Feline parvovirus monoclonal antibody and application thereof

Abstract

The application provides a feline parvovirus antibody and application, a heavy chain variable region amino acid sequence of the feline parvovirus antibody 3E8 is shown as SEQ ID NO:1; a light chain variable region amino acid sequence is shown as SEQ ID NO:2.The monoclonal therapy group of the parvovirus antibody obtained in the application has a 60% higher cure rate than the adjuvant therapy group, further indicating that the feline parvovirus monoclonal antibody injection has a better treatment effect on the test cat infected with FPV.The monoclonal antibody has very high neutralization activity through in vitro cell level neutralization test and animal protection test, can specifically treat FPV, and is simple to prepare and convenient to operate, and has great significance for preventing and treating the disease.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a feline parvovirus antibody, and especially relates to a feline parvovirus antibody and a specific application thereof. Background Art

[0002] Feline panleukopenia (FP), also known as feline distemper and infectious feline enteritis, is a highly contagious disease of cats caused by the feline parvovirus (FPV). FPV is a linear, single-stranded DNA virus with a genome length of approximately 4.5 to 5.5 kb. It encodes VP1, VP2, and VP3 proteins, all of which are regulated by the P38 promoter. VP2, the primary antigenic protein encoded by FPV, plays a crucial role in immune responses, receptor recognition, and tissue infection.

[0003] Cats infected with FPV develop typical clinical symptoms, including biphasic fever, depression, loss of appetite, diarrhea, vomiting, and dehydration. A significant decrease in leukocytes is a specific indicator of the disease. Depending on the onset of clinical manifestations and severity, the mortality rate in affected cats ranges from 25% to 100%. FPV is the most widespread and pathogenic of the carnivorous parvovirus genus, causing an acute, highly contagious disease characterized by high fever, vomiting, enteritis, and severe leukopenia, with a mortality rate generally ranging from 50% to 60%. The disease occurs worldwide and has been reported in many areas of my country, posing a significant threat to cats and other felines. Currently, vaccination is the most effective preventive and treatment option. There is no specific drug for FPV infection, and clinical practice primarily relies on a combination of symptomatic and supportive care. A highly specific FPV treatment for feline panleukopenia is urgently needed. Moreover, the diagnostic reagents for this disease are also uneven. Therefore, establishing a rapid, accurate, specific and sensitive diagnostic method is not only important for the purification of the cat population epidemic, but also conducive to promoting the process of experimental animalization of experimental cats in my country. Summary of the Invention

[0004] To address the above-mentioned deficiencies, the present invention provides a feline parvovirus monoclonal antibody and a method for preparing the same, as well as an application of the antibody in preparing a drug for treating feline parvovirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1CPE produced by isolated viruses on F81 cells, A: normal F81 cells (40×); B: CPE produced by BJX09 strain on F81 cells (40×);

[0006] Figure 2 PCR test results, where M is Marker DL2000; FPV identification (1: BJX09 virus liquid; 2: negative control; 3: positive control); FHV identification (4: BJX09 virus liquid; 5: negative control; 6: positive control); FCV identification (7: BJX09 virus liquid; 8: negative control; 9: positive control)

[0007] Figure 3 Western-Blot results, including: 1. Protein purified from FPV BJX09 strain; 2. Protein purified from F81 cells. DETAILED DESCRIPTION

[0008] The present invention will be further described below with reference to the accompanying drawings and examples. The following examples are only used to explain the content of the present invention and are not used to limit the scope of protection of the present invention.

[0009] Example 1 Isolation and Identification of Feline Parvovirus BJX09 Strain

[0010] The specimens were obtained from anal swabs of cats suspected of being infected with FPV in a Beijing pet hospital; F81 cells were passaged and preserved by Beijing Broadly Biotechnology Co., Ltd.

[0011] DMEM medium was purchased from Gibco; bovine serum was purchased from Lanzhou Minhai Bioengineering Co., Ltd.; trypsin was purchased from Hyclone; viral genomic DNA / RNA extraction kit and RNA reverse transcription kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; PCR and RT-PCR amplification kits were purchased from Bao Bioengineering (Dalian) Co., Ltd.

[0012] After thoroughly shaking and mixing the anal swab fluid that was positive for FPV identified by PCR, centrifuge at 8000r / min for 8-10 minutes, remove the supernatant and sterilize it by 0.22μm filtration. Inoculate F81 cells simultaneously and add DMEM culture medium containing 2% bovine serum. Incubate the cells in a 37℃, 5% CO2 incubator for observation. When the cytopathic effect (CPE) reaches 80%, freeze and thaw twice, harvest the virus fluid, and pass it according to this method. Under the microscope, typical lesions such as swelling, rounding, shedding, and stringing of cells can be observed. Figure 1 .

[0013] Using the nucleic acid of BJX09 strain as template, PCR amplification was performed using FPV, FHV and FCV specific primers. The specific primers for FPV, FHV and FCV are shown in Table 1. The electrophoresis results showed that only FPV was positive. Figure 2 . It showed that a FPV strain was successfully isolated and named BJX09.

[0014] Table 1 Primer information

[0015]

[0016] Example 1 Preparation and Biological Characterization of Feline Parvovirus Monoclonal Antibodies

[0017] Feline parvovirus BJX09 strain was isolated, identified and stored by Beijing Broadly Biotechnology Co., Ltd. as described in Example 1.

[0018] SP2 / 0 myeloma cells and F81 cells were passaged and stored by Beijing Broadly Biotechnology Co., Ltd.

[0019] DMEM medium was purchased from Gibco; bovine serum was purchased from Lanzhou Minhai Bioengineering Co., Ltd.; viral genomic DNA / RNA extraction kit and RNA reverse transcription kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; PCR and RT-PCR amplification kits were purchased from Bao Bioengineering (Dalian) Co., Ltd.; Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), polyethylene glycol fusion agent (PEG 2000), HAT, HT culture medium, and mouse monoclonal antibody typing kit were purchased from Sigma; horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; antibody purification kit (Protein A) was purchased from abcam; and BCA protein concentration determination kit was purchased from Beijing Solebold Technology Co., Ltd.

[0020] Experimental animals: Female BALB / c mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0021] Hybridoma cell preparation

[0022] Preparation of feline parvovirus solution: The BJX09 strain of feline parvovirus was inoculated onto F81 cells simultaneously, cultured and observed in a 37°C, 5% CO2 incubator. When the cell pathological rate reached about 80%, the virus solution was harvested, frozen and thawed twice, and stored at -70°C for later use.

[0023] The virus liquid was centrifuged at 8000 r / min for 30 minutes at 4°C to remove cell debris, and then the supernatant was ultracentrifuged at 40000 r / min for 3 hours. The precipitate was resuspended with 1% PBS of the original volume and stored for use. Sucrose solutions with concentrations of 60%, 50%, 40%, 30% and 20% were prepared, and 0.8 ml of each sucrose solution was added to a 5 ml centrifuge tube from bottom to top. Then, 1 ml of the resuspended virus was added, and the mixture was ultracentrifuged at 40000 r / min for 3 hours at 4°C. The purified virus band was collected and stored at -70°C for use as an antigen. The TCID was calculated according to the Reed-Muench method. 50 The protein content was determined by the BCA method.

[0024] Six to eight-week-old female BALB / c mice were selected. The purified FPV BJX09 strain antigen solution 100 μg 150 μl was emulsified with an equal volume of Freund's complete adjuvant, and then the BALB / c mice were injected intraperitoneally. Two weeks and four weeks after the first immunization, the purified virus was emulsified with an equal volume of Freund's incomplete adjuvant for the second and third immunizations. After the third immunization, the serum titer was measured every week by ELISA. When the ELISA titer reached more than 1:10000, the BALB / c mice were injected intraperitoneally with 100 μg 150 μl of FPV antigen without adjuvant for booster immunization. Three days later, the mouse spleen was taken for fusion.

[0025] Cell fusion: Preparation of immune mouse spleen cells: On the third day after booster immunization, the immune mouse spleen was taken aseptically and placed in a dish containing DMEM medium. The attached connective tissue and fat were removed by gentle washing. The spleen was cut into small pieces with scissors and ground on a 200-mesh copper screen with a syringe core. The spleen cells were washed into the solution by passing through the mesh. The spleen cell solution was transferred to a centrifuge tube and centrifuged at 1000 r / min for 5 minutes. The supernatant was discarded. The cells were washed once by the same method. Then the cells were resuspended in DMEM medium. The cell suspension was counted and stored for use.

[0026] Preparation of SP2 / 0 myeloma cells: 36-48 hours before fusion, the SP2 / 0 cells were subcultured in 75 cm 2 cell bottles. On the day of fusion, the SP2 / 0 cells with good morphology and logarithmic growth were gently blown off the bottle wall and collected in a centrifuge tube. The cells were centrifuged at 1000 r / min for 5 minutes, and then resuspended in an appropriate amount of DMEM medium for cell fusion.

[0027] Preparation of feeder cells: On the day of cell fusion or one day before, blood was collected from the eyeballs of unimmunized BALB / c mice (8-12 weeks old), and the mice were killed by cervical dislocation and immersed in 75% alcohol for 5 minutes. The mice were placed in a wax tray on a clean bench and fixed with the abdomen facing upwards. The skin of the BALB / c mice was lifted with sterilized tweezers, and the abdominal skin was carefully cut open to separate the skin and peritoneum, fully exposing the peritoneum. An appropriate amount of DMEM culture medium was injected into the abdominal cavity with a sterile syringe, and the abdomen was massaged. After the peritoneal cells and the injected DMEM culture medium were fully mixed, the intraperitoneal fluid was extracted and added to a sterile centrifuge tube. The washing was repeated 2-3 times. The obtained liquid was centrifuged at 1000 r / min for 5 minutes, and the supernatant was discarded. An appropriate amount of DMEM culture medium was added to the precipitate to resuspend the cells. After counting the above cell suspension, the cells were suspended in culture medium containing HAT to adjust the cell concentration to 1-2×10 5 The cells were added into a 96-well cell culture plate (100 μl / well) and cultured in a 37° C., 5% CO 2 incubator until ready for use.

[0028] Cell fusion: Splenocytes from immunized mice were mixed with SP2 / 0 cells in the logarithmic growth phase at a ratio of 5-10:1 in a centrifuge tube. The cells were centrifuged at 1000 rpm for 8 minutes, the supernatant discarded, and the bottom of the tube gently tapped to loosen the pellet into a paste. 1 ml of 50% PEG 2000 (pH 8.0) solution preheated to 37°C was slowly added dropwise to initiate fusion. 25 ml of DMEM medium was added to the centrifuge tube to terminate fusion. The fused cells were centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells were resuspended in HAT-containing medium preheated in a 37°C water bath. The cells were then added to a 96-well culture plate containing feeder cells at a rate of 100 μl / well and incubated in a 37°C, 5% CO2 incubator. Cell growth was recorded daily. On day 4 after fusion, the medium was replaced by half with 1% HAT selection medium. Approximately 7-10 days after fusion, the medium was replaced by half with 1% HT selection medium and continued to be cultured.

[0029] Cloning of positive hybridoma cells: After half-volume medium exchange, observe cell growth daily. If cloned cells are observed, wait until the cell colonies grow to 1 / 3 of the bottom of the well. Collect the cell supernatant and perform antibody detection by ELISA. Hybridoma cells with positive test results should be cloned promptly. Otherwise, the positive hybridoma cells will be subject to competitive inhibition. Cells that do not secrete antibodies will inhibit the growth of the positive hybridoma cells, resulting in the loss of hybridoma cells that secrete specific antibodies. Cloning is also an important step in obtaining a single cell population and pure, uniform monoclonal antibodies. Therefore, cloning can be used to ensure the long-term and stable secretion of monoclonal antibodies by hybridoma cells.

[0030] This experiment uses the limiting dilution method to clone cells. The specific method is as follows:

[0031] (1) Prepare feeder cells one day in advance.

[0032] (2) Select cells that are in good condition and are single-cell colonies for cloning. Use a pipette to aspirate the cells into a new 24-well cell culture plate and wash them as clean as possible.

[0033] (3) After counting, 3 cells were counted per well and added to a 96-well cell culture plate for culture.

[0034] (4) Observe the cells. When the cells cover 1 / 3 of the bottom of the well, perform ELISA on the cell supernatant to detect whether there are specific antibodies in the supernatant, and mark the corresponding cell wells to detect their positive rate.

[0035] (5) Similarly, cells with good condition and single-cell colonies should be selected for the next cloning. In order to avoid cell contamination, the cloned cells should be frozen in time.

[0036] (6) After three cloning cycles, if the positive rate of the cell supernatant reaches 100%, it can be determined that the positive hybridoma cell line can stably secrete monoclonal antibodies. If it does not reach 100%, cloning should be continued until the positive rate reaches 100%, and the positive wells should be expanded and cultured.

[0037] After the spleen cells of immunized mice were fused with hybridoma cells, a hybridoma cell line that stably secreted anti-FPV monoclonal antibodies was obtained through HAT / HT selection culture, screening, identification and subcloning, and was named 3E8.

[0038] Large-scale preparation and purification of monoclonal antibodies

[0039] The screened positive hybridoma cell lines are cultured for expansion or ascites induction in syngeneic mice to prepare monoclonal antibodies.

[0040] Preparation of monoclonal antibodies by cell expansion culture method: hybridoma cells are cultured in large quantities using a bioreactor fermentation culture method, the cultured hybridoma cell supernatant is collected, centrifuged at 3000 rpm for 10 minutes, and the supernatant is taken or the collected cell supernatant is concentrated by ultrafiltration.

[0041] Preparation of monoclonal antibody ascites in syngeneic mice: BALB / c mice weighing more than 20 g (8-10 weeks old) were selected and sterilized liquid paraffin was injected into the peritoneal cavity of each mouse 0.5 mL; 14-18 days later, hybridoma cells (0.5-1×10 6 cells / mouse); starting from the 7th day after cell implantation, the abdomen of the mouse was observed every day. When the abdomen of the mouse was obviously swollen, 9 #The abdominal cavity is punctured with a needle to collect ascites, usually 3 to 5 times in a row. The ascites is centrifuged at 3000 rpm for 10 minutes to remove oil and precipitate. The supernatant is collected and the antibody titer is measured. Neutralization test results show that the titer of the ascites is approximately 1:35500.

[0042] Purification: Purification by Protein A affinity chromatography and anion-cation exchange chromatography.

[0043] Biological characterization of monoclonal antibodies

[0044] Monoclonal antibody subtype identification: Identification was performed according to the instructions of the Mouse Monoclonal Antibody Identification Kit. The results showed that the subtype of monoclonal antibody 3E8 was IgG1, as shown in Table 2.

[0045] Table 2 Monoclonal antibody subclass identification results

[0046]

[0047] Specificity identification of monoclonal antibodies: Feline parvovirus (BJX09 strain), feline herpesvirus (BJS01 strain) and feline calicivirus (BJH13 strain) were diluted to 200 TCID in DMEM cell culture medium containing 4% bovine serum. 50 After 0.1 ml, the samples were mixed with an equal volume of monoclonal antibodies secreted by 3E8 cells, neutralized at 37°C for 1 hour, and inoculated into 96-well cell culture plates (① for samples neutralized with FPV: 100 μl of F81 cell suspension containing 2% bovine serum was added to each well; ② for samples neutralized with FHV: inoculated onto CRFK cells that had grown into a monolayer; ③ for samples neutralized with FCV: inoculated onto F81 cells that had grown into a monolayer), with each sample inoculated into 4 wells, 100 μl per well; normal cell controls and virus controls (virus (200 TCID 50 1 ml (0.1 ml) was mixed with an equal volume of DMEM medium and neutralized at 37°C for 1 hour in four wells. The cells were then cultured in a 37°C, 5% CO2 incubator and observed for 4-7 days. The results showed that the monoclonal antibody secreted by 3E8 cells did not exhibit CPE in the FPV-neutralized wells, but did exhibit CPE in the FHV-neutralized wells and the FCV-neutralized wells. No CPE was observed in the normal cell control wells, but CPE was observed in the virus control wells. These results demonstrate that the monoclonal antibody is specific for FPV.

[0048] To determine the stability of monoclonal antibody secretion, hybridoma cells were serially passaged to passage P20 and cryopreserved in liquid nitrogen for 3, 6, 9, and 12 months before thawing. When cells reached confluence, the culture medium was centrifuged and the supernatant was collected for analysis according to the "Neutralization Test" in the Appendix of the current Chinese Veterinary Pharmacopoeia. The results showed that the neutralization titer of hybridoma cells serially passaged to passage P20 ranged from 1:316 to 501. After 12 months of storage in liquid nitrogen, the antibody titer of 3E8 cells at passage P20 was 1:398 to 501, indicating that hybridoma cells secrete stable antibodies. The results are shown in Tables 3 and 4.

[0049] Table 3 Neutralization titer determination results of hybridoma cell supernatants at different passages

[0050]

[0051] Table 4 Neutralization titer determination results of hybridoma cell supernatants of 3E8 P20 generation stored in liquid nitrogen for different time periods

[0052]

[0053] Western Blot Identification of Monoclonal Antibodies: Purified FPV BJX09 strain and F81 cells purified by the same method were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. Western Blot analysis was performed with the monoclonal antibody secreted by the 3E8 hybridoma cells. The 3E8 monoclonal antibody reacted with the FPV BJX09 strain to produce a clear, single band at approximately 65 kD, but did not react with F81 cells. Figure 3 The results of the study showed that the 3E8 monoclonal antibody is a neutralizing monoclonal antibody specific for FPV-VP2, which has neutralizing activity against FPV infection and produces antigen-antibody reactions with cell tissues.

[0054] Chromosome number analysis of hybridoma cells: Take the hybridoma cells in the logarithmic growth phase, add colchicine at 0.1 μg / ml in the culture medium, and continue to culture at 37°C, 5% CO2 cell incubator for 6-10 hours; blow the cells with DMEM medium, centrifuge at 1000 r / min for 10 minutes, discard the supernatant, add 5 ml of 37°C preheated 0.075 mol / L KCl solution, mix well, and place at 37°C for 30 minutes; add 1 ml of freshly prepared acetic acid:methanol (1:3) fixing solution to the cell suspension, mix well, and fix the cell surface slightly to prevent cell adhesion into a group; centrifuge at 1000 r / min for 10 minutes, discard the supernatant, add 5-10 ml of fixing solution, mix well, and stand at room temperature for 30 minutes; repeat the fixing for 3 times, finally add 5 ml of fixing solution to suspend the cells and mix well, and stand at 4°C overnight; the next day, discard part of the supernatant, add 0.5-1 ml of fixing solution to resuspend the cells, add 1-2 drops of cell suspension to a pre-cooled glass slide, and dry at room temperature; after staining with Giemsa staining solution, select cells with well-dispersed chromosomes, no overlapping, and no loss under a microscope for observation and analysis, and record the number of chromosomes. The chromosome number of normal BALB / c mouse spleen cells is 40, and the chromosome number of myeloma cells is 60-70, so the chromosome number of the fused cells should be between 100-110. The test results show that the chromosome number of 3E8 hybridoma cells changes in the range, with an average of 107, which meets the expected number.

[0055] Identification of the purity of the monoclonal antibody: the purified monoclonal antibody is identified by SDS-PAGE for its purity, and the above monoclonal antibody is sent to Huada (Beijing) Company for sequencing, and the amino acid sequence of the heavy chain variable region is: QVQLQLPGGTELVKGGLVQPGSPGASVRMSCKASGYAFTSYWIHAVKRLGKSAGTQGLEWIGEAYLQINPSGRSNSNKFK TKALTVDVWGPGVYLSTAYMQSSLHTSEDSLYYARTKLGPKLYLFPLWGTLTVLAAK (SEQ ID NO. 1).

[0056] The amino acid sequence of the light chain variable region is: DIQMTSPQFSLSKALLSASVGESLGDTVTVIYQTITCRSYKRLIYAALEWLQQLVYNAKHQAEDVATLAGKVLPSRIGSL SGTLKRQGDKPNSLQPEDFGYGTKLYAASENHYSPYFGGTLMK (SEQ ID NO. 2).

[0057] Example 3 Therapeutic effect of feline parvovirus monoclonal antibody on cats artificially infected with feline parvovirus virulent strain

[0058] Feline parvovirus BJX09 strain was isolated, identified and preserved in our laboratory as described in Example 1.

[0059] The 3E8 feline parvovirus monoclonal antibody injection prepared in Example 2 (neutralizing antibody titer of 1:1024, properties, pH value, sterility, mycoplasma, and exogenous virus tests all passed) was prepared in this laboratory.

[0060] Cats aged 2 to 3 months (FPV, FHV, and FCV antigens negative, FPV neutralizing antibody titer no higher than 1:2) were purchased from a pet market in Gu'an.

[0061] Take FPV BJX09 strain (virus content is 10 4.0 TCID 50 Twelve cats were challenged with 4 ml of the drug orally. Anal swabs were collected daily to measure FPV antigen and blood was collected to measure white blood cell count. Mental status, appetite, and clinical symptoms such as diarrhea, retching, vomiting, and death were recorded.

[0062] According to the disease criteria, 10 cats with the disease were selected and randomly divided into two groups, 5 cats in each group. The first group received monoclonal antibody treatment, while the second group received adjuvant treatment without treatment.

[0063] The criteria for onset of the disease are: ① Depression and loss of appetite, lasting for more than 2 days; ② Diarrhea symptoms: watery stools; loose stools, yellow or grayish white; bloody stools, pink or coal tar-like; ③ Dry heaves and vomiting; ④ Significant decrease in white blood cells, with a white blood cell count of <4000 / mm 3 If ④ appears and any one of ①, ② and ③ appears, it is considered to be onset.

[0064] Monoclonal antibody treatment group: Feline parvovirus monoclonal antibody injection was injected intramuscularly at a dose of 1 mL / kg, and ceftiofur sodium was injected subcutaneously at a dose of 5 mg / kg. Appropriate fluid replacement therapy (0.9% saline and 5% glucose solution) was also given once a day for 5 consecutive days.

[0065] Adjuvant treatment group: Ceftiofur sodium was injected subcutaneously at a dose of 5 mg / kg and appropriate fluid replacement therapy (0.9% saline, 5% glucose solution) was given once a day for 5 consecutive days.

[0066] On the 10th day after challenge, the cure rate of cats in the monoclonal antibody treatment group was 80% (4 / 5) after 5 days of treatment, and the cure rate of cats in the adjuvant treatment group was 20% (1 / 5).

[0067] The cure criteria are: ① normal spirit and appetite; ② no diarrhea; ③ no retching or vomiting; ④ normal white blood cell count, ≥ 4000 cells / mm 3Patients who meet the above clinical manifestations are considered cured.

[0068] The results showed that the cure rate in the monoclonal antibody treatment group was 60% higher than that in the adjuvant therapy group, further demonstrating the effectiveness of the feline parvovirus monoclonal antibody injection in treating FPV-infected cats. In vitro cell-based neutralization tests and animal protection studies have demonstrated that this monoclonal antibody has high neutralizing activity and is specifically effective in treating FPV. Its simple preparation and ease of use make it of great significance for the prevention and treatment of the disease.

[0069] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody 3E8 against feline parvovirus, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3E8 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. A composition comprising the monoclonal antibody 3E8 according to claim 1.

3. Use of the monoclonal antibody 3E8 as claimed in claim 1 in the preparation of a therapeutic agent for treating feline parvovirus infection.

4. Use of the monoclonal antibody 3E8 according to claim 1 in the preparation of a reagent for detecting feline parvovirus.

5. A kit for detecting feline parvovirus, characterized in that: The kit comprises the monoclonal antibody 3E8 according to claim 1.

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