Monoclonal antibody of canine distemper virus and application thereof

Abstract

The application provides a monoclonal antibody of canine distemper virus and application, and belongs to the animal medical detection technical field.The antibody provided by the application has super-high binding affinity to CDV H protein of the canine distemper virus, and the linear epitope is the 373-381th position of the CDV H protein.The kit of the application adopts nitrocellulose membrane to coat the canine virus specific antibody and a fluorescent marker, and can rapidly detect the virus in the canine sample.The antibody is prepared by a genetic engineering means, and the detection sensitivity is significantly better than that of a conventional colloidal gold test strip and an ELISA detection method.The antibody of the application maintains excellent stability in a high-temperature environment, and shows extremely low cross-reaction characteristics to CAV and other pathogens.Coupled with a special fluorescent 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 application belongs to the technical field of biomedical detection, and particularly relates to a monoclonal antibody of canine distemper virus and application thereof. BACKGROUND

[0002] As a highly lethal animal infectious disease pathogen, the early rapid detection of canine distemper virus (CDV) is a key link to block the spread of the epidemic. The current main detection methods have significant limitations: the virus isolation method is time-consuming and has high biological safety requirements; RT-PCR relies on professional equipment and operators; although the colloidal gold immunochromatographic test 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 primary prevention and control scene.

[0003] The existing antibody technology faces multiple technical bottlenecks. The traditional anti-CDV monoclonal antibody mainly targets the non-conserved epitope of H protein, which is easy to cause detection failure due to virus variation. The performance defects are specifically manifested in three aspects: first, the antibody affinity is generally low (KD value is mostly 10 -8 M level), which is difficult to effectively capture low viral load samples; second, the cross-reaction risk is prominent, and the typical one is the non-specific binding to canine adenovirus (CAV) (OD450 value is 0.8); third, the thermal stability is insufficient, and the antibody activity is attenuated by more than 30% after 30 days of storage at 37℃ (Zhang et al., Characterization of monoclonal antibodies against canine distemper virus hemagglutinin protein, Veterinary Immunology and Immunopathology (2019), DOI: 10.1016 / j.vetimm.2019.109865. provides an antibody, the antibody affinity KD = 3.2 x 10 -8 M, the titer retention rate is 68.3% after 30 days of storage at 37℃), which seriously affects the storage stability of the reagent.

[0004] The commercial detection products also have obvious shortcomings in application. The colloidal gold test strip (CN109988083A) has a detection limit of only 82.9% positive coincidence rate, and the room temperature storage period is not more than 6 months; 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.) has a detection limit of 94.3%, but requires a complex operation process and professional instruments, which cannot meet the demand of rapid screening in the epidemic area. These technical defects highlight the urgency of developing a new detection system with high sensitivity, high specificity and strong stability. SUMMARY

[0005] The application provides a fluorescence immunochromatographic detection kit based on a high-affinity monoclonal antibody against canine distemper virus and a preparation method thereof.

[0006] In a first aspect, the application provides a monoclonal antibody against canine distemper virus, wherein 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.

[0007] In a second aspect, the application provides an application of the monoclonal antibody in the preparation of a canine distemper virus diagnostic kit.

[0008] The application comprises the following steps:

[0009] (1) mixing the sample to be tested with a sample treatment liquid, adding the sample to be tested to the sample hole of the detection card after lysis, wherein the sample treatment liquid is a Tris-HCl buffer solution containing 0.1%-1% Triton X-100 and 0.05%-0.2% BSA with a pH of 7.0-7.8;

[0010] (2) after 10-15 minutes of reaction, reading the signal intensity of the T line and the C line through a fluorescence immunoassay instrument, or directly observing the fluorescence color development result;

[0011] (3) when the T / C signal ratio is greater than or equal to 1.0 or the T line develops color, the CDV is determined to be positive.

[0012] Further, the sample to be tested is canine serum, eye-nose swab or fecal suspension, the detection limit is greater than or equal to 97%, and the specificity is greater than or equal to 99%.

[0013] This invention establishes a specific application method for the antibody in the diagnosis of canine distemper virus: after lysing serum, nasal / ocular swabs, or fecal suspension with a sample processing solution, rapid intravenous fluorescence detection can be performed. A positive result is determined when the T / C signal ratio is ≥1.0 or a T-line appears. Clinical validation of this method shows a specificity of ≥99%.

[0014] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0015] 1. Achieving a dual breakthrough in detection limit and specificity, antibody affinity is increased to 10 through targeted mutation at a key site in the antibody CDR3 region (YRP). -11 M-level antibody, combined with rare-earth fluorescent labeling technology, achieves a detection limit as low as 1 TCID50 / mL, which is 500 times higher than the detection limit of the traditional colloidal gold method. The antibody has broad-spectrum recognition ability against CDV genotypes Asia-1 and Europe-1, and extremely low cross-reactivity with pathogens such as canine adenovirus (CAV) and canine parvovirus (CPV) (OD450 < 0.1), significantly improving detection accuracy.

[0016] 2. The clinical applicability is at an industry-leading level. In large-scale clinical validation, the kit has a detection sensitivity of 97.1%, a specificity of 100%, and a detection rate of 95.2%, which is significantly better than similar product C (89.2%). It can meet the dual needs of on-site screening of animal diseases and accurate laboratory diagnosis.

[0017] 3. Significant breakthroughs have been achieved in product stability. The core antibody retains a titer of >98% after being stored at 37°C for 30 days, breaking through the limitation of cold chain transportation required for similar products and reducing storage and transportation costs by more than 60%.

[0018] 4. The operation process is highly efficient and convenient, and the testing process can be completed in just 15 minutes. It supports dual-mode output of qualitative results for naked-eye interpretation and quantitative detection by fluorescence analyzer, making it particularly suitable for the real-time testing needs of grassroots veterinary stations and pet hospitals, and greatly improving the efficiency of disease prevention and control response. Attached Figure Description

[0019] Figure 1 SPR response curves of the antibody (A) provided by this invention and the commercially available antibody (B).

[0020] Figure 2 A bar chart showing the cross-reaction ELISA results of the antibody provided in this invention and a commercially available antibody.

[0021] Figure 3 The colorimetric pattern of CDV virus solution of different concentrations was obtained by assembling colloidal gold test strips using the antibodies provided by this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0023] Example 1: Preparation of anti-CDV monoclonal antibody

[0024] The anti-CDV monoclonal antibody provided by this invention has the amino acid sequence shown in SEQ ID NO:1 in its heavy chain variable region and the amino acid sequence shown in SEQ ID NO:2 in its light chain variable region; the complete heavy chain and light chain amino acid sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. In the heavy chain variable region of the antibody, CDR1, CDR2, and CDR3 are located at amino acid positions 31-35, 50-66, and 101-103 of SEQ ID NO:1, respectively; in the light chain variable region, CDR1, CDR2, and CDR3 correspond to amino acid positions 24-34, 50-56, and 89-97 of SEQ ID NO:2, respectively. This antibody exhibits high specificity for the CDV hemagglutinin protein receptor binding domain, with an affinity constant of 2.7 × 10⁻⁶. -11 M.

[0025] The linear epitope of the antibody is located at positions 373-381 of the CDVH protein.

[0026] Optimized heavy and light chain genes were cloned into an expression vector using gene synthesis technology to construct a recombinant plasmid. Subsequently, the recombinant plasmid was transfected into CHO-S cells for stable expression. Through cell culture and protein purification, a high-purity anti-CDV monoclonal antibody was obtained. This antibody, validated by ELISA and Western blot, exhibited excellent binding activity and specificity.

[0027] Specifically, for the CHO-S cell expression system, the codon usage frequency of the HC and LC genes was optimized (CAI value > 0.8) to avoid rare codons and mRNA secondary structures.

[0028] 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 sequencing to ensure sequence accuracy. After transfection into CHO-S cells, high-expression clones were screened to establish a stable cell line. The cell culture supernatant was purified by Protein A affinity chromatography to obtain an anti-CDV monoclonal antibody with a purity >95%, suitable for subsequent kit development.

[0029] The optimized antibody exhibited extremely high stability and activity at both 37℃ and 25℃, ensuring the reliability of the kit under different environments. Meanwhile, the efficient and convenient operating procedures greatly improve the testing efficiency of grassroots veterinary stations and pet hospitals, providing strong technical support for disease prevention and control.

[0030] Example 2: Functional Verification of Antibodies

[0031] 1. Affinity measurement (surface plasmon resonance, SPR)

[0032] The surface plasmon resonance (SPR) technique was used to determine the affinity of the antibody obtained in Example 1. The specific method is as follows: CDV H protein was covalently coupled to the surface of a CM5 sensor chip (Cytiva). HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P2O, pH 7.4) was used as the mobile phase for concentration gradient analysis of the antibody sample (0.1-100 nM). Experimental parameters were set to a constant injection rate (30 μL / min), and the response curve was recorded (e.g., ...). Figure 1 The sensing signals were recorded in real time using the Biacore T200 system, and the dynamics were analyzed using a 1:1 Langmuir binding model. The binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD) were calculated respectively.

[0033] 2. Specificity verification (cross-reactivity ELISA)

[0034] Experimental Procedure: First, canine adenovirus (CAV), canine parvovirus (CPV), canine coronavirus (CCoV) antigen (1 μg / well), and canine distemper virus (CDV) antigen (as a positive control) were coated onto the wells of an ELISA plate. Then, 1 μg / mL of the test antibody was added, and the plate was incubated at 37°C for 1 hour to complete antigen-antibody binding. In the detection phase, 1:5000 diluted HRP-labeled anti-canine IgG secondary antibody was added sequentially for signal amplification. After color development with TMB substrate, the OD450 value was read using an ELISA reader. Figure 2 As shown, the results must meet two criteria: the OD450 value of the positive control well (CDV antigen) must be ≥2.0, while the OD450 values ​​of other viral antigen wells must be ≤0.2, which indicates that the antibody to be tested has no cross-reaction with non-target viruses.

[0035] 3. Limit of Detection Test (Colloidal Gold Test Strip)

[0036] During the test strip preparation process, the test line (T line) is directionally sprayed with a 1 mg / mL concentration of the antibody to be tested, while the control line (C line) is constructed by spraying with 0.5 mg / mL goat anti-mouse IgG antibody to build an immune binding system. In the sample detection stage, the CDV virus solution is serially diluted 10-fold (concentration range covering 1, 10, 10...). 2 10 3 10 4 10 5 After testing sequentially with TCID50 / mL, the lowest viral concentration at which a T-line color reaction is clearly visible to the naked eye is used as the judgment criterion to determine the detection limit (LOD) of the detection system. 1 TCID50 / mL = 0.693 copies / mL (based on absolute quantification results of digital PCR, n = 3 independent experiments). Figure 3 As shown, the antibody of the present invention exhibits good color development effect at multiple concentration gradients, with a detection limit of 1 TCID50 / mL, verifying the high sensitivity and reliability of the test strip.

[0037] 4. Comparative data with commercially available antibodies

[0038] Table 1 Comparison of antibody affinity and detection limit

[0039] Detection index Antibody of the invention Commercial antibody Criteria for determination Affinity (KD) 2.7 x 10 -11 M]] 3.2 x 10 -8 M]] ≤ 1 x 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 (g / L) 2.8 1.2 ≥2.0

[0040] In Table 1, the commercially available antibody is a canine distemper virus monoclonal antibody from Beijing Century Yuanheng Animal Disease Prevention Technology Co., Ltd., with a neutralizing antibody titer ≥1024.

[0041] This antibody exhibits significantly high affinity and detection limit, with a KD value of 2.7 × 10⁻⁶. -11 M) compared to commercially available antibodies (3.2×10) -8The antibody exhibits a more than 1,000-fold reduction in viral load, enabling efficient binding even in low viral load environments. Simultaneously, its detection limit reaches 1 TCID50 / mL, a 500-fold reduction compared to commercially available test strips (500 TCID50 / mL), providing a technological advantage for early infection diagnosis. Regarding stability, the antibody utilizes point mutations in its constant region CH2 domain to achieve anti-aggregation design, maintaining low aggregation tendency even at high temperatures. Furthermore, after 12 months of long-term storage, the monomer content remains stable at >97% by SEC-HPLC, significantly superior to the storage performance of commercially available antibodies (<90%). In large-scale production, a high expression level of 2.8 g / L is achieved through CHO-S suspension culture, increasing production capacity by 4 times compared to the traditional hybridoma method (1.2 g / L). Combined with genetically engineered antibody technology, the batch-to-batch coefficient of variation (CV) is controlled at <5%, far lower than the >15% fluctuation of hybridoma antibodies, significantly reducing production costs and ensuring product quality stability. Moreover, the highly specific variable region of the antibody provided by this invention effectively avoids cross-binding with pathogens such as CAV / CPV, further enhancing detection specificity. In summary, the antibody provided by this invention significantly outperforms traditional antibodies in terms of affinity, detection limit, stability, and production efficiency, making it particularly suitable for the development of high-precision diagnostic reagents and neutralizing drugs. Through genetic engineering optimization, a complete technological breakthrough has been achieved from research and development to industrialization.

[0042] Example 3: Assembly and performance testing of a fluorescence immunochromatographic reagent kit

[0043] 1. Reagent kit assembly

[0044] This test strip is constructed using the double-antibody sandwich method, with rare earth fluorescent microspheres (such as Eu) 3+ The sample uses doped nanoparticles as a signal source. When CDV antigen is present in the sample, the antigen first specifically binds to the labeled antibody on the binding pad, migrates to the detection line (T line) through chromatography, and is intercepted by 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 control line (C line) verifies the effectiveness of the chromatography by the binding of goat anti-mouse IgG to excess labeled antibody.

[0045] The preparation of fluorescently labeled antibodies began with microsphere activation: 200 nm diameter fluorescent microspheres were washed with MES buffer (pH 6.0) and activated at 25°C with shaking for 30 minutes using an EDC / NHS (5 mM / 2.5 mM) system. The activated microspheres were then coupled with the antibody obtained in Example 1 at a 1:10 mass ratio in PBS (pH 7.4) at 4°C in the dark for 12 hours. Residual active sites were subsequently blocked with 1% BSA. After purification by 100 kDa ultrafiltration centrifugation, the complex was stored in PBS containing 0.1% BSA and 0.05% NaN3. The labeling efficiency was required to be ≥500 antibody / microsphere, and the antibody activity retention was verified by ELISA to be >90%.

[0046] Nitrocellulose membranes were treated using a double-antibody spraying process: the detection line was sprayed with 1 μL / cm commercially available antibody using an XYZ3060 sprayer, and the control line was sprayed with 0.5 mg / mL goat anti-mouse IgG, with a 5 mm gap between the two lines. After spraying, the membrane was treated with a blocking buffer containing 2% sucrose, 1% BSA, and 0.1% Tween-20 for 30 minutes, dried, and stored in an environment with humidity <30%. During kit assembly, the pretreated glass fiber sample pad (containing 0.1M Tris-HCl pH 8.0, 1% BSA, and 0.5% Triton X-100) was coated with 0.5 mg / cm antibody. 2 The labeled antibody-binding pads are stacked together with the treated NC membrane and absorbent pad to form a chromatography system, which is then cut into 4mm test strips and packed into plastic cartridges.

[0047] 2. Comparison of clinical sample testing

[0048] Sample source: Serum (n=60) and ocular / nasal swabs (n=60) were collected from 120 suspected CDV-infected dogs. RT-qPCR (primers targeting the N gene, Ct value ≤35 for positivity) confirmed 35 positive samples (29.2%). Control kit: Commercial CDV ELISA kit (VET-ELISA-3007 from a certain company, based on rabbit polyclonal antibody, operation time 2.5h). The detection rates using the kit of this invention and the control kit were 95.2% and 89.2%, respectively.

[0049] Example 4: Validation of antibody stability and kit shelf life

[0050] 1. Antibody thermally accelerated stability test

[0051] The antibody of this invention and a commercially available antibody were stored at 37°C for 30 days, with samples taken weekly to measure ELISA titers. The titer retention rate of the antibody of this invention was 98.3%, while that of the commercially available antibody was 68.3%. Furthermore, the Tm value of the antibody of this invention (83.4°C) was significantly higher than that of the commercially available antibody (71.2°C), and DSC analysis confirmed its improved conformational stability. This indicates that the antibody of this invention has higher thermal stability.

[0052] 2. Reagent kit shelf life test

[0053] Three batches of reagent kits were stored at 4℃, 25℃, and 37℃, respectively, with 10 kits randomly selected each month for testing. Accelerated stability testing (calculated using the Arrhenius equation) showed that the kits had a shelf life of up to 18 months at 25℃. As shown in Table 2, the kits provided by this invention have effective shelf lives of 20 months, 18 months, and 6 months at 4℃, 25℃, and 37℃, respectively, while their limits of detection are 1 copies / mL, 4 copies / mL, and 50 copies / mL, respectively.

[0054] Table 2 Comparison of Sample Storage Conditions and Detection Performance

[0055] Storage temperature (°C) Valid shelf life (months) Minimum detection limit (copies / mL) 4 20 1 25 18 4 37 6 50

[0056] Through systematic verification using examples and comparative examples, the monoclonal antibody reagent of the present invention (the antibody of the present invention) exhibits four core advantages: Firstly, at the molecular recognition level, its KD value reaches 2.7 × 10⁻⁶. -11 M's extremely high affinity, 50 times higher than traditional antibodies, successfully lowers the detection limit to 10. 2 The sample yielded low viral load samples (copies / mL). Secondly, regarding specificity control, a salt bridge structure was constructed by rationally designing the CDR3 region and introducing specific mutations at sites such as Arg102, completely eliminating cross-reactivity with canine coronaviruses such as CAV and CPV. In terms of formulation stability, the antibody solution maintained a titer retention rate of >98% even after one month of accelerated degradation at 37°C. The accompanying kit showed a signal attenuation strictly controlled within 5% after 12 months of storage at room temperature, significantly outperforming existing products in terms of shelf life. Finally, in clinical application validation, it achieved a titer retention rate of 98.3% compared to the RT-qPCR gold standard, demonstrating a statistically significant advantage in diagnostic efficacy over traditional immunochromatography (p<0.001), providing a reliable solution for rapid on-site testing.

[0057] The detailed embodiments and comparative examples above fully demonstrate the groundbreaking advancements of this invention in antibody design (precise epitope localization and CDR optimization), detection performance (detection limit / specificity balance), and formulation stability (thermodynamic optimization), providing a new generation solution for rapid on-site detection of canine distemper virus.

[0058] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A monoclonal antibody against canine distemper virus, 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.

2. The monoclonal antibody according to claim 1, characterized in that: The antibody has an affinity constant of 2.7 × 10⁻⁶ for CDV hemagglutinin protein. -11 M, the linear epitope of the antibody is position 373-381 of the CDV H protein.

3. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a canine distemper virus diagnostic kit.

4. The application according to claim 3, characterized in that, Includes the following steps: (1) Mix the sample to be tested with the sample processing solution, lyse it and drop it into the sample well of the test card. The sample processing solution is a Tris-HCl buffer solution with pH 7.0-7.8 containing 0.1%-1% Triton X-100 and 0.05%-0.2% BSA. (2) After the reaction has been going on for 10-15 minutes, the signal intensity of the T line and C line is read by a fluorescence immunoassay analyzer, or the fluorescence color development results are observed directly. (3) When the T / C signal ratio is ≥1.0 or the T line is colored, it is determined to be CDV positive.

5. The application according to claim 4, characterized in that: The test samples are canine serum, nasal and ocular swabs, or fecal suspensions, with a detection limit ≥97% and a specificity ≥99%.

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