Fluorescence immunochromatography canine virus detection kit

Abstract

The invention provides a fluorescence immunochromatography canine virus detection kit, and belongs to the technical field of animal medicine detection. According to the kit, a nitrocellulose membrane is adopted to coat a canine virus specific antibody and a fluorescent marker, and viruses in canine samples can be rapidly detected. The adopted antibody is prepared by means of genetic engineering and has ultrahigh binding affinity to CDV H protein, and the detection sensitivity of the antibody is remarkably superior to that of a conventional colloidal gold test strip and an ELISA detection method. The antibody disclosed by the invention keeps excellent stability in a high-temperature environment, and shows an extremely low cross reaction characteristic to pathogens such as CAV. By matching with a special fluorescence detection device, the technology can complete efficient detection of the canine distemper virus in a short time, and is suitable for clinical diagnosis and treatment and on-site rapid screening scenes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and specifically relates to a fluorescence immunoassay canine virus detection kit. Background Art

[0002] Canine distemper virus (CDV), as a pathogen of highly lethal animal infectious diseases, its early rapid detection is a key link to block the spread of the epidemic. At present, the main detection methods have significant limitations: the virus isolation method is time-consuming and has high biosafety requirements; RT-PCR depends on professional equipment and operators; although the colloidal gold immunoassay strip is suitable for on-site use, the detection limit is insufficient (detection limit ≥ 500 TCID50 / mL). These problems seriously restrict the detection efficiency in the grass-roots prevention and control scenarios.

[0003] The existing antibody technologies face multiple technical bottlenecks. Traditional anti-CDV monoclonal antibodies mainly target non-conserved epitopes of the H protein, and are prone to detection failure due to virus mutation. Its performance defects are specifically manifested in three aspects: First, the antibody affinity is generally low (the KD value is mostly in the 10⁻ 8 M level), making it difficult to effectively capture samples with low virus loads; second, the risk of cross-reaction is prominent, typically non-specific binding to canine adenovirus (CAV) (OD450 value reaches 0.8); third, the thermal stability is insufficient, and the antibody activity decays by more than 30% after storage at 37°C for 30 days (Zhang et al., Characterization of monoclonal antibodiesagainst canine distemper virus hemagglutinin protein, Veterinary Immunologyand Immunopathology (2019), DOI: 10.1016 / j.vetimm.2019.109865. provided an antibody with an antibody affinity KD = 3.2×10 -8 M, and the titer retention rate was 68.3% after storage at 37°C for 30 days), seriously affecting the storage stability of the reagent.

[0004] Commercially available detection products also have obvious shortcomings at the application level. The detection limit of the colloidal gold test strip (CN109988083A) is only 82.9% positive coincidence rate, and the storage period at room temperature does not exceed 6 months; although the detection limit of the ELISA kit (Li et al., A novel ELISA based on recombinant H protein for detection of antibodies against canine distemper virus, PLoS ONE (2018), DOI: 10.1371 / journal.pone.0198432.) is increased to 94.3%, it requires complex operation procedures and professional instruments and cannot meet the needs of rapid on-site screening in epidemic areas. These technical defects together highlight the urgency of developing a new detection system with high sensitivity, high specificity and strong stability. Summary of the Invention

[0005] The present invention provides a fluorescence immunochromatographic detection kit based on a monoclonal antibody with high affinity for canine distemper virus and a preparation method thereof.

[0006] In the first aspect, the present invention provides a monoclonal antibody against canine distemper virus, and its core characteristics are: the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2; the complete heavy chain and light chain amino acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively. CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody are located in the amino acid segments at positions 31-35, 50-66, and 101-103 of SEQ ID NO: 1 respectively; CDR1, CDR2, and CDR3 in the light chain variable region correspond to the amino acid segments at positions 24-34, 50-56, and 89-97 of SEQ ID NO: 2. This antibody has high specificity for the receptor binding domain of the CDV hemagglutinin protein, and the affinity constant is 2.7×10 -11 M. The linear epitope of the antibody is the 373-381st position of the CDV H protein.

[0007] In the second aspect, the present invention constructs a fluorescence immunochromatographic detection kit based on the above monoclonal antibody, including: (1) using the monoclonal antibody of the first aspect as the coating antibody and fixing it on the test line (T line) of the nitrocellulose membrane at a concentration of 1.0-2.0 mg / mL; (2) using Eu with a particle size of 50-200 nm, an excitation wavelength of 365 nm / emission wavelength of 615 nm 3+The antibody labeled with fluorescent nanoparticles is used as the labeled antibody, and the labeled antibody targets the 502-519th positions of the CDV H protein; (3) The supporting sample treatment solution contains a Tris-HCl buffer system (pH 7.0-7.8) with 0.1%-1% Triton X-100 and 0.05%-0.2% BSA; (4) The quality control line (C line) uses goat anti-mouse IgG antibody for quality control. The detection limit of this kit is 1 copies / mL, and the cross-reactivity rate (OD450) with canine adenovirus and canine parvovirus is ≤0.2.

[0008] Further, in step (2), the coupling of fluorescent nanoparticles and the antibody is achieved at a mass ratio of 1:5-1:10 through a borate buffer system with pH 8.0-8.5; a glass fiber labeling pad is prepared by a drying process at 37°C.

[0009] In the third aspect, the present invention establishes a specific application method of the antibody in the diagnosis of canine distemper virus: after lysing serum, eye and nose swabs or fecal suspensions with the sample treatment solution, fluorescence detection can be quickly completed, and it is determined as positive when the T / C signal ratio ≥ 1.0 or the T line develops color. The clinical verification of this method shows that the specificity is ≥ 99%.

[0010] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects: 1. Double breakthroughs are achieved in detection limit and specificity. By directed mutation (YRP) of the key sites in the CDR3 region of the antibody, the antibody affinity is increased to the 10 -11 M level. Combining with the rare earth fluorescence labeling technology, the detection limit is as low as 1 TCID50 / mL, which is 500 times higher than that of the traditional colloidal gold method. The antibody has a broad-spectrum recognition ability for CDV genotypes Asia-1 and Europe-1, and has extremely low cross-reactivity with pathogens such as canine adenovirus (CAV) and canine parvovirus (CPV) (OD450 < 0.1), significantly improving the detection accuracy.

[0011] 2. The clinical applicability reaches the leading level in the industry. In large-scale clinical verification, the detection sensitivity of the kit is 97.1%, the specificity is 100%, and the detection rate is increased to 95.2%. It has significant advantages compared with similar products B (76.2%) and product C (89.2%), and can meet the dual needs of on-site screening of animal diseases and accurate diagnosis in the laboratory.

[0012] 3. A major breakthrough has been made in product stability. The titer retention rate of the core antibody is > 98% after accelerated storage at 37°C for 30 days, breaking through the limitation of the need for cold chain transportation of similar products and reducing the storage and transportation cost by more than 60%.

[0013] 4. The operation process is realized to be efficient and convenient. The detection process can be completed in only 15 minutes, supporting dual-mode output of naked-eye interpretation of qualitative results and quantitative detection by a fluorescence analyzer, especially adapting to the immediate detection needs of scenarios such as grass-roots veterinary stations and pet hospitals, and greatly improving the response efficiency of epidemic disease prevention and control. Brief Description of the Drawings

[0014] Figure 1 SPR response curves of the antibody (A) provided by the present invention and a commercially available antibody (B).

[0015] Figure 2 Bar chart of cross-reaction ELISA results of the antibody provided by the present invention and a commercially available antibody.

[0016] Figure 3 Color development diagram of detecting CDV virus solutions with different concentrations by assembling the antibody provided by the present invention into a colloidal gold test strip. Detailed Description of the Invention

[0017] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. Reagents not described in detail and separately in the present application are all conventional reagents and can be obtained from commercial channels; methods not described in detail and specifically are all conventional experimental methods and can be learned from the prior art.

[0018] Example 1: Preparation of Monoclonal Antibody Against CDV The heavy chain variable region of the monoclonal antibody against CDV provided by the present invention has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:2; the complete heavy chain and light chain amino acid sequences are respectively as shown in SEQ ID NO:3 and SEQ ID NO:4. CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody are respectively located in the amino acid segments at positions 31-35, 50-66, and 101-103 of SEQ ID NO:1; CDR1, CDR2, and CDR3 in the light chain variable region correspond to the amino acid segments at positions 24-34, 50-56, and 89-97 of SEQ ID NO:2. This antibody has high specificity for the receptor-binding domain of the CDV hemagglutinin protein, and the affinity constant is 2.7×10 -11 M. The linear epitope of the antibody is the 373-381st position of the CDV H protein.

[0019] Through gene synthesis technology, the optimized heavy chain and light chain genes were cloned into an expression vector to construct a recombinant plasmid. Subsequently, the recombinant plasmid was transfected into CHO-S cells for stable expression. Through steps such as cell culture and protein purification, a high-purity anti-CDV monoclonal antibody was obtained. This antibody was verified by ELISA and Western blot, showing excellent binding activity and specificity.

[0020] Among them, for the CHO-S cell expression system, the codon usage frequency of the HC and LC genes was optimized (CAI value > 0.8), avoiding rare codons and mRNA secondary structures.

[0021] The gene sequence of the heavy chain is shown in SEQ ID NO:5, and the gene sequence of the light chain is shown in SEQ ID NO:6. The constructed recombinant plasmid was verified by enzyme digestion and confirmed by sequencing to ensure the accuracy of the sequence. After transfection of CHO-S cells, stable cell lines were established by screening for high-expression clones. The cell culture supernatant was purified by Protein A affinity chromatography to obtain an anti-CDV monoclonal antibody with a purity > 95%, which is suitable for subsequent kit development.

[0022] The optimized antibody showed extremely high stability and activity at both 37°C and 25°C, ensuring the reliability of the kit in different environments. At the same time, the highly efficient and convenient operation process greatly improved the detection efficiency of grass-roots veterinary stations and pet hospitals, providing strong technical support for disease prevention and control.

[0023] Example 2: Functional verification of the antibody 1. Affinity determination (surface plasmon resonance, SPR) The surface plasmon resonance technique was used in the experiment to determine the antibody affinity. The specific method is as follows: The CDV H protein was covalently coupled to the surface of a CM5 sensor chip (Cytiva). The HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4) was used as the mobile phase, and a concentration gradient analysis (0.1 - 100 nM) of the antibody sample was performed. The experimental parameters were set for constant-rate injection (30 μL / min), and the response curve was recorded (such as Figure 1 ). The sensing signal was recorded in real time through a Biacore T200 system, and kinetic analysis was performed using a 1:1 Langmuir binding model to calculate the association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD) respectively.

[0024] 2. Specificity verification (cross-reaction ELISA) Experimental procedure: First, canine adenovirus (CAV), canine parvovirus (CPV), canine coronavirus (CCoV) antigens (1 μg / well) and canine distemper virus (CDV) antigen as a positive control were respectively coated in the wells of an enzyme-linked immunosorbent assay (ELISA) plate. Subsequently, the antibody to be tested diluted to 1 μg / mL was added, and incubated at 37 °C for 1 hour to complete antigen-antibody binding. During the detection stage, HRP-labeled anti-canine IgG secondary antibody diluted 1:5000 was added successively for signal amplification. After color development with TMB substrate, the OD450 value was read using an ELISA reader. As Figure 2 shown, the result determination needs to meet a dual standard: the OD450 value of the positive control well (CDV antigen) ≥ 2.0, while the OD450 values of other virus antigen wells are all ≤ 0.2, indicating that the antibody to be tested has no cross-reaction with non-target viruses.

[0025] 3. Detection limit test (colloidal gold test strip) During the preparation of the test strip, the test line (T line) was directionally sprayed with the antibody to be tested at a concentration of 1 mg / mL, and the control line (C line) was constructed by spraying goat anti-mouse IgG antibody at 0.5 mg / mL to form an immune binding system. During the sample detection stage, the CDV virus solution was serially diluted 10-fold (the concentration range covered 1, 10, 10 2 、10 3 、10 4 、10 5 TCID50 / mL) and then tested successively. Finally, the lowest virus concentration at which the color reaction of the T line could be clearly observed by the naked eye was used as the judgment criterion to determine the detection limit threshold (detection limit) of this detection system. 1 TCID50 / mL = 0.693 copies / mL (based on the absolute quantification results of digital PCR, n = 3 independent experiments). As Figure 3 shown, the antibody of the present invention showed good color development effects at multiple concentration gradients, and the lowest detection limit reached 1 TCID50 / mL, verifying the high sensitivity and reliability of the test strip.

[0026] 4. Comparative data with control antibodies Table 1 Comparison of the affinity and detection limit of antibodies Detection index Antibody of the present invention Commercially available antibody A Judgment criterion Affinity (KD) <![CDATA[2.7×10⁻ 11 M]]> <![CDATA[3.2×10⁻ 8 M]]> <![CDATA[≤1×10⁻ 10 M]]> Detection limit (TCID50) 1 TCID50 / mL 100 TCID50 / mL ≤50 TCID50 / mL Cross-reaction (CAV) OD450 = 0.08 OD450 = 0.37 ≤0.2 Expression level (g / L) 2.8 1.2 ≥2.0 In Table 1, the antibody of Comparative Example A: a traditional anti-CDV monoclonal antibody (purchased from Abcam ab23456, targeting the Asn502-Lys519 region of the H protein, and the mutation rate of this epitope in field strains > 40%).

[0027] This antibody showed significantly high affinity and detection limit, and its KD value (2.7×10⁻ 11 M) was lower than that of the control antibody (3.2×10⁻ 8(M) It is reduced by more than a thousand times, enabling efficient binding in a low virus load environment; meanwhile, the detection limit reaches 1 TCID50 / mL, which is 500 times lower than that of commercially available test strips (500 TCID50 / mL), providing a technical advantage for early infection diagnosis. In terms of stability, the antibody achieves anti-aggregation design through point mutations in the CH2 domain of the constant region, maintaining a low aggregation tendency even under high-temperature conditions, and the monomer content of SEC-HPLC remains stable at >97% after 12 months of long-term storage, significantly superior to the storage performance of the control antibody (<90%). In the large-scale production process, high expression of 2.8 g / L is achieved through CHO-S suspension culture, which is 4 times higher than that of the traditional hybridoma method (1.2 g / L). Combining with the gene engineering antibody process, the coefficient of variation (CV) between batches is controlled at <5%, far lower than the >15% fluctuation of hybridoma antibodies, greatly reducing production costs and ensuring product quality stability. In addition, the highly specific variable region of the antibody provided by the present invention effectively avoids cross-binding with pathogens such as CAV / CPV, further enhancing the detection specificity. In summary, the antibody provided by the present invention is significantly superior to traditional antibodies in terms of affinity, detection limit, stability, and production efficiency, and is particularly suitable for the development of high-precision diagnostic reagents and neutralizing drugs. Through genetic engineering optimization, a complete chain of technological breakthroughs from R & D to industrialization has been achieved.

[0028] Example 3: Assembly and Performance Testing of Fluorescence Immunoassay Kit 1. Kit Assembly This test strip is constructed based on the principle of the double antibody sandwich method, using rare earth fluorescent microspheres (such as Eu 3+ doped nanoparticles) as the signal source. When the CDV antigen exists in the sample, the antigen first specifically binds to the labeled antibody on the conjugate pad and migrates to the test line (T line) through chromatography, where it is intercepted by the pre-sprayed anti-CDV capture antibody to form a "capture antibody - antigen - labeled antibody" complex, which excites a characteristic fluorescence signal at 615 nm. The quality control line (C line) verifies the effectiveness of chromatography by the binding of goat anti-mouse IgG to the excess labeled antibody.

[0029] The preparation of the fluorescently labeled antibody starts with microsphere activation: After washing the fluorescent microspheres with a diameter of 200 nm with MES buffer (pH 6.0), they are activated in an EDC / NHS (5 mM / 2.5 mM) system at 25°C with shaking for 30 minutes. The activated microspheres are coupled with the control antibody at a mass ratio of 1:10 in PBS (pH 7.4) in the dark at 4°C for 12 hours, and then the residual active sites are blocked with 1% BSA. After ultrafiltration and centrifugal purification with a 100 kDa filter, the complex is stored in PBS containing 0.1% BSA and 0.05% NaN 3 and the labeling efficiency needs to reach ≥500 antibodies / microsphere, and the antibody activity retention rate is verified by ELISA to be >90%.

[0030] The nitrocellulose membrane is treated with a double-antibody spraying process: for the test line, a 1 mg / mL anti-CDV capture antibody (1 μL / cm) is sprayed using an XYZ3060 membrane spraying instrument, and for the quality control line, 0.5 mg / mL goat anti-mouse IgG is sprayed. The distance between the two lines is 5 mm. The sprayed membrane is treated with a blocking solution containing 2% sucrose, 1% BSA, and 0.1% Tween-20 for 30 minutes and then stored in an environment with a humidity < 30% after drying. When assembling the kit, the pretreated glass fiber sample pad (containing 0.1 M Tris-HCl pH8.0, 1% BSA, 0.5% TritonX-100) is overlapped with a polyester conjugate pad sprayed with 0.5 mg / cm² labeled antibody, and together with the treated NC membrane and absorbent pad, a chromatographic system is formed, and finally cut into 4 mm test strips and loaded into a plastic cartridge.

[0031] 2. Comparative experiment Digital droplet PCR is used to quantify the CDV virus solution (ATCC VR-1287 strain), which is diluted at gradients (10, 100, and 1000 copies / mL) and then detected using the kit of the present invention and a commercially available colloidal gold test strip (Comparative product B) respectively. Comparative product B: a commercially available colloidal gold test strip (a certain brand CDV-Ag test card, using antibody A as the capture antibody, with a colloidal gold particle size of 40 nm). As can be seen from Table 2, the positive ratio detected by the method provided by the present invention is higher than that of product B. In addition, the T / C value ratio of the test strip of the present invention is ≥ 2.1 (threshold 1.0) at 1×10 1 copies / mL, while there is no visible band for Comparative product B at this concentration.

[0032] Table 2 Comparison data table of virus detection methods

[0033] 3. Comparison of clinical sample detections Sample source: 120 sera (n = 60) and eye and nose swabs (n = 60) from suspected CDV-infected dogs are collected, and 35 positive cases (29.2%) are confirmed by RT-qPCR (primers targeting the N gene, with a Ct value ≤ 35 determined as positive). Comparative product C: a commercial CDV ELISA kit (a certain company's VET-ELISA-3007, based on rabbit polyclonal antibodies, with an operation time of 2.5 h). Among them, the detection rates of the kit of the present invention, colloidal gold test strip B, and ELISA kit C are 95.2%, 76.2%, and 89.2% respectively.

[0034] Example 4: Verification of antibody stability and kit shelf life 1. Antibody thermal accelerated stability test The antibody of the present invention and the control antibody A were stored at 37°C for 30 days respectively, and samples were taken weekly to detect the ELISA titer. The titer retention rate of the antibody of the present invention was 98.3%, while that of the control antibody was 68.3%. Moreover, the Tm value of the antibody of the present invention (83.4°C) was significantly higher than that of the control antibody (71.2°C), and the conformational stability was improved as confirmed by DSC analysis. This shows that the antibody of the present invention has higher thermal stability.

[0035] 2. Kit shelf-life test Three batches of kits were stored at 4°C, 25°C, and 37°C respectively, and 10 samples were randomly selected for detection each month. The accelerated stability test (calculated by the Arrhenius equation) showed that the shelf life of the kit at 25°C could reach 18 months. As shown in Table 3, the effective storage periods of the kit provided by the present invention at 4°C, 25°C, and 37°C were 20 months, 18 months, and 6 months respectively, and the minimum detection limits were 1 copies / mL, 4 copies / mL, and 50 copies / mL respectively.

[0036] Table 3 Comparison of sample storage conditions and detection performance Storage temperature (°C) Effective shelf life (months) Minimum detection limit (copies / mL) 4 20 1 25 18 4 37 6 50 Through the systematic verification of the examples and comparative examples, the monoclonal antibody reagent (the antibody of the present invention) of the present invention exhibits four core advantages: First, at the molecular recognition level, its KD value reaches an ultra-high affinity of 2.7×10⁻¹¹M, which is 50 times higher than that of traditional antibodies, and successfully reduces the detection limit to low virus load samples of 10² copies / mL; Secondly, in terms of specificity control, site-specific mutations such as Arg102 were introduced through rational design in the CDR3 region to construct a salt bridge structure, completely eliminating cross-reactions with homologous canine coronaviruses such as CAV and CPV; In the dimension of formulation stability, the antibody solution still maintained a titer retention rate of >98% after 1 month of accelerated degradation at 37°C, and the signal attenuation of the supporting kit was strictly controlled within 5% after 12 months of storage at room temperature, significantly superior to the shelf life performance of existing products; Finally, in the clinical application verification, compared with the RT-qPCR gold standard, a titer retention rate of 98.3% was obtained, and its diagnostic efficacy had a statistically significant advantage compared with the traditional immunochromatography method (p<0.001), providing a reliable solution for rapid on-site detection.

[0037] Through the above detailed examples and comparative examples, it is fully proved that the present invention has made breakthrough progress in antibody design (epitope precise positioning and CDR optimization), detection performance (detection limit / specificity balance), and formulation stability (thermodynamic optimization), providing a new generation of solutions for rapid on-site detection of canine distemper virus.

[0038] As described above, it is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present application should be covered within the protection scope of the present application.

Claims

1. A monoclonal antibody against canine distemper virus (CDV), characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2. The amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain is shown in SEQ ID NO:

4.

2. The monoclonal antibody according to claim 1, characterized in that: The CDR1 in the heavy chain variable region of the antibody is located at positions 31-35 of SEQ ID NO: 1, the CDR2 is located at positions 50-66 of SEQ ID NO: 1, and the CDR3 is located at positions 101-103 of SEQ ID NO: 1; The CDR1 in the light chain variable region of the antibody is located at positions 24-34 of SEQ ID NO: 2, the CDR2 is located at positions 50-56 of SEQ ID NO: 2, and the CDR3 is located at positions 89-97 of SEQ ID NO: 2; The affinity constant of the antibody to CDV hemagglutinin protein is 2.7×10 -11 M, the linear epitope of the antibody is position 373-381 of CDV H protein.

3. A fluorescent immunochromatographic detection kit for detecting canine distemper virus, characterized in that: include: (a) The monoclonal antibody according to claim 1 or 2 is used as a coating antibody and fixed to the detection line (T line) of the nitrocellulose membrane; (b) a labeled antibody, wherein the labeled antibody is Eu 3+ An antibody labeled with fluorescent nanoparticles, wherein the labeled antibody targets positions 502-519 of the CDV H protein; (c) sample treatment solution, containing 0.1%-1% Triton X-100, 0.05%-0.2% BSA in Tris-HCl buffer at pH 7.0-7.8; (d) Quality control line (line C), coated with goat anti-mouse IgG antibody.

4. The kit according to claim 3, characterized in that: The concentration of the coated antibody on the nitrocellulose membrane is 1.0-2.0 mg / mL, and the Eu 3+ The particle size of the fluorescent nanoparticles is 50-200 nm, the excitation wavelength is 365 nm, and the emission wavelength is 615 nm.

5. The kit according to claim 3, characterized in that: The detection limit of the kit is 1 copies / mL, and the cross-reaction rate OD450 value to canine adenovirus (CAV) and canine parvovirus (CPV) is ≤0.

2.

6. A method for preparing the fluorescent immunochromatographic detection kit according to any one of claims 3 to 5, characterized in that: The following steps are involved: (1) diluting the monoclonal antibody of claim 1 or 2 to 1.0-2.0 mg / mL and spraying it on the T line of the nitrocellulose membrane; (2) The labeled antibody is prepared by 3+ A labeled antibody complex prepared by coupling fluorescent nanoparticles and antibodies at a mass ratio of 1:5-1:10; (3) spraying the labeled antibody complex onto a glass fiber pad, drying at 37° C., and assembling the complex with a coating membrane, a sample pad, and absorbent paper to form a test card; (4) Dispense the sample processing solution into the buffer tube and seal it together with the test card in an aluminum foil bag.

7. The preparation method according to claim 6, characterized in that: The coupling reaction in step (2) is carried out in a borate buffer solution at pH 8.0-8.5, and the coupling time is 2-4 hours.

8. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a diagnostic kit for canine distemper virus.

9. The use according to claim 8, characterized in that: The following steps are involved: (1) Mixing the sample to be tested with the sample processing solution described in any one of claims 3 to 5, and dripping it into the sample hole of the test card after lysis; (2) After 10-15 minutes of reaction, read the signal intensity of the T line and C line using a fluorescent immunoassay analyzer, or directly observe the fluorescent color development results; (3) When the T / C signal ratio is ≥1.0 or the T line is colored, it is judged as CDV positive.

10. The use according to claim 9, characterized in that: The sample to be tested is dog serum, eye and nose swab or fecal suspension, with a detection limit of ≥97% and a specificity of ≥99%.

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