Hybridoma cell strain 3D7 capable of generating canine parvovirus monoclonal antibody and application of hybridoma cell strain 3D7

Abstract

The invention discloses a hybridoma cell strain 3D7 capable of generating a canine parvovirus monoclonal antibody and application of the hybridoma cell strain 3D7, and belongs to the technical field of biological detection. The preservation number of the hybridoma cell strain 3D7 is CCTCC (China Center For Type Culture Collection) NO: C2024216. The monoclonal antibody generated by the hybridoma cell strain 3D7 provided by the invention is taken as a capture antibody to prepare a colloidal gold test strip for the canine parvovirus disease, so that the canine parvovirus disease can be accurately detected, the specificity is good, the sensitivity is high, various subtype CPV strains of CPV-2, CPV-2a, CPV-2b and CPV-2c can be identified, the broad spectrum is good, and the test strip can be used for detecting the canine parvovirus disease. The lowest detection quantity is superior to that of a commercialized colloidal gold test strip sold in the market. The test strip has a high coincidence rate with PCR. The invention provides a technical means for rapid on-site diagnosis of CPV.

Description

Technical Field

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

[0002] Canine parvovirus disease (CPVD) is an acute and highly contagious disease widely spread in dogs caused by canine parvovirus (CPV). CPV has a wide range of hosts and can infect almost all mammals, including raccoons, cats, and wolves. It can be directly transmitted through the fecal-oral route or indirectly transmitted through parasites contaminated by feces. It recognizes mesenteric lymph nodes and thymic lymphoid tissues as target tissues for viral replication and spreads to the intestinal crypts of the small intestine through the blood. However, the spread of the virus is not limited to the intestines, but also includes the lungs, spleen, liver, heart, bone marrow, kidneys, and brain. It is a systemic disease. The virus completes mutation by changing the amino acids of the VP2 protein that binds to the transferrin receptor of host cells. So far, the antigenic subtypes of CPV circulating globally include CPV-2, CPV-2a, CPV-2b, CPV-2c, new CPV-2a, and new CPV-2b.

[0003] CPVD develops rapidly, spreads quickly, has a high mortality rate, and sick dogs die quickly after onset. Rapid diagnosis is very important for the prevention and control of CPVD. At present, many 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, they are time-consuming and require professional instruments. They can only be tested in the laboratory by professionals and are not suitable for on-site rapid diagnosis of canine parvovirus disease.

[0004] Colloidal gold immunochromatographic test strips are fast, simple, inexpensive, and intuitive, and do not require additional equipment. Currently, colloidal gold immunochromatographic test strips for rapid detection of canine parvovirus are available on the market. However, the test strips on the market have problems such as incomplete coverage of strains, low sensitivity, and low consistency with PCR test results.

[0005] Due to the long-term evolution and mutation of CPV, different subtypes of CPV-2, CPV-2a, CPV-2b and CPV-2c have appeared in China. However, there are no effective test strips in China that can broadly identify the above-mentioned multiple subtypes. Therefore, based on the current domestic epidemic trend of CPV, it is of great significance to screen monoclonal antibodies that can effectively identify multiple subtypes such as CPV-2, CPV-2a, CPV-2b and CPV-2c, and then broadly identify the four strains of CPV, quickly diagnose animals suspected of CPV infection, and break the technical monopoly of imported test strips. Summary of the invention

[0006] The purpose of the present invention is to provide a hybridoma cell line 3D7 that produces canine parvovirus monoclonal antibodies and its application to solve the problems existing in the above-mentioned prior art. The canine parvovirus disease colloidal gold test strip is prepared using the monoclonal antibody produced by the hybridoma cell line 3D7 provided by the present invention as the capture antibody, which can accurately detect canine parvovirus, has good specificity and high sensitivity, and can identify CPV strains of multiple subtypes such as CPV-2, CPV-2a, CPV-2b and CPV-2c, has good broad spectrum, and is superior to commercial colloidal gold test strips on the market in terms of the minimum detection amount.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a hybridoma cell line 3D7 that produces canine parvovirus monoclonal antibodies. The hybridoma cell line 3D7 was deposited in the China Center for Type Culture Collection on October 18, 2024, with a deposit number of CCTCC NO: C2024216.

[0009] The present invention also provides the use of the hybridoma cell line 3D7 in preparing canine parvovirus monoclonal antibodies.

[0010] The invention also provides a canine parvovirus monoclonal antibody produced by the hybridoma cell line 3D7.

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

[0012] The present invention also provides a colloidal gold test strip for canine parvovirus disease, which consists of the following components: a sample pad, a gold-labeled antibody binding pad, absorbent paper and a nitrocellulose membrane; the nitrocellulose membrane is provided with 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 sheep anti-mouse antibody.

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

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

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

[0016] Furthermore, the gold spraying volume of the colloidal gold-labeled antibody 2A10 is 5 μL / cm.

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

[0018] The present invention uses hybridoma cell technology to screen 8 hybridoma cell strains that can secrete anti-CPV monoclonal antibodies. The antibody subclass identification results show that 3D7, 1B3, 6C3, 5D4 and 1D3 are IgG subtypes. The obtained 5 IgG subtype monoclonal antibodies are used with the commercial antibody 2A10 for antibody cross-matching test, and 3D7 is screened as the best capture antibody and 2A10 is the best gold label antibody. The colloidal gold immunochromatographic test strip prepared in this way can accurately detect canine parvovirus, has good specificity and high sensitivity, and can identify CPV strains of multiple subtypes of CPV-2, CPV-2a, CPV-2b and CPV-2c, has good broad spectrum, and is superior to commercial colloidal gold test strips on the market in terms of the minimum detection amount. The test strips and PCR are used to detect 70 clinical samples respectively. The results showed that the positive coincidence rate between the two was 91.8% (45 / 49), the negative coincidence rate was 100% (21 / 21), and the total coincidence rate was 94.3%, which was higher than the PCR coincidence rate. The present invention provides a technical means for rapid on-site diagnosis of CPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Sucrose density gradient centrifugation SDS-PAGE analysis (A) and Western blot detection for CPV virus to verify immunogenicity (B);

[0021] Figure 2 Serum titers were measured by indirect ELISA after immunization of 6 mice with CPV;

[0022] Figure 3 The antibody titers in hybridoma cell culture supernatant (A) and ascites fluid (B);

[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 This is the Western blot identification result of anti-CPV monoclonal antibody;

[0026] Figure 7 Preliminary screening results for antibody cross-matching;

[0027] Figure 8 This is the optimization result of the best gold spraying amount;

[0028] Fig. 9 This is the optimization result of the best coating amount of 3D7 capture antibody T line;

[0029] Fig.10 The results of optimizing the best coating amount for sheep anti-mouse antibody C line;

[0030] Fig.11 It is the test strip specific test result;

[0031] Fig.12 The sensitivity test results of the test strips to different strains of CPV are shown in Figure 1. A is the sensitivity test result of the test strips to CPV-2; B is the sensitivity test result of the test strips to CPV-2a; C is the sensitivity test result of the test strips to CPV-2b; D is the sensitivity test result of the test strips to CPV-2c;

[0032] Fig.13 This is the test strip stability test result;

[0033] Fig.14 Comparison of the minimum detection amounts of CPV-2 (A), CPV-2a (B), CPV-2b (C) and CPV-2c (D) detected by homemade colloidal gold test strips and commercial test strips. The first row of each figure is the homemade test strip, and the second row is the commercial test strip.

[0034] Biological Deposit

[0035] Hybridoma cell line 3D7.

[0036] Deposit number: CCTCC NO: C2024216;

[0037] Deposit date: October 18, 2024;

[0038] Depository: China Center for Type Culture Collection;

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

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The CPV virus used as an antigen in the embodiments of the present invention, different subtypes of CPV-2, CPV-2a, CPV-2b, CPV-2c viruses, and canine coronavirus (CCV), canine distemper virus (CDV), canine parainfluenza virus (CPIV) and canine adenovirus (CAV) used for specific detection are all preserved by the National Key Laboratory of Agricultural Microbiological Resources Exploration and Utilization of Huazhong Agricultural University, and are promised to be distributed to the public within 20 years from the date of application. Canine nasopharyngeal swabs were provided by the Experimental Dog Base of Wuhan Keqian Biological Co., Ltd. in Hubei Province, with a total of 70 clinical samples. PCR experiments were performed to detect CPV according to the primer sequences provided in standard number GB / T 34746-2017, of which 49 were positive and 21 were negative. The primers were synthesized by Wuhan Qingke Biological Co., Ltd. Other experimental materials, unless otherwise specified, can be obtained through conventional purchase.

[0046] Example 1 Preparation of CPV monoclonal antibody

[0047] 1. Preparation and purification of CPV antigen

[0048] CPV was inoculated into a monolayer of F81 cells in good growth state for amplification culture. After culturing for 3-4 days, when the cytopathic effect (CPE) reached 80%, the virus was collected by repeated freezing and thawing three times. The collected virus liquid was placed in a centrifuge tube by ultracentrifugation, and the centrifuge tube was placed in a sleeve, and centrifuged at 28000rpm and 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. 2 mL of sucrose solution with mass concentration of 20%, 30%, 40%, 50% and 60% was added to the ultracentrifuge tube in sequence. The ultracentrifuge concentrated virus solution was added to the top layer. The centrifuge tube was placed in a casing and centrifuged at 28000 rpm and 4°C for 2 h. After the centrifugation, the virus was distributed in each sucrose concentration layer. The virus solution distributed in each layer was sucked out and added to an ultracentrifuge tube, which was balanced with PBS solution and ultracentrifuged again for sugar removal. After the centrifugation, the supernatant was discarded and the lower precipitate was dissolved with a small amount of PBS.

[0050] The virus solution of each sucrose layer was verified by SDS-PAGE to check the distribution of virus protein in each sucrose layer. The results are as follows: Figure 1 A shows that the viral protein mainly accumulates in the 20-30% sucrose layer, and the virus purification effect is good. Then Western blot is performed to verify the recognition of viral proteins in each layer and verify its immunogenicity. The results are as follows Figure 1 B shows that the main immunogenic protein VP2 exists in the purified CPV antigen, and the virus layer with the most viral protein and the VP2 protein that recognizes the virus is selected as the immunogen for immunizing mice.

[0051] 2. Animal immunization

[0052] Six healthy BALB / c mice aged 6-8 weeks were immunized with purified and concentrated CPV virus solution. The first immunization was an equal mixture of CPV and Freund's complete adjuvant, and 0.2 mL / mouse was injected intraperitoneally. The second and third immunizations were emulsified with CPV and Freund's incomplete adjuvant, with the same dose as the first immunization, and the interval was two weeks. Eye blood was collected in the sixth week after the third CPV immunization, and the mouse serum was diluted at 1:10 and 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 The titer was determined by indirect ELISA. The results are shown in Table 1 and Figure 2 As shown, the serum titers of the six mice after immunization were all greater than 10 5 , the antibody levels in vivo have reached the fusion standard, and the mice can be boosted with immunization before cell fusion.

[0053] Table 16 Determination of cell serum titer of mice

[0054]

[0055] The mice with higher indirect ELISA detection values ​​were boosted with intraperitoneal injection of CPV virus solution without adjuvant at a dose of 0.2 mL / mouse, and cell fusion was performed 3 days after the boosted immunization.

[0056] 3. Cell fusion

[0057] Two days before cell fusion, non-immunized mice were taken to prepare feeder layer cells. The mice were killed by eye removal and blood collection, and negative serum was collected. The mice were soaked in 75% anhydrous ethanol for disinfection for 10 minutes, and then their limbs were fixed on a disinfected foam board. A small incision was carefully made in the skin of the mouse abdomen to avoid damaging the peritoneum. After cutting, the skin was pulled in both directions to fully expose the peritoneum. 5mL of HAT culture medium containing 20% ​​serum (RPMI-1640 containing 2% HAT, 20% FBS and 1% penicillin-streptomycin double antibody) was drawn with a syringe and injected into the mouse peritoneum. The abdomen was massaged to fully mix the culture medium with the abdominal contents; the peritoneum was lifted with tweezers, and the fluid in the mouse peritoneal cavity was extracted and placed in a 50mL centrifuge tube. This step was repeated three times, and the cell suspension was spread flat in a 96-well culture plate and cultured in a 37°C, 5% CO2 incubator.

[0058] The mice with high serum titers detected by indirect ELISA were blooded from their eyes, killed by cervical dislocation, and disinfected with 75% anhydrous ethanol for 10 minutes. The mice were then fixed on a disinfected fixing plate, the spleen was removed, and the spleen was placed on a cell sieve and ground with a syringe plunger. After the spleen was ground clean, RPMI-1640 basal culture medium was aspirated to carefully rinse the cells on the cell sieve, and the prepared spleen cell suspension was centrifuged at 1000r / min for 10 minutes, the supernatant was discarded, and the spleen cells were resuspended for counting.

[0059] Fusion process: spleen cell suspension and SP2 / 0 myeloma cells in logarithmic growth phase were mixed in a ratio of 5:1, centrifuged at 1000r / min for 10min, 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 placed on the edge of the clean bench and scraped several times to keep the centrifuged cells loose and improve the contact efficiency with PEG. After the cells were loose, 1mL of preheated 50% PEG was slowly added to the mixed cells in a 37℃ water bath. Stir continuously during the addition process, stir continuously for 30s, and stand still for 30s until it is completely added. Then add preheated RPMI-1640 basal medium to terminate, and centrifuge at 1000r / min for 10min. The cells were suspended with HAT medium, and then evenly spread on the feeder layer cells, about 200μL / well, and cultured in a cell culture incubator.

[0060] 4. Hybridoma cell screening

[0061] ELISA plates were coated with optimized antigen concentration (6.8 μg / mL). When the hybridoma cells grew into single clones and the color of the culture medium turned slightly yellow, the hybridoma cells were screened by indirect ELISA method. A portion of the hybridoma cells that tested positive for antibodies were expanded and cultured, and the other portion was subcloned (limiting dilution method) to prevent the loss of hybridoma cell lines due to cell contamination.

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

[0063] 5. Identification of hybridoma cell antibody subtypes

[0064] According to the immunoglobulin typing detection kit, CPV antigen was coated on the ELISA plate, and the obtained 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 antibodies for indirect ELISA experiments, 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 subtypes and 3 are IgM subtypes. The heavy chains of 3D7, 6C3, and 5D4 are IgG2a subtypes, the heavy chains of 1B3 and 1D3 are IgG1 subtypes, and the heavy chains of 3A9, 1D9, and 1H2 are IgM subtypes; 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 28 Subtype identification of hybridoma cell lines

[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 each mouse was intraperitoneally injected with 500 μL of Freund's incomplete adjuvant. One week later, well-growing hybridoma cells were collected and resuspended in RPMI-1640 medium. Cell counting was performed using a cell counting plate. Each mouse was intraperitoneally injected with 10 6 The mice were fed for 1 week and the abdominal condition was observed. When ascites was produced, the ascites could be drawn out with a syringe and stored at -20°C after aliquoting.

[0069] The cell supernatants of 8 hybridomas were diluted 2-fold and the ascites were diluted 10-fold. The ascites and cell culture supernatants were used as primary antibodies and the titers of the monoclonal antibodies were determined by indirect ELISA.

[0070] By indirect ELISA method, the dilution ratios of hybridoma cell culture supernatant were 1:2 and 1:2 respectively. 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 The dilution multiples of hybridoma cell mouse ascites were 1:10 and 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 The titer was determined by dilution. The results are shown in Table 3 and Figure 3 As shown, the titer of the supernatant of the 8 hybridoma strains ranged from 2 4 ~2 12 , ascites titer range is 10 3 ~10 8 .

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

[0072]

[0073] 7. Indirect immunofluorescence identification of monoclonal antibodies

[0074] CPV-2, CPV-2a, CPV-2b and CPV-2c were inoculated into the monolayer of F81 cells with good growth status, cultured at 37℃ for 72h, the culture supernatant was discarded, the cells were fixed with methanol for 15min, and washed slowly and carefully with PBS 3 times, 5min each time (be careful not to wash away the cells), and the culture supernatant of the hybridoma cells to be tested was added and incubated at 37℃ for 1h. At the same time, the supernatant of SP2 / 0 cells was used as the negative control well. After washing, goat anti-mouse FITC-IgG and FITC-IgM (1:3000) were added and incubated at 37℃ for 1h in the dark. The test results were observed under an inverted fluorescence microscope and photographed for storage. 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 had no specific fluorescence reaction ( Figure 4 B and D), and the SP2 / 0 supernatant control and cell control had no fluorescence.

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

[0076] Dilute the virus to a concentration of 100 TCID 50 , and ascites diluted with 8 monoclonal antibodies (1:2, 1:2 2 , 1:2 3 , 1:2 4 , 1:2 5 , 1:2 6) were mixed in equal volumes, incubated at 37°C, 5% CO2 for 1h, and then inoculated with F81 cells, 100μL / well, and repeated 4 times in parallel. At the same time, cell wells with only culture medium and virus solution were set as negative and positive controls. The CPE of the cells was observed after 4 days of culture, and the neutralizing activity of MAb was detected. It was verified that only the antibody secreted by 3D7 had neutralizing activity. The concentrations of 3D7 that completely neutralized CPV-2, CPV-2a, CPV-2b and CPV-2c viruses were 0.39μg / mL, 0.69μg / mL, 0.70μg / mL and 0.69μg / mL, respectively.

[0077] 9. Purification of monoclonal antibody from ascites and testing of its efficacy

[0078] The five IgG subtype monoclonal antibodies screened were purified from the collected ascites by Protein A+G affinity chromatography. The protein concentration of the purified antibodies was detected by a spectrophotometer or a BCA kit and stored in aliquots at -20°C.

[0079] Take 20 μL of purified antibody and use SDS-PAGE to verify the purification effect. Figure 5 As shown, after the five monoclonal antibodies were purified and concentrated, they were identified by SDS-PAGE. The band at 50kD was the heavy chain of the monoclonal antibody, and the band between 20-25kD was the light chain of the monoclonal antibody. There were no obvious mixed bands, and the purity was good.

[0080] 10. Analysis of viral proteins recognized by MAbs

[0081] The five IgG monoclonal antibodies 1D3, 6C3, 5D4, 1B3, and 3D7 were used to verify their recognition of viral proteins by Western blot. Figure 6 As shown, 3D7, 6C3, 5D4, 1B3, and 1D3 have specific bands at 70KD, which identify the VP2 protein of the CPV virus.

[0082] Example 2 Preliminary establishment of canine parvovirus colloidal gold immunochromatographic detection method

[0083] 1. Determination of the minimum labeling amount and optimal pH of colloidal gold detection antibodies

[0084] The five 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 subjected to square titration of gold-labeled antibodies. 0, 5, 10, 15 and 20 μL of 0.2 mol / L K2CO3 solution were added to adjust the pH of the colloidal gold solution, the antibody was pre-diluted to 0.5 mg / mL, and 1:2, 1:2, and 2:3 titrations were performed. 2 , 1:2 3 , 1:2 4 , 1:2 5 , 1:2 6 , 1:2 7 Dilute by 100 times, add to colloidal gold solution, mix thoroughly and measure OD 524nm The results are shown in Table 4. The minimum stable amount of antibody labeling for 3D7 is 0.125 mg / mL, and the minimum stable amount of antibody labeling for 6C3, 5D4, 1B3, 1D3, and 2A10 is 0.065 mg / mL. During the experiment, the minimum stable amount can be increased by 20%; the amount of K2CO3 solution added for 6C3 and 1D3 is 5 μL, 1B3 is 10 μL, and 5D4, 3D7, and 2A10 is 15 μL.

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

[0086]

[0087] The gold-labeled antibodies were prepared according to the minimum stable amount and optimal pH of the above 6 monoclonal antibody colloidal gold-labeled antibodies. 2 mL of colloidal gold solution was added to 6 15 mL centrifuge tubes, and the solution was shaken for 10 min on a rotator. The pH was adjusted by adding the corresponding volume of 0.2 mol / L K2CO3 solution according to the optimal pH of each monoclonal antibody, and the solution was shaken for 10 min on a rotator. The 6 monoclonal antibodies were added to 6 centrifuge tubes according to the optimized conditions of the colloidal gold labeled antibody amount, and the solution was mixed for 10 min on a rotator and then allowed to stand at room temperature for 30 min to allow the colloidal gold particles to fully combine with the antibody. 10% PEG-6000 solution was added to the 6 centrifuge tubes to a final concentration of 1%, and the solution was mixed for 10 min on a rotator and then allowed to stand at room temperature for 30 min for blocking. The blocked colloidal gold solution was placed in a high-speed refrigerated centrifuge at 8000 r / min and centrifuged at 4°C for 20 min, and then taken out steadily to prevent the precipitate from resuspension. The supernatant was carefully aspirated with a pipette and discarded, and 200 μL of gold resuspension solution was added to each tube and gently blown to fully resuspend the precipitate, thereby obtaining 10× gold-labeled antibody, which was placed at 4°C for standby use.

[0088] 2. Screening of the best gold-labeled antibody and the best capture antibody

[0089] In order to determine the best gold-labeled antibody and the best capture antibody, the above 6 CPV monoclonal antibodies (6C3, 5D4, 1B3, 3D7, 1D3 and 2A10) were arranged and combined with the above 6 antibodies labeled with colloidal gold (Au-6C3, Au-5D4, Au-1B3, Au-3D7, Au-1D3 and Au-2A10), paired in pairs, and prepared into test strips, with a total of 36 combinations. 6 TCID 50 / mL high concentration CPV-2 virus samples were preliminarily screened, and the T line signal value of the test strip was read and recorded using a colloidal gold immunoassay quantitative analyzer. Figure 7 As shown in Table 5. 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-matching

[0091]

[0092] Then the virus titer was 10 4 Low-concentration CPV-2, CPV-2a, CPV-2b and CPV-2c virus samples with TCID50 / mL were further screened, and a negative control was set up to observe the color development of the T line of the test strip, and record the T value results read by the colloidal gold immunoquantitative 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 color development effect of the test strip for detecting different strains is the best, the T line signal value is the highest, and the specificity is good, so 3D7 is finally determined as the capture antibody and 2A10 is used as the gold-labeled antibody.

[0093] Table 6 Antibody cross-pairing for further screening

[0094]

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

[0096] Prepare a test strip, in which the concentration of the test line coated with 3D7 antibody is 2.0 mg / mL, and the concentration of the quality control line coated with goat anti-mouse antibody is 4.0 mg / mL. Set the 2A10 gold-labeled antibody spraying amount of 1 μL / cm, 2 μL / cm, 3 μL / cm, 4 μL / cm, 5 μL / cm, and 6 μL / cm to prepare a gold-labeled pad. Use the prepared test strip to detect the same negative sample and positive sample, repeat 3 times, and use an immunoassay to detect the colloidal gold signal intensity of the T line and C line. Select the amount of gold-labeled probe added when the signal-to-noise ratio is the highest as the optimal addition volume. According to multiple measurements, when the colloidal gold immunoassay reads a T value less than 90, there is no visible band on the test strip, so 90 is set as the critical value for result judgment.

[0097] Test results such as Figure 8 As shown in the figure, when the gold-spraying amount of the gold-labeled antibody 2A10 is 5 μL / cm, the color development effect of the T line is the best, the signal value of the T line is the highest, and the T value of the negative control sample is lower than 90. In summary, the optimal gold-spraying amount of the gold-labeled antibody 2A10 is 5 μL / cm.

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

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

[0100] Test results such as Fig. 9 As shown, it can be seen that the signal value of the T line increases with the increase of the C line antibody concentration. When the T line antibody concentration is 2.5 mg / mL, the T line signal value is the highest, and the T value of the negative control sample is less than 90. In summary, the optimal T line antibody concentration is 2.5 mg / mL.

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

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

[0103] Test results such as Fig.10 As shown by Fig.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. In summary, the optimal C line antibody concentration is 4.5 mg / mL.

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

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

[0106] The test strip was streaked, the capture antibody 3D7 and the goat anti-mouse antibody were diluted to the optimal concentrations determined above, the cleaned BIODOT streaking machine was loaded, the machine parameters were modified, the Y-axis position was set to 4.0 mm, the streaking amount was 1 μL per centimeter, and the streaking was completed and dried in a 37°C oven for 4 h; the gold-labeled antibody binding pad was treated (the gold spraying amount of the fixed gold-labeled antibody 2A10 was 5 μL / cm), the binding pad treatment solution was prepared, the binding pad was soaked with the binding pad treatment solution, and dried in a 37°C oven overnight; the sample pad was treated, the sample pad treatment solution was prepared, the sample pad was soaked with the sample pad treatment solution, and dried in a 37°C oven overnight. Assemble the test strips, cut the sample pad, binding pad, NC membrane (CN95 nitrocellulose membrane), and absorbent paper into appropriate widths in turn, and stick them to the PVC base plate in turn, with each original overlapping by about 2 mm to ensure the flow of sample liquid; cut the test strips, use a strip cutter, and set the test strip width in the system parameters to 4.0 mm; assemble the test strips, install the cut test strips in the plastic card holder in the correct direction for the next experiment.

[0107] (2) Specificity detection

[0108] In order to detect the specificity of colloidal gold test strips in recognizing antigens, canine coronavirus (CCoV), canine distemper virus (CDV), canine parainfluenza virus (CPIV), canine adenovirus (CAV) and F81 cell supernatant were used to test the test strips, and positive and negative controls were set up. 100 μL of each sample was added dropwise to the sample slot of the test strip card, and the color development results were observed after standing at room temperature for 15 minutes.

[0109] Specific test results such as Fig.11 As shown, the test strip only has obvious specific bands when detecting CPV positive control, but no bands when detecting other common canine viruses, F81 cell supernatant and negative control. In summary, the specificity of the test strip is good.

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

[0111] To detect the broad-spectrum property of CPV colloidal gold test strips, four subtypes of CPV strains, CPV-2, CPV-2a, CPV-2b, and CPV-2c, were used for the test. Each subtype of virus was diluted in two-fold ratios, and the reaction of the test strips to each subtype of CPV virus was observed, and the detection limits of the four strains were determined.

[0112] The sensitivity test results of this test strip for CPV-2 are as follows Fig.12 As shown in A, it can be seen from the figure that the minimum detection amount of CPV-2 by this test strip is 7.8×10 3 TCID 50 / mL (1:1280); The sensitivity test results of this test strip for CPV-2a are as follows Fig.12 As shown in B, it can be seen from the figure that the minimum detection amount of the test strip for CPV-2a strain is 6.25×10 3 TCID 50 / mL (1:160); The sensitivity test results of this test strip for CPV-2b are as follows Fig.12 As shown in C, it can be seen from the figure that the minimum detection amount of the test strip for CPV-2b strain is 7.8×10 3 TCID 50 / mL (1:1280); The sensitivity test results of this test strip for CPV-2c are as follows Fig.12 As shown in D of D, it can be seen from the figure that the minimum detection amount of the test strip for CPV-2c strain is 7.8×10 3 TCID 50 / mL(1:1280).

[0113] 7. Repeatability within and between test strips

[0114] Prepare multiple batches of colloidal gold test strips, randomly select 2 batches of test strips, and test the same virus sample with gradient dilutions of 1:100, 1:200, and 1:400, respectively, repeat 3 times, and record the T / C value. Calculate the intra-batch and inter-batch coefficients of variation of the two batches of test strips. When the intra-batch and inter-batch coefficients of variation (CV) of the samples are less than 15%, it shows that the precision of the test strips is high. Coefficient of variation (CV) = standard deviation (SD) / mean value.

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

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

[0117] In summary, the intra-group coefficient of variation of the test strip is less than 10%, and the inter-group coefficient of variation is less than 15%, so the intra-group and inter-group repeatability of the test strip is good.

[0118] Table 7 Intra-group repeatability test results

[0119]

[0120] Table 8 Results of repeatability test between groups

[0121]

[0122] 8. Test strip stability test

[0123] In order to test the stability of the homemade colloidal gold test strips, the homemade colloidal gold test strips were placed in a 37°C environment for stability tests. The homemade test strips were taken out at 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days, and the virus samples diluted 100 times were tested, and the test was repeated 3 times each time. The T / C signal intensity was detected by an immunoassay analyzer to analyze the stability of the test strips.

[0124] The test strip stability results are as follows Fig.13 As shown by Fig.13 It can be seen that there is no significant difference in T / C value when the test strips stored within 30 days detect the same sample. Therefore, the homemade colloidal gold test strips have good stability.

[0125] 9. Comparison between homemade test strips and commercial test strips

[0126] Homemade colloidal gold test strips and commercial canine parvovirus colloidal gold test strips (Korea Anjie CPV antigen detection colloidal gold test strips) were used to detect CPV-2, CPV-2a, CPV-2b and CPV-2c viruses that were diluted twice (each virus had been pre-diluted 10 times), and the minimum detection amounts of homemade colloidal gold test strips and imported brand colloidal gold test strips for CPV-2, CPV-2a, CPV-2b and CPV-2c strains were compared.

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

[0128] In summary, the homemade colloidal gold test strips are superior to the commercially available colloidal gold test strips in terms of the minimum detection amount.

[0129] 10. Clinical sample testing

[0130] In order to verify the consistency of the test strip and PCR, 70 suspected canine parvovirus samples were tested using the homemade test strip and PCR method. The PCR test results are shown in Table 9. PCR detected 49 positive samples and 21 negative samples. The consistency results are shown in Table 9. The positive consistency of the test strip and PCR is 91.8% (45 / 49), the negative consistency is 100% (21 / 21), and the total consistency is 94.3%. Therefore, the consistency of the homemade test strip and PCR is good.

[0131] Table 9 The coincidence rate between the homemade colloidal gold test strip and PCR

[0132]

[0133] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A hybridoma cell line 3D7 producing canine parvovirus monoclonal antibodies, characterized in that: The hybridoma cell line 3D7 was deposited in the China Center for Type Culture Collection on October 18, 2024, with the deposit number being CCTCC NO: C2024216.

2. Use of the hybridoma cell line 3D7 as claimed in claim 1 in the preparation of canine parvovirus monoclonal antibodies.

3. A canine parvovirus monoclonal antibody, characterized in that: Produced by the hybridoma cell line 3D7 according to claim 1.

4. Use of the canine parvovirus monoclonal antibody as claimed in claim 3 in the preparation of a colloidal gold detection kit for canine parvovirus disease.

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

6. The colloidal gold test strip for canine parvovirus disease according to claim 5, characterized in that: The gold-labeled antibody conjugate pad is coated with colloidal gold-labeled antibody 2A10.

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

8. The colloidal gold 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 test strip for canine parvovirus disease according to claim 6, characterized in that: The gold spraying volume of the colloidal gold-labeled antibody 2A10 was 5 μL / cm.

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