 3A9, 1D9, and 1H2 can specifically bind to CPV-2, CPV-2a, CPV-2b, and CPV-2c, producing fluorescence. Figure 4 A, C, E, F, G, and H), 1B3, and 5D4 showed no specific fluorescent reaction. Figure 4 (B and D), and SP2 / 0 supernatant control and cell control showed no fluorescence.

[0075] 8. Identification of the neutralizing activity of monoclonal antibodies

[0076] Dilute the virus concentration to 100 TCID 50 Ascites fluid serially diluted with 8 monoclonal antibodies (1:2, 1:2) 2 1:2 3 1:2 4 1:2 5 1:2 6) mixed with equal volume, incubated at 37℃, 5% CO2 for 1h, then inoculated with F81 cells, 100 μL / well, 4 parallel repeats, at the same time, set up cells with medium only and cells with virus only as negative and positive controls, cultured for 4d, observed CPE of cells, and detected neutralization activity of MAb. It was verified that only 3D7 secreted antibody had neutralization activity. The concentration of 3D7 completely neutralizing CPV-2, CPV-2a, CPV-2b and CPV-2c virus was 0.39 μg / mL, 0.69 μg / mL, 0.70 μg / mL and 0.69 μg / mL respectively.

[0077] 9. Ascites purification of monoclonal antibody and effect test

[0078] The ascites of the 5 IgG subtype monoclonal antibodies screened were purified by Protein A+G affinity chromatography, and the protein concentration of the obtained antibody after purification was detected by spectrophotometer or BCA kit, and stored at -20℃.

[0079] 20 μL of the purified antibody was taken and SDS-PAGE was performed to verify the purification effect. The results are shown in Figure 5 After purification and concentration, the 5 monoclonal antibodies were identified by SDS-PAGE, and the 50 kD band was the heavy chain of the monoclonal antibody, and the 20-25 kD band was the light chain of the monoclonal antibody, without obvious impurity band, with good purity.

[0080] 10. Analysis of virus proteins recognized by MAb

[0081] The 5 IgG monoclonal antibodies 1D3, 6C3, 5D4, 1B3 and 3D7 were verified by Western blot to identify the virus proteins. The results are shown in Figure 6 It can be seen that 3D7, 6C3, 5D4, 1B3 and 1D3 appeared specific bands at 70KD, which recognized the VP2 protein of CPV virus.

[0082] Example 2 Preliminary establishment of colloidal gold immunochromatographic assay for canine parvovirus

[0083] 1. Determination of minimum labeling amount and optimum pH of colloidal gold detection antibody

[0084] The 5 IgG subtype monoclonal antibodies 1D3, 6C3, 5D4, 1B3 and 3D7 prepared in Example 1 and the commercial CPV monoclonal antibody 2A10 (Parvovirus, Canine, antibody 3PV16-2A10) purchased from HyTest were respectively subjected to gold tag antibody square array titration. The pH of the colloidal gold solution was adjusted by adding 0, 5, 10, 15 and 20 μL of 0.2 mol / L K2CO3 solution respectively, the antibody was pre-diluted to 0.5 mg / mL, and was subjected to 1:2, 1:2 2 , 1:2 3 , 1:2 4 , 1:2 5 , 1:2 6 , 1:2 7 , 1:2 fold dilution, and was added to the colloidal gold solution. After mixing well, the OD 524nm value was determined. The results are shown in Table 4. The minimum stable amount of antibody labeling of 3D7 was 0.125 mg / mL, and the minimum stable amount of antibody labeling of 6C3, 5D4, 1B3, 1D3 and 2A10 was 0.065 mg / mL. During the experiment, 20% more than the minimum stable amount could be added. The amount of K2CO3 solution added for 6C3 and 1D3 was 5 μL, for 1B3 was 10 μL, and for 5D4, 3D7 and 2A10 was 15 μL.

[0085] Table 4 Determination of minimum stable amount and optimum pH of MAb labeling

[0086]

[0087] The gold tag antibody was prepared according to the minimum stable amount and optimum pH of the 6 monoclonal colloidal gold labeled antibodies. In 6 15 mL centrifuge tubes, 2 mL of colloidal gold solution was added, and was shaken well with a rotary instrument for 10 min. The pH was adjusted by adding a corresponding volume of 0.2 mol / L K2CO3 solution according to the optimum pH of each monoclonal antibody, and was shaken well with a rotary instrument for 10 min. The 6 monoclonal antibodies were added to the 6 centrifuge tubes according to the optimized conditions of colloidal gold labeled antibody, and were mixed well with a rotary instrument for 10 min, and were then placed at room temperature for 30 min to allow the colloidal gold particles to fully combine with the antibodies. 10% PEG-6000 solution was added to the 6 centrifuge tubes to a final concentration of 1%, and was mixed well with a rotary instrument for 10 min, and was then placed at room temperature for 30 min for blocking treatment. The colloidal gold solution after blocking was placed in a high-speed refrigerated centrifuge, and was centrifuged at 8000 r / min at 4°C for 20 min. The precipitate was resuspended by carefully pipetting the supernatant and discarding it, and adding 200 μL of gold resuspension solution to each tube. After the precipitate was fully resuspended by gently blowing, 10x gold tag antibody was obtained, and was stored at 4°C.

[0088] 2. Screening of optimum gold tag antibody and optimum capture antibody

[0089] In order to determine the best gold-labeled antibody and the best capture antibody, the above-mentioned 6 strains of CPV monoclonal antibodies (6C3, 5D4, 1B3, 3D7, 1D3 and 2A10) were arranged in combination with the above-mentioned 6 strains of gold-labeled antibodies (Au-6C3, Au-5D4, Au-1B3, Au-3D7, Au-1D3 and Au-2A10), paired two by two, and prepared into test strips, a total of 36 combinations. A high-concentration CPV-2 virus sample with a virus titer of 10 6 TCID 50 / mL was used for preliminary screening, and the T-line signal value of the test strip was read by a colloidal gold immunometric analyzer and recorded. The results are shown in Table 5 and Table 6. Figure 7 From the experimental results, it was preliminarily determined that the best capture antibody was one of 3D7, 2A10, 5D4, 6C3 and 1D3, and the best gold-labeled antibody was one of 2A10, 3D7 and 5D4.

[0090] Table 5 Preliminary screening of antibody cross-pairing

[0091]

[0092] Then, a low-concentration CPV-2, CPV-2a, CPV-2b and CPV-2c virus sample with a virus titer of 10 4 TCID50 / mL was used for further screening, with a negative control, to observe the color development of the T-line of the test strip, and record the T value read by the colloidal gold immunometric analyzer. The results are shown in Table 6. Considering the broad spectrum, sensitivity and specificity of the test strip, it can be seen from the experimental results that when 3D7 is used as the capture antibody and 2A10 is used as the gold-labeled antibody, the test strip for detecting different strains has the best color development effect and the highest T-line signal value, and has good specificity. Therefore, it is finally determined that 3D7 is used as the capture antibody and 2A10 is used as the gold-labeled antibody.

[0093] Table 6 Further screening of antibody cross-pairing

[0094]

[0095] 3. Determination of the best gold spraying amount

[0096] A test strip was prepared, in which the detection line was coated with 3D7 antibody at a concentration of 2.0 mg / mL, and the quality control line was coated with goat anti-mouse antibody at a concentration of 4.0 mg / mL. Gold spraying was performed to prepare a gold-labeled pad by setting 1 μL / cm, 2 μL / cm, 3 μL / cm, 4 μL / cm, 5 μL / cm and 6 μL / cm of 2A10 gold-labeled antibody gold spraying amount, respectively. The same negative sample and positive sample were detected by using the prepared test strip, repeated 3 times, and the colloidal gold signal intensity of T line and C line was detected by using an immunization analyzer. The gold-labeled probe addition amount at the highest signal-to-noise ratio was selected as the best addition volume. According to multiple determinations, when the T value read by the colloidal gold immunization analyzer was less than 90, there was no visible band on the test strip, so 90 was set as the critical value for result determination.

[0097] The detection results are shown in Table 1. Figure 8 As shown in Table 1, when the gold-labeled antibody 2A10 gold spraying amount was 5 μL / cm, the color development effect of the T line was the best, and the signal value of the T line was the highest, and the T value of the negative control sample was less than 90. In summary, the best gold spraying amount of the gold-labeled antibody 2A10 was 5 μL / cm.

[0098] 4. Determination of the optimal T line antibody concentration

[0099] The quality control line was coated with goat anti-mouse antibody at a concentration of 4.0 mg / mL, and the gold-labeled antibody 2A10 was gold sprayed at a gold spraying amount of 5 μL / cm. The purified 3D7 antibody at a concentration gradient of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mg / mL was used as the T line antibody for scribing. The same positive sample was detected by using test strips with different T line coating concentrations, repeated 3 times, and the colloidal gold signal intensity of the T line was detected by using an immunization analyzer. The minimum 3D7 antibody coating concentration required for the T line signal to reach stability was the optimal detection line coating antibody amount.

[0100] The detection results are shown in Table 2. Figure 9 As shown in Table 2, the signal value of the T line increased with the increase of the C line antibody concentration. When the T line antibody concentration was 2.5 mg / mL, the signal value of the T line was the highest, and the T value of the negative control sample was less than 90. In summary, the optimal T line antibody concentration was 2.5 mg / mL.

[0101] 5. Determination of the optimal C line antibody concentration

[0102] The detection line was coated with 2.5 mg / mL 3D7 antibody, and the gold-sprayed amount of gold-labeled antibody 2A10 was 5 μL / cm. Goat anti-mouse antibody concentrations of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 mg / mL were used as the C-line antibody concentrations for streaking. The same negative sample was tested using test strips with different C-line coating concentrations, repeated three times. The colloidal gold signal intensity of the C-line was measured using an immunoassay analyzer. The minimum goat anti-mouse antibody coating concentration required for the C-line signal to stabilize is the optimal antibody amount for the control line.

[0103] Test results as follows Figure 10 As shown, by Figure 11 It can be seen that the signal value of the C line increases with the increase of the C line antibody concentration. When the C line antibody concentration is 4.5 mg / mL, the C line signal value is the largest. Therefore, the optimal C line antibody concentration is 4.5 mg / mL.

[0104] 6. Test strip specificity, broad spectrum and sensitivity.

[0105] (1) Assembly and preparation of test strips

[0106] For the test strip streaking, the capture antibody 3D7 and goat anti-mouse antibody were diluted to the optimal concentration determined above. After sample loading and cleaning, the BIODOT streaking machine was used. The machine parameters were modified, the Y-axis position was set to 4.0 mm, and the streaking volume was 1 μL per centimeter. After streaking, the strips were dried in an oven at 37°C for 4 hours. The gold-labeled antibody conjugate pads were treated (the gold-spraying volume of the gold-labeled antibody 2A10 was fixed at 5 μL / cm). The conjugate pad treatment solution was prepared, and the conjugate pads were soaked in the treatment solution and dried overnight in an oven at 37°C. The sample pads were treated by preparing the sample pad treatment solution, soaking the sample pads in the solution, and drying overnight in an oven at 37°C. Assemble the test strips by cutting the sample pad, conjugate pad, NC membrane (CN95 nitrocellulose membrane), and absorbent paper to appropriate widths and pasting them onto the PVC base plate in sequence, overlapping each component by about 2mm to ensure sample flow. Cut the test strips using a strip cutter, setting the strip width to 4.0mm in the system parameters. Assemble the test strips by placing the cut strips in the plastic sleeves in the correct orientation for the next step of the experiment.

[0107] (2) Specific detection

[0108] To test the specificity of the colloidal gold test strip in recognizing antigens, the test strip was tested with canine coronavirus (CCoV), canine distemper virus (CDV), canine parainfluenza virus (CPIV), canine adenovirus (CAV), and F81 cell supernatant, with positive and negative controls included. 100 μL of sample was added to the sample compartment of the test strip card, and the colorimetric results were observed after standing at room temperature for 15 minutes.

[0109] The specific detection results are shown in Figure 11 As shown in the figure, the test strip has obvious specific bands only when detecting CPV positive control, and has no bands when detecting other common viruses of dogs, F81 cell supernatant and negative control. In summary, the specificity of the test strip is good.

[0110] (3) Broad spectrum and sensitivity of the test strip

[0111] The broad spectrum of the CPV colloidal gold test strip was tested by using CPV-2, CPV-2a, CPV-2b and CPV-2c four subtypes of CPV strains. Each subtype of virus was diluted by two-fold ratio, the reaction of the test strip to each subtype of CPV virus was observed, and the detection limit of the four strains was determined.

[0112] The sensitivity of the test strip to CPV-2 is shown in A of Figure 12 As shown in the figure, the minimum detection amount of the test strip for CPV-2 is 7.8 x 10 3 TCID 50 / mL (1:1280); the sensitivity of the test strip to CPV-2a is shown in B of Figure 12 As shown in the figure, the minimum detection amount of the test strip for CPV-2a is 6.25 x 10 3 TCID 50 / mL (1:160); the sensitivity of the test strip to CPV-2b is shown in C of Figure 12 As shown in the figure, the minimum detection amount of the test strip for CPV-2b is 7.8 x 10 3 TCID 50 / mL (1:1280); the sensitivity of the test strip to CPV-2c is shown in D of Figure 12 As shown in the figure, the minimum detection amount of the test strip for CPV-2c is 7.8 x 10 3 TCID 50 / mL (1:1280).

[0113] 7. Intra-group and inter-group repeatability of the test strip

[0114] Multiple batches of colloidal gold test strips were prepared, and two batches of test strips were randomly selected. The same virus sample diluted by 1:100, 1:200 and 1:400 was detected, and the detection was repeated for three times. The T / C value was recorded. The intra-group and inter-group variation coefficients of the two batches of test strips were calculated. When the sample intra-group and inter-group variation coefficients (CV) were less than 15%, the precision of the test strip was high. The variation coefficient (CV) = standard deviation (SD) / average value.

[0115] The results of the intra-group repeatability tests are shown in Table 7. As can be seen from Table 7, the intra-group coefficient of variation for the self-made colloidal gold test strip for high-concentration samples was 9.63%, for medium-concentration samples it was 5.26%, and for low-concentration samples it was 5.98%.

[0116] The results of the inter-group repeatability test are shown in Table 8. As can be seen from the table, the inter-group coefficient of variation for the self-made colloidal gold test strip for high-concentration samples was 8.59%, for medium-concentration samples it was 9.11%, and for low-concentration samples it was 8.33%.

[0117] In summary, the coefficient of variation within groups of this test strip is less than 10%, and the coefficient of variation between groups is less than 15%, indicating good repeatability within and between groups.

[0118] Table 7 Results of Intra-group Repeatability Tests

[0119]

[0120] Table 8 Results of intergroup repeatability tests

[0121]

[0122] 8. Test strip stability test

[0123] To test the stability of the self-made colloidal gold test strips, stability tests were conducted at 37°C. The test strips were removed on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30, and tested with 100-fold diluted viral samples. Each test was repeated three times. The T / C signal intensity was measured using an immunoassay analyzer to analyze the test strip stability.

[0124] The stability results of the test strip are as follows Figure 13 As shown, by Figure 13 It is evident that the T / C values ​​of the test strips stored for 30 days showed no significant difference when testing the same sample. Therefore, the self-made colloidal gold test strips exhibit good stability.

[0125] 9. Comparison of homemade test strips and commercially available test strips

[0126] A self-made colloidal gold test strip and a commercially available canine parvovirus colloidal gold test strip (Korean Anje CPV antigen detection colloidal gold test strip) were used to detect CPV-2, CPV-2a, CPV-2b, and CPV-2c viruses that had been serially diluted 10 times (each virus was pre-diluted 10 times). The lowest detection limits of the self-made colloidal gold test strip and the imported brand colloidal gold test strip for CPV-2, CPV-2a, CPV-2b, and CPV-2c strains were compared.

[0127] The minimum detection limits for CPV-2 for the two test strips are as follows: Figure 14 As shown in Figure A, the lowest detectable amount of the homemade test strip for detecting CPV-2 is 7.8 × 10⁻⁶. 3 TCID 50 / mL (1:1280), the lowest detection limit of commercially available test strips is 1.56×10⁻⁶. 4 TCID 50 / mL (1:640); the minimum detectable dose results for CPV-2a for the two test strips are as follows: Figure 14 As shown in B, the lowest detectable amount of the homemade test strip when detecting CPV-2a is 6.25 × 10⁻⁶. 3 TCID 50 / mL (1:160), the lowest detection limit of commercially available test strips is 1.25×10⁻⁶. 4 TCID 50 / mL (1:80); the minimum detectable levels for CPV-2b for the two test strips are as follows: Figure 14 As shown in C, the lowest detectable amount of the homemade test strip when detecting CPV-2b is 7.8 × 10⁻⁶. 3 TCID 50 / mL (1:1280), the lowest detection limit of commercially available test strips is 3.12×10⁻⁶. 4 TCID 50 / mL (1:320); the minimum detectable levels for CPV-2c for the two test strips are as follows: Figure 14 As shown in D, the lowest detectable amount of the homemade test strip when detecting CPV-2c is 7.8 × 10⁻⁶. 3 TCID 50 / mL (1:1280), the lowest detection limit of commercially available test strips is 7.8 × 10⁹ / mL. 3 TCID 50 / mL (1:1280). Therefore, the sensitivity of the self-made colloidal gold test strip for detecting CPV-2 and CPV-2a strains is twice that of the commercial test strip, the sensitivity of the self-made colloidal gold test strip for detecting CPV-2b strain is four times that of the commercial test strip, and the sensitivity of the self-made colloidal gold test strip for detecting CPV-2c strain is consistent with that of the commercial test strip.

[0128] In summary, the homemade colloidal gold test strips outperform commercially available colloidal gold test strips in terms of minimum detection limits.

[0129] 10. Clinical sample testing

[0130] To verify the coincidence rate of the test strip and PCR, 70 suspected canine parvovirus samples were detected by the self-made test strip and PCR respectively. The PCR detection results are shown in Table 9. PCR detected 49 positive samples and 21 negative samples. The coincidence rate results are shown in Table 9. The positive coincidence rate of the test strip and PCR was 91.8% (45 / 49), the negative coincidence rate was 100% (21 / 21), and the total coincidence rate was 94.3%. Therefore, the coincidence rate of the self-made test strip and PCR is good.

[0131] Table 9 Coincidence rate of the self-made colloidal gold test strip and PCR

[0132]

[0133] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A hybridoma cell line 3D7 producing a monoclonal antibody against canine parvovirus, characterized in that, The hybridoma cell strain 3D7 is preserved in China Center for Type Culture Collection on October 18, 2024, and the preservation number is CCTCC NO: C2024216.

2. The use of the hybridoma cell strain 3D7 of claim 1 in the preparation of a canine parvovirus monoclonal antibody.

3. A canine parvovirus monoclonal antibody, characterized in that, The hybridoma cell strain 3D7 of claim 1 is produced.

4. The use of the canine parvovirus monoclonal antibody of claim 3 in the preparation of a colloidal gold detection kit for canine parvovirus disease.

5. A colloidal gold detection test strip for canine parvovirus disease, characterized in that, The test strip is composed of the following components: sample pad, gold-labeled antibody binding pad, absorbent paper, and nitrocellulose membrane; the nitrocellulose membrane has a quality control line and a detection line, the detection line is coated with the canine parvovirus monoclonal antibody of claim 3, and the quality control line is coated with goat anti-mouse antibody.

6. The colloidal gold detection test strip for canine parvovirus disease according to claim 5, characterized in that, The gold-labeled antibody binding pad is coated with colloidal gold-labeled Parvovirus, Canine, antibody 3PV16-2A10.

7. The colloidal gold detection test strip for canine parvovirus disease according to claim 5, characterized in that, The coating concentration of the goat anti-mouse antibody is 4.5 mg / mL.

8. The colloidal gold detection test strip for canine parvovirus disease according to claim 5, characterized in that, The coating concentration of the canine parvovirus monoclonal antibody is 2.5 mg / mL.

9. The colloidal gold detection test strip for canine parvovirus disease according to claim 6, characterized in that, The gold-labeled Parvovirus, Canine, antibody 3PV16-2A10 is sprayed at a volume of 5 μL / cm. The gold-labeled Parvovirus, Canine, antibody 3PV16-2A10 is sprayed at a volume of 5 μL / cm.

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