Monoclonal antibody pair for detecting feline calicivirus, hybridoma cell strain and application thereof

Abstract

The application provides a pair of monoclonal antibodies for detecting feline calicivirus, hybridoma cell strains and application, specifically, the application provides a pair of hybridoma cell strains, one of the hybridoma cell strains is named FCV-13#, the preservation number is CGMCC NO.45747, and the preservation date is November 21, 2023, the other hybridoma cell strain is named FCV-11#, the preservation number is CGMCC NO.45746, and the preservation date is November 21, 2023. Further, the application provides a pair of monoclonal antibodies produced by the hybridoma cell strains and application in detecting feline calicivirus. The monoclonal antibody pair for detection has high sensitivity and good specificity, can accurately and rapidly detect feline calicivirus, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a monoclonal antibody pair, hybridoma cell line, and application for detecting feline calicivirus. Background Technology

[0002] Feline calicivirus (FCV) belongs to the family Caliciviridae and the genus Calicivirus. FCV is a single-stranded positive-sense RNA virus with a stereosymmetrical viral particle structure, lacking an envelope, and prone to genetic mutation and antigenic variation. FCV is highly infectious and transmissible in felines, exhibiting no obvious seasonality, with a slightly higher incidence in winter than summer. Transmission primarily occurs through contact with the oral and nasal secretions of infected cats and through contaminated environments. Feline calicivirus infection mainly causes oral and upper respiratory symptoms, including fever (39.5–42°C), decreased appetite, lethargy, oral ulcers, swollen and red gums, increased eye discharge, nasal discharge, sneezing, and in severe cases, lung infection.

[0003] FCV was first isolated by Fastier in 1957 and subsequently discovered worldwide. FCV has only one serotype, and different variants exhibit varying virulence and cross-antigenicity. In recent years, highly pathogenic FCV strains (VSD) have emerged. FCV outbreaks have occurred in several countries. This strain can cause systemic multi-systemic lesions in infected cats, with a high mortality rate. As a highly variable RNA virus, monitoring circulating strains and identifying variant strains of FCV play an important role in its genetic evolution analysis.

[0004] Vaccination is the most effective way to prevent and control this disease. However, due to the low homology (70-80%) between strains, existing vaccines offer limited protection, especially against the highly lethal FCV-VSD strain. Furthermore, FCV is highly variable, and the frequent emergence of variant strains is a major factor in immunization failure. Treatment for feline calicivirus disease primarily involves symptomatic and supportive therapies. Interferon and broad-spectrum antibiotics are commonly used in clinical treatment. Interferon can inhibit viral replication and infection, while broad-spectrum antibiotics can prevent secondary bacterial infections. However, there is a lack of specific treatments for FCV. Therefore, the fundamental measures for controlling this disease lie in immunization and quarantine.

[0005] Given the high variability of FCV and the limited protective effect of vaccines, early diagnosis and timely prevention are crucial. Therefore, developing a highly specific and broad-spectrum feline calicivirus monoclonal antibody is of great significance for both the diagnosis and prevention of this disease. Summary of the Invention

[0006] To improve the sensitivity and specificity of feline calicivirus (FCV) detection and establish a rapid detection method for FCV, and to overcome the deficiencies of existing technologies, this invention provides a monoclonal antibody pair, a hybridoma cell line, and their applications for detecting FCV. Specifically, this invention provides the following technical solution: In a first aspect, the present invention provides a pair of hybridoma cell lines that produce monoclonal antibodies against feline calicivirus (FCV), wherein one hybridoma cell line is deposited under the name FCV-13#, accession number CGMCC NO.45747, and accession date November 21, 2023, and the other hybridoma cell line is deposited under the name FCV-11#, accession number CGMCC NO.45746, and accession date November 21, 2023.

[0007] In a second aspect, the present invention provides a monoclonal antibody pair for feline calicivirus, the monoclonal antibody pair being composed of monoclonal antibodies FCV-13 and FCV-11, wherein monoclonal antibody FCV-13 is produced by hybridoma cell line FCV-13# and monoclonal antibody FCV-11 is produced by hybridoma cell line FCV-11#.

[0008] In one embodiment, the monoclonal antibody FCV-13 is a detection antibody and the monoclonal antibody FCV-11 is a capture antibody, or the monoclonal antibody FCV-13 is a capture antibody and the monoclonal antibody FCV-11 is a detection antibody.

[0009] In a preferred embodiment, the monoclonal antibody FCV-11 is used as a detection antibody and the monoclonal antibody FCV-11 is used as a capture antibody.

[0010] A third aspect of the present invention provides the use of the above-described hybridoma cell line or monoclonal antibody pair in the preparation of a product for detecting feline calicivirus.

[0011] In one embodiment, the product is a formulation, a kit, and / or a test strip.

[0012] A fourth aspect of the present invention provides a test strip for detecting feline calicivirus, the test strip comprising: a sample pad, a colloidal gold pad, absorbent filter paper, and a nitrocellulose membrane; wherein the nitrocellulose membrane is coated with a monoclonal antibody FCV-11, and the colloidal gold pad is coated with a colloidal gold-labeled monoclonal antibody FCV-13; or the nitrocellulose membrane is coated with a monoclonal antibody FCV-13, and the colloidal gold pad is coated with a colloidal gold-labeled monoclonal antibody FCV-11.

[0013] A fifth aspect of the present invention provides a method for preparing the above-mentioned test strip, the method comprising the following steps: (1) Preparation of gold-labeled compounds Place the colloidal gold solution on a stirrer and stir at 300 g / min. Adjust the pH to 8.5 with 0.2 mol / L potassium carbonate solution. Slowly add monoclonal antibody FCV-13 or FCV-11 dropwise to a final concentration of 8–12 μg / ml. Let stand at room temperature. Add 10% BSA solution to the colloidal gold solution containing the monoclonal antibody to a final concentration of 1%. Mix well and block for 30 minutes or overnight. Centrifuge at 7500 rpm for 20 minutes, discard the supernatant, and resuspend the centrifuged precipitate with gold-labeled buffer to a volume of 1 / 10 of the original volume. This is the gold-labeled conjugate. (2) Preparation of gold-labeled conjugate pads Add 300 μl of gold-labeled antigen resuspension to each 1 ml of colloidal gold obtained by centrifugation, mix well, spray onto a glass fiber membrane, place in a 37°C forced-air drying oven for 5 hours, and after drying, cut the gold-labeled conjugate pad into strips. (3) Coating of nitrocellulose membrane Dilute FCV-11 or FCV-13 with membrane coating solution and spray it onto nitrocellulose membrane as the detection line; dilute goat anti-mouse IgG with membrane coating solution and spray it onto nitrocellulose membrane at a rate of 1.0 μg / cm 5 mm below the detection line as the quality control line; dry the coated nitrocellulose membrane at 37°C for 3 hours and then seal and store it.

[0014] Compared with the prior art, the present invention has the following significant advantages: We performed cross-pairing experiments on three FCV monoclonal antibodies with good affinity obtained through screening, further identifying the optimal pairing method using FCV-11 monoclonal antibody as the detection antibody and FCV-13 monoclonal antibody as the capture antibody. The antibody pair provided by this invention exhibits good binding ability to feline calicivirus. When applied to the detection of feline calicivirus, this antibody pair does not show a positive reaction with other viruses causing common clinical diseases in cats, and when the virus is diluted to 10... 3.0 / TCID 50 It can still detect positive results at a concentration of 0.1 ml, demonstrating excellent specificity and sensitivity. It can be used as a detection antibody and capture antibody in colloidal gold test strips, and has advantages such as good specificity, high sensitivity and good stability. This establishes a rapid detection method for feline calicivirus, filling the gap in FCV colloidal gold test strips, and is of great significance for the prevention and control of feline calicivirus epidemics. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 PAGE image of the purified FCV-VP1 strain; Figure 2 The results are from serum titer ELISA assays. Figure 3 The results were identified by Western blotting. Figure 4 Homology comparison diagram of 7 FCV strains; Figure 5 These are indirect immunofluorescence detection results, where A: 4#; B: 11#; C: 13#; D: FCV commercial antibody; E: negative control; Figure 6 These are the results of immunohistochemical testing, where A: a tongue sample from a cat that died from FCV infection; B: a negative sample. Figure 7 These are the results of direct immunofluorescence detection, where A: FITC-labeled antibody; B: cell control; Figure 8 To examine the structure of the test strip; Figure 9 The results are for testing samples diluted 10-fold. In the following ratios: 1: Antibody #11 is the detection antibody, and antibody #13 is the capture antibody; 2: Antibody #11 is the detection antibody, and antibody #4 is the capture antibody; 3: Antibody #4 is the detection antibody, and antibody #13 is the capture antibody; 4: Antibody #4 is the detection antibody, and antibody #11 is the capture antibody; 5: Antibody #13 is the detection antibody, and antibody #11 is the capture antibody; 6: Antibody #13 is the detection antibody, and antibody #4 is the capture antibody. Figure 10 The test results were obtained after the samples were diluted 100-fold. In the following ratios: 1: Antibody #11 was used as the detection antibody, and antibody #13 was used as the capture antibody; 2: Antibody #11 was used as the detection antibody, and antibody #4 was used as the capture antibody; 3: Antibody #4 was used as the detection antibody, and antibody #13 was used as the capture antibody; 4: Antibody #4 was used as the detection antibody, and antibody #11 was used as the capture antibody; 5: Antibody #13 was used as the detection antibody, and antibody #11 was used as the capture antibody; 6: Antibody #13 was used as the detection antibody, and antibody #4 was used as the capture antibody. Figure 11 For specific detection results, 1: feline calicivirus cell culture; 2: feline coronavirus swab treatment solution; 3: feline parvovirus cell culture; 4: feline herpesvirus cell culture; 5: F81 cell culture medium; Detailed Implementation The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0016] It should be noted that in this article, "FCV-11" and "11# antibody" both refer to monoclonal antibodies secreted by 11# hybridoma cells, and so on.

[0017] Example 1: Construction of recombinant baculovirus FCV-VP1 strain 1. Experimental Materials 1.1 Sf9 insect cells were preserved by Changchun Xinuo Biotechnology Co., Ltd.

[0018] 1.2 Main reagents: Sf-900II SFM medium and DMEM medium were purchased from Gibco; fetal bovine serum was purchased from Gibco.

[0019] 2 Experimental Methods 2.1 Construction of recombinant baculovirus FCV-VP1 strain By inserting two optimized FCV VP1 genes into the transfer vector pFastBacDual, recombinant baculoviruses were obtained after transforming competent cells containing the baculovirus genome. After transfecting Sf9 cells, recombinant baculovirus expressing FCV VP1 was successfully rescued and named the recombinant baculovirus FCV-VP1 strain.

[0020] 2.2 Culture of recombinant baculovirus FCV-VP1 strain Sf9 cells were cultured in Sf-900 II SFM medium containing 0.5% penicillin antibiotics at 27°C and 120 r / min until the cell count reached approximately 2 × 10⁻⁶. 6 When the cell count was 10 cells / ml, the recombinant baculovirus FCV-VP1 strain was inoculated at an MOI of 0.1 and cultured at 27°C. After 96 hours, the cell culture was harvested and stored at -70°C.

[0021] 2.3 Purification of recombinant baculovirus FCV-VP1 strain (1) The cell culture was freeze-thawed once at room temperature and -20℃, and centrifuged at 3000 rpm, 30 min, and 4℃ to collect the supernatant.

[0022] (2) Centrifuge the supernatant at 20000, 1.5 h, 4℃ and resuspend the precipitate with PBS.

[0023] (3) The virus strips were further purified by sucrose density gradient centrifugation with sucrose densities of 10%, 20%, 30%, 40% and 50%, respectively. The centrifugation was carried out at 21,000 rpm for 1.5 h at 4℃. The virus strips were resuspended in STE and centrifuged at 28,000 rpm for 1.5 h at 4℃ to remove sucrose. The precipitate was resuspended in a small amount of saturated EDTA and stored at 4℃ for later use.

[0024] 2.4 SDS-PAGE identification: Take 20 μl of the purified recombinant baculovirus FCV-VP1 strain, add 5 μl of 5× loading buffer, mix well, boil for 5 minutes, and then use it for SDS-PAGE detection.

[0025] 3 Results 3.1 SDS-PAGE Identification Samples were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining analysis. The results showed that the target protein was concentrated around 60 kDa. (See attached image for details.) Figure 1 .

[0026] Example 2: Screening and Application of FCV Monoclonal Antibody Cell Lines 1. Materials 1.1 Cells and virus strains SP2 / 0 myeloma cells, F81 cells, and FCV-VLP protein were preserved by Changchun Xinuo Biotechnology Co., Ltd.

[0027] 1.2 The main reagents were: RPMI-1640 medium purchased from GIBCO; DMSO, HAT, and HT purchased from Sigma; semi-solid HAT hybridoma cell screening medium purchased from STEMCELL; PEG1500 purchased from Roche; monoclonal antibody typing kit purchased from Southern Biotech; and HRP-labeled goat anti-mouse IgG antibody and FITC-labeled goat anti-mouse IgG antibody purchased from Bioworld.

[0028] 2 methods 2.1 Animal immunization and serum titer detection 2.1.1 Animal Immunization Four SPF female mice were immunized subcutaneously with FCV-VLP protein. The initial immunization was 60 μg / mouse, followed by a booster immunization of 30 μg / mouse, with booster immunizations every two weeks for a total of four immunizations. Blood samples were collected one week after the fourth immunization to measure serum titer. Based on serum titer, mice were selected for pre-cell fusion intraperitoneal shock therapy with an immunization dose of 50 μg / mouse.

[0029] 2.1.2 Serum titer detection The serum titer of mice was detected by indirect ELISA. 2 μg / ml FCV-VLP protein was used as the coating antigen, and the plate was coated overnight at 4°C. 2% skim milk powder was used for blocking at 37°C for 2 h. Serum was serially diluted 2-fold starting from 1:200. PBS was used as the blank control, and negative control was a 200-fold dilution of negative serum.

[0030] 2.2 Cell Fusion (1) Gently blow the healthy sp2 / 0 cells off the culture flask wall and aspirate them into a 50 ml centrifuge tube.

[0031] (2) Blood was collected from the eyes of the mice, and they were then euthanized by dislocation of the neck and soaked in 75% alcohol for 5 minutes.

[0032] (3) Pour a small amount of serum-free RPMI-1640 into a petri dish, and place the cell strainer and syringe core into the petri dish. Remove the spleen from the mouse with scissors and tweezers and place it on the cell strainer. Gently crush the spleen thoroughly with the syringe core, and aspirate the crushed cells into a centrifuge tube containing sp2 / 0. Centrifuge at 1500 rpm for 5 min.

[0033] (4) Remove the mouse thymus with scissors and tweezers and crush it. Transfer the crushed thymus cells to a 15 ml centrifuge tube, add 1 ml of HAT, and place it in a cell culture incubator for later use.

[0034] (5) After centrifuging, discard the supernatant, carefully and gently mix the cells with serum-free RPMI-1640, and centrifuge (1500 rpm, 5 min).

[0035] (6) Discard as much of the cell supernatant as possible after centrifugation. Tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, and slowly add 1 ml of PEG over 1 minute. After adding the PEG, let it stand in the warm water for 1 minute. Then, slowly add 2 ml of serum-free RPMI-1640 over 2 minutes, followed by 8 ml of serum-free RPMI-1640 over another 2 minutes. Centrifuge at 1000 rpm for 5 minutes.

[0036] (7) Discard the supernatant, add 10 ml of serum, carefully blow the cells evenly, and pour them into the prepared thymocytes. Add 25 ml of sterile semi-solid HAT hybridoma cell selection medium and mix thoroughly. Then pour evenly into 30 cell culture dishes. Place the cell culture dishes in a cell culture incubator for culture.

[0037] 2.3 Screening of positive hybridoma cell lines (1) Pick 30 cell clones from the cell culture dishes in 2.2 and culture them in 96-well cell culture plates (pre-coated with thymocytes, 100 μl / well).

[0038] (2) After 6 days of cell culture, the cell culture supernatant was removed for screening.

[0039] (3) Dilute FCV-VLP protein with coating buffer to a final concentration of 2 μg / ml, add 100 μl / well to ELISA plate, and incubate overnight at 4°C.

[0040] (4) After washing with PBST three times, add 2% skim milk powder for sealing, 200 μl / well, and incubate at 37°C for 2 h.

[0041] (5) After washing three times with PBST, add 100 μl of primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times with PBS) to each well, and incubate at 37°C for 1 h.

[0042] (6) After washing three times with PBST, add PBS diluted 20,000 times to the secondary antibody, 100 μl / well, and incubate at 37℃ for 1 h.

[0043] (7) After washing with PBST 3 times, use colorimetric solution to develop color, 100 μl / well, for 5 min.

[0044] (8) Add 50 μl of stop solution to each well to stop the color development.

[0045] (9) Use dual wavelengths (450 nm, 630 nm) to measure absorbance and record and save the data.

[0046] 2.4 Identification of Monoclonal Antibody Subclasses (1) Dilute the capture antibody to 0.5 μg / ml with 100 mM PBS (pH 7.4), 100 μl per well, and incubate overnight at 4°C.

[0047] (2) After washing three times with PBST, add 200 μl of blocking solution to each well and incubate at 37°C for 2 h.

[0048] (3) After washing three times with PBST, add 100 μl of hybridoma supernatant to each well and incubate at 37°C for 1 h.

[0049] (4) After washing three times with PBST, add 100 μl of HRP-labeled antibody diluted 1:10000 (κ, λ) or 1:20000 (other) with blocking buffer to each well and incubate at 37°C for 1 h.

[0050] (5) After washing with PBST three times, add 50 μl of substrate solution to each well and measure the absorbance at dual wavelengths (450 nm, 630 nm) within 10 to 20 min. Record and save the data.

[0051] 2.5 Large-scale preparation of monoclonal antibodies 2.5.1 Preliminary Experiment One week before inoculation with hybridoma cells secreting monoclonal antibodies, 6-8 week old female Balb / c mice were intraperitoneally injected with sterile liquid paraffin, 0.5 mL per mouse.

[0052] 2.5.2 Hybridoma Cell Preparation: Cultured hybridoma cells were blown off, centrifuged at 2000 rpm and 25°C for 10 min, and then the cell pellet was resuspended and mixed with PBS buffer (pH 7.2). After trypan blue staining for viable cell counting, the cell density was adjusted to 2–4 × 10⁻⁴ cells / mL. 6 per mL.

[0053] 2.5.3 Intraperitoneal injection of hybridoma cells: The injection site in the mouse abdomen was disinfected with alcohol swabs. Hybridoma cells were injected intraperitoneally, with each mouse receiving 0.5 mL of cell suspension containing approximately 5 × 10⁻⁶ cells. 5 Each cell.

[0054] 2.5.4 Observe the condition of mice after inoculation by collecting ascites fluid. After about 7-10 days, the mice's abdomen will be noticeably distended and they will have difficulty moving. At this time, disinfect the skin of the lower abdomen with an alcohol swab, and insert a 20 mL syringe needle into the abdominal cavity. Pale yellow ascites fluid will flow out. Collect the fluid in a 2 mL sterile centrifuge tube, centrifuge at 3500 rpm for 10 min at 4℃, and the supernatant is the ascites fluid. Aliquot the ascites fluid into 2 mL centrifuge tubes, label them with the number of collections, and store at -80℃. After 2-3 days, when the ascites fluid has regenerated and accumulated, collect it again using the same method. Generally, 3-5 collections can be performed on one mouse.

[0055] 2.6 Purification of Monoclonal Antibodies 2.6.1 Connect both ends of the Protein G column to the chromatography system.

[0056] 2.6.2 Turn on the peristaltic pump and first use 10 ml of 20 mM pH7.0 PBS to equilibrate the protein column and remove the original 20% ethanol in the protein column. Be careful not to let any gas enter. Adjust the pump speed to 1 mL / min.

[0057] 2.6.3 Add the sample to 20 mM pH 7.0 PBS at a 1:1 ratio and filter using a 0.45 μm filter membrane. Determine the protein concentration of the sample. The protein saturation of the protein column is 25 mg, thus determining the sample loading volume. After loading the sample, continue to equilibrate the protein column with 5–10 ml of 20 mM pH 7.0 PBS. At this point, the measurement parameters of the UV detector will rise from 0 to the tens and then drop back to 0. Collect all the liquid flowing out at this point and aliquot it into small samples; this is the breakthrough peak.

[0058] 2.6.4 Once the UV detector reading reaches 0, elute with 0.1M pH 2.7 Gly-HCl. When the reading rises rapidly, collect the sample in 1.5 ml centrifuge tubes pre-filled with 100 μl of 1M pH 9.0 Tris-HCl, filling each tube to 1 ml. Stop collecting when the reading decreases.

[0059] 2.6.5 Dialysis was performed using a 10 KD dialysis bag with 20 mM pH 7.0 PBS as the dialysate, and the solvent was replaced at 4°C.

[0060] 2.6.6 Determine the final protein concentration of the sample, aliquot it, and store it at -20℃ for later use.

[0061] 2.7 Identification and Application of Monoclonal Antibodies 2.7.1 Western blotting detection (1) After the purified FCV VP1 was subjected to SDS-PAGE, it was then subjected to wet transfer NC membrane.

[0062] (2) After the transfer is completed, use 5% skim milk powder to seal at room temperature for 2 hours.

[0063] (3) Cut the NC membrane into strips and add different monoclonal antibodies (200-fold dilution) to each strip. At the same time, use positive mouse serum (1000-fold dilution) as a positive control and incubate at room temperature for 2 h.

[0064] (4) Wash the membrane 5 times with PBST.

[0065] (5) Add HRP-labeled goat anti-mouse IgG (5000 times dilution) and incubate at room temperature for 1 hour.

[0066] (6) Wash the membrane 5 times with PBST.

[0067] (7) Add ECL chemiluminescent substrate in the dark and develop color for 5-10 min in the dark.

[0068] 2.7.2 Indirect immunofluorescence detection (1) Passage F81 cells in the logarithmic growth phase, seed them in 96-well cell culture plates, and inoculate 17 FCV strains simultaneously with 100 μl / well. A negative control well is also provided.

[0069] (2) After the F81 cells showed obvious cytopathic effects, the cell culture medium was discarded, and the cells were fixed with 80% cold acetone, 200 μl / well, and incubated at room temperature for 30 min.

[0070] (3) Wash the plate three times with PBST.

[0071] (4) Add the monoclonal antibody (200-fold dilution) to a 96-well plate and use commercial FCV monoclonal antibody (200-fold dilution) as a positive control, 40 μl / well, and incubate at 37°C for 1 h.

[0072] (5) Wash the plate three times with PBST.

[0073] (6) Dilute the FITC-labeled goat anti-mouse secondary antibody with PBST (200-fold dilution), and add 200-fold diluted Evans blue, 50 μl / well, and incubate at 37°C in the dark for 1 h.

[0074] (7) Wash the plate 3 times with PBST.

[0075] (8) Observation under a fluorescence microscope.

[0076] 2.7.3 Double Antibody Sandwich ELISA Detection (1) Coating: Monoclonal antibodies secreted by strains 4#, 11# and 13# released by coating solution, 0.1 μg / well (100 μl / well), overnight at 4℃.

[0077] (2) Washing: Discard the liquid, wash the plate 5 times with PBST, 5 min each time.

[0078] (3) Sealing: Discard the liquid, add 5% skim milk powder, 150 μl / well, 37℃ for 1h.

[0079] (4) Incubation with antigen: Discard the solution, add the antigen to be tested, 100 μl / well, incubate at 37℃ for 1 h. At the same time, add F81 cell supernatant as a negative control.

[0080] (5) Washing: Discard the liquid, wash the plate 5 times with PBST, 5 min each time.

[0081] (6) Antibody detection: Discard the liquid, add 100 μl of purified feline FCV positive serum (2000-fold dilution) diluted with 5% skim milk powder, and incubate at 37°C for 1 h.

[0082] (7) Washing: Discard the liquid, wash the plate 5 times with PBST, 5 min each time.

[0083] (8) Incubate secondary antibody: Discard the liquid, add HRP-labeled secondary antibody diluted with 5% skim milk powder (15000 times dilution), 100 μl / well, 37℃ for 1h.

[0084] (9) Washing: Discard the liquid, wash the plate 5 times with PBST, 5 min each time.

[0085] (10) Color development: Discard the liquid, add TMB color development solution (Sigma) in the dark, 100 μl / well, and develop color for 3 min in the dark.

[0086] (11) Terminate color development: Add 0.5M H2SO4, 50 μl / well.

[0087] (12) Reading: Use the machine to read the OD value immediately.

[0088] 2.7.4 Immunohistochemistry (1) Dewaxing and hydration: Paraffin sections were prepared from the tongues of cats infected with FCV that died, and the sections were placed in a staining box for dewaxing. The dewaxed staining racks were removed, drained, and placed in a staining box containing anhydrous ethanol. The sections were then placed in 3% H2O2 for 3 min to eliminate the activity of endogenous peroxidase in the tissue. The sections were then placed in PBS at pH 7.4 and allowed to stand.

[0089] (2) High-pressure antigen retrieval: Heat the water in a pressure cooker to 100°C and place the staining rack inside.

[0090] (3) Blocking: Block with 10% goat serum and place in a humidified box at room temperature for 30 min.

[0091] (4) Primary antibody incubation: Discard the blocking solution, add 13# FCV monoclonal antibody diluted with 1% BSA, and incubate overnight at 4°C. Place the slides in a staining rack and wash four times in PBST.

[0092] (5) Secondary antibody incubation: Add 1% BSA diluted goat anti-mouse IgG to the slide and incubate at 37°C for 1 hour in a humidified chamber. Place the slide in a staining rack and wash four times in PBST.

[0093] (6) DAB staining: Add DAB staining solution to the tissue sections and place them in a humidified chamber at room temperature for 10-15 min. Place the sections in a staining rack and wash them twice in PBS for 5 min each time.

[0094] (7) Hematoxylin staining: Add hematoxylin staining solution to the tissue section and place it in a humidified box at room temperature for 30 min.

[0095] (8) Dehydration and mounting: Wash and soak the staining rack in clean water for 30 min, then dry it in an oven. Add neutral resin to the tissue sections and cover them with coverslips.

[0096] (9) Observe and photograph under a microscope. 2.7.5 FITC Marking and Detection (1) The purified 13# FCV monoclonal antibody was replaced with a pH 9-9.5 carbonate buffer using a G25 column.

[0097] (2) Aseptically take an appropriate amount of DMSO and add FITC powder to make a final concentration of 1 mg / ml FITC-DMSO solution.

[0098] (3) Calculate based on adding 0.05 mg of FITC label per mg of antibody. Add FITC-DMSO solution dropwise to the dialyzed antibody while stirring with a magnetic stirrer. After adding the solution, stir at room temperature in the dark for 4 h.

[0099] (4) Use a 10 KD ultrafiltration tube to concentrate the bound antibody to about 2 ml.

[0100] (5) Replace the concentrated fluorescent antibody solution with 10 mmol / L pH 7.2 PBS using a G25 column.

[0101] (6) F81 cells were seeded into 96-well cell culture plates and FCVs were seeded simultaneously. The labeled antibodies were detected by direct immunofluorescence, and negative cell controls were also provided.

[0102] 3 Results 3.1 Serum titer detection One week after the fourth immunization, the serum titer of mice was measured using ELISA. The antibody titer reached 1:12800. The results are shown in Table 1. Figure 2 FJIN-1 mice were selected for shock experiments to induce cell fusion.

[0103] Table 1. Serum titer ELISA detection OD values 3.2 Screening of positive hybridoma cell lines After one cell fusion, 10 cell clones × 93 were selected and preliminarily screened for positive hybridoma cell lines using indirect ELISA, yielding 24 positive hybridoma cell lines. These 24 positive hybridoma cell lines were then subjected to a second screening using indirect ELISA, ultimately resulting in 6 positive hybridoma cell lines: hybridoma cell lines #4, #11, #12, #13, #15, and #16. The results are shown in Tables 2 and 3.

[0104] Table 2 Preliminary screening results of positive hybridoma cell lines (OD values) Table 3. Results of the second screening of positive hybridoma cell lines (OD values) 3.3 Results of Monoclonal Antibody Subclass Identification The monoclonal antibody subtypes of strains 4#, 11#, 12#, 13#, 15#, and 16# were identified. The results showed that strains 4#, 11#, and 15# were IgG1, strain 16# was IgG2a, and strains 12# and 13# were IgG2b, with the light chain type being κ chain in all cases. The results are shown in Table 4. Hybridoma cell line 13# was deposited at the China General Microbiological Culture Collection Center (CGMCC) under the name FCV-13#, accession number CGMCC NO.45747, and deposit date November 21, 2023; hybridoma cell line 11# was also deposited at the same center under the name FCV-11#, accession number CGMCC NO.45746, and deposit date November 21, 2023.

[0105] Table 4. Identification results of monoclonal antibody subclasses 3.4 Results of Monoclonal Antibody Specificity Identification 3.4.1 Western blotting Western blotting analysis of monoclonal antibodies from strains 4#, 11#, 12#, 13#, 15#, and 16# showed that all six monoclonal antibodies specifically reacted with FCV protein. Figure 3 ).

[0106] 3.4.2 Results of indirect immunofluorescence detection For 7 FCV strains (homology comparison see...) Figure 4 Indirect immunofluorescence was used to detect monoclonal antibodies against strains 4#, 11#, and 13#. The results showed that all three monoclonal antibodies could specifically react with most FCV strains, with strains 4# and 13# showing the best efficacy. The results are shown in Table 5. Figure 5 .

[0107] Table 5 Results of indirect immunofluorescence of monoclonal antibodies Note: "×" indicates no fluorescent focus; "+" indicates fluorescence intensity, and more "+" indicates stronger fluorescence.

[0108] 3.4.3 Results of Double Antibody Sandwich ELISA The double-antibody sandwich ELISA method was used to detect 7 FCV strains. The coating was performed with monoclonal antibodies #4, #11, and #13. The results showed that the three monoclonal antibodies could detect most FCV strains, with monoclonal antibody #13 showing the best effect. The results are shown in Table 6.

[0109] Table 6 Results of Double Antibody Sandwich ELISA 3.4.4 Immunohistochemical detection Immunohistochemical staining of the tongues of cats that died from FCV infection using the 13# FCV monoclonal antibody revealed scattered positive substances in epithelial cells, muscle fibers, and subepithelial connective tissue in the tongue samples, while no visible positive substances were found in the negative samples. (See details...) Figure 6 .

[0110] 3.4.5 FITC Marking and Detection FITC-labeled monoclonal antibody #13 was detected by direct immunofluorescence. The results showed that the FITC-labeled antibody could specifically bind to FCV. Figure 7 ).

[0111] Example 3: Preparation of a colloidal gold test strip for detecting feline calicivirus 1. Materials 1.1 The main reagent, goat anti-mouse IgG antibody, was purchased from Boyin Biotechnology.

[0112] 2 methods 2.1 Paired assay for coating and detection antibodies Three FCV monoclonal antibodies, 4#, 11#, and 13#, were labeled with colloidal gold at the same concentration (10 μg / μl), coated with NC membranes at the same concentration, dried at 37°C for 5 hours, and then cross-paired. Positive control was added, and the combination with the strongest signal intensity was selected as the optimal combination of capture antibody and coating antibody.

[0113] 2.2 Preparation of Colloidal Gold Take 1g of chloroauric acid, add purified water to a final volume of 100ml, filter through a 0.22μm filter membrane, and store in a clean glass container in the refrigerator, protected from light. Add 1% chloroauric acid solution to an appropriate amount of purified water to make the concentration of the chloroauric acid solution 0.01%. Add 0.01% chloroauric acid solution to a silanized glass container, place it on a magnetic stirrer, boil for 3 minutes, and then quickly add 1.2ml of 1% trisodium citrate aqueous solution while stirring. Continue heating for 6-8 minutes, cool to room temperature, and then reconstitute the volume with purified water to 100ml. Filter through a 0.22μm filter membrane and store in a clean glass bottle. Store the colloidal gold solution at 2-8℃. Before use, visually inspect the colloidal gold; it should be wine-red in color, with no oily floating matter on the surface and no black particulate matter agglomerated at the bottom of the container.

[0114] 2.3 Preparation of gold-labeled conjugates Place the colloidal gold solution on a magnetic stirrer and stir at 300 g / min. Adjust the pH to 8.5 with 0.2 mol / L potassium carbonate solution. Slowly add feline calicivirus monoclonal antibody 11# dropwise to a final concentration of 8–12 μg / ml. Let stand at room temperature for 120 minutes.

[0115] Add 10% BSA solution to the colloidal gold solution containing the monoclonal antibody to achieve a final concentration of 1%. Mix well and block for 30 minutes or overnight. Centrifuge at 7500 rpm for 20 minutes, discard the supernatant, and rinse with gold-labeled buffer (pH 8.5 Tris). The centrifuged precipitate was resuspended with HCl, 1% BSA, 1% sucrose and 0.5% trehalose to a volume of 1 / 10 of the original volume. This is the gold standard conjugate, which was then refrigerated for later use.

[0116] 2.4 Preparation of gold-labeled conjugate pads Add 300 μl of gold-labeled antigen resuspension (0.02 mol / L Tris) to each 1 ml of gold-labeled conjugate obtained by centrifugation of colloidal gold. HCl (containing 1% BSA, 5% sucrose, 5% trehalose, pH 8.5) was mixed thoroughly and sprayed onto a glass fiber membrane (the gold standard conjugate coating amount was 25 μl / cm). 2 Place the sample in a 37℃ forced-air drying oven for 5 hours. After drying, cut the gold-labeled bonding pad into strips of 4mm × 300mm and seal them for storage.

[0117] 2.5 Coating of nitrocellulose membranes Feline calicivirus monoclonal antibody 13# was diluted with membrane coating buffer (0.02 mol / L PBS, 1.5% sucrose) and sprayed onto a nitrocellulose membrane at a concentration of 1.0 μg / cm as the test line. Goat anti-mouse IgG was diluted with membrane coating buffer (0.02 mol / L PBS, 1.5% sucrose) and sprayed onto a nitrocellulose membrane at a concentration of 1.0 μg / cm 5 mm below the test line as the control line. The coated nitrocellulose membranes were dried at 37°C for 3 hours and then sealed for storage.

[0118] 2.6 Preparation of large plates according to Figure 8 As shown, attach each component (sample pad, colloidal gold pad, absorbent filter paper, NC membrane, etc.) to a clean PVC base plate in sequence. After attaching, add a desiccant and seal for storage.

[0119] 2.7 Finished Product Assembly Cut the large plate into 4mm wide test strips. Assemble the strips by fitting them into the grooves of the plastic card base, cover with the top cover, press firmly, and then use a pressing machine to press the card to form the test strips.

[0120] 2.8 Sensitivity Test The feline calicivirus cell culture medium was diluted 1:10 and 1:100 (the 100-fold dilution resulted in a 10...). 3.0 / TCID 50 (0.1 ml), each dilution was used as the sample to be tested, and each dilution was tested twice.

[0121] 2.9 Specificity test Specific controls were tested. Feline coronavirus swabs (swabs that tested positive by PCR), feline parvovirus cell culture, and feline herpesvirus cell culture were reconstituted with 1 ml of sample processing solution and then tested with this test strip. F81 cell culture medium was melted and then tested directly with this test strip. Each specific control was tested twice.

[0122] 3 Results 3.1 Antibody pairing test and sensitivity test FCV monoclonal antibodies #4, #11, and #13 were cross-paired to prepare colloidal gold test strips for detection. Feline calicivirus cell culture medium was diluted 1:10 and 1:100 (100x dilution yields 10...). 3.0 / TCID 50 / 0.1ml), each dilution was tested twice. When antibody #11 was used as the detection antibody and antibody #13 as the capture antibody, both 1:10 and 1:100 dilutions yielded positive results, as shown in Table 7. Figure 9 , 10 .

[0123] Table 7. Test results after sample dilution Note 1: 1: Antibody #11 is the detection antibody, and antibody #13 is the capture antibody; 2: Antibody #11 is the detection antibody, and antibody #4 is the capture antibody; 3: Antibody #4 is the detection antibody, and antibody #13 is the capture antibody; 4: Antibody #4 is the detection antibody, and antibody #11 is the capture antibody; 5: Antibody #13 is the detection antibody, and antibody #11 is the capture antibody; 6: Antibody #13 is the detection antibody, and antibody #4 is the capture antibody.

[0124] Note 2: "+" indicates a positive result; "-" indicates a negative result. The more "+" signs, the stronger the positive result.

[0125] 3.2 Specificity Detection Using antibody #11 as the detection antibody and antibody #13 as the capture antibody, a colloidal gold test strip was prepared to detect its specificity. The strip was used to detect feline calicivirus, feline parvovirus, feline herpesvirus cell cultures, feline coronavirus swab treatment solution, and F81 cell culture medium. Each sample was tested twice. The results showed that the test was positive for feline calicivirus cell cultures, while the other samples were negative. (See details...) Figure 11 .

[0126] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A pair of hybridoma cell lines producing monoclonal antibodies against feline calicivirus (FCV), characterized in that, One of the hybridoma cell lines is named FCV-13#, with accession number CGMCC NO.45747 and accession date November 21, 2023. The other hybridoma cell line is named FCV-11#, with accession number CGMCC NO.45746 and accession date November 21, 2023.

2. A monoclonal antibody pair against feline calicivirus, characterized in that, The monoclonal antibody pair consists of monoclonal antibodies FCV-13 and FCV-11, wherein monoclonal antibody FCV-13 is produced by the hybridoma cell line FCV-13# as described in claim 1, and monoclonal antibody FCV-11 is produced by the hybridoma cell line FCV-11# as described in claim 1.

3. The monoclonal antibody pair as described in claim 2, characterized in that, The monoclonal antibody FCV-13 is a detection antibody and the monoclonal antibody FCV-11 is a capture antibody, or the monoclonal antibody FCV-13 is a capture antibody and the monoclonal antibody FCV-11 is a detection antibody.

4. The monoclonal antibody pair as described in claim 2 or 3, characterized in that, The monoclonal antibody FCV-11 is used as the detection antibody, and the monoclonal antibody FCV-13 is used as the capture antibody.

5. The use of the hybridoma cell line that produces a pair of monoclonal antibodies against feline calicivirus (FCV) as described in claim 1, or the monoclonal antibody pair against feline calicivirus as described in any one of claims 2-4, in the preparation of a product for detecting feline calicivirus.

6. The application as described in claim 5, characterized in that, The products are formulations, kits, and / or test strips.

7. A test strip for detecting feline calicivirus, characterized in that, The test strip comprises the following components: a sample pad, a colloidal gold pad, absorbent filter paper, and a nitrocellulose membrane; the nitrocellulose membrane is coated with the monoclonal antibody FCV-11 as described in claim 2, and the colloidal gold pad is coated with the colloidal gold-labeled monoclonal antibody FCV-13 as described in claim 2; or the nitrocellulose membrane is coated with the monoclonal antibody FCV-13 as described in claim 2, and the colloidal gold pad is coated with the colloidal gold-labeled monoclonal antibody FCV-11 as described in claim 2.

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