A monoclonal antibody combination against the nucleocapsid protein of canine distemper virus and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a combination of monoclonal antibodies against nucleocapsid protein of canine distemper virus and application. The combination is composed of monoclonal antibodies 1C8 and 1G8, and the sequences of the heavy chain and light chain variable region complement determining regions are clear (as shown in SEQ ID NO. 1-12, respectively), which endow the combination with high affinity and specificity. It has been verified that the combination can specifically recognize CDV-NP, and has no cross reaction with irrelevant antigens such as canine parainfluenza virus, canine coronavirus and canine parvovirus, thereby guaranteeing the specificity of detection from the source. The antibody combination can be used for preparing colloidal gold detection test strips, kits and other detection tools. The colloidal gold detection test strip based on the double antibody sandwich method uses 1C8 as a capture antibody and 1G8 as a labeled antibody, and is simple to operate, intuitive in results, does not require professional equipment, and is suitable for rapid screening in the field.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies against canine distemper virus nucleocapsid protein and their application. Background Technology

[0002] Canine distemper virus (CDV) is a single-stranded, negative-sense RNA virus belonging to the genus Morbillivirus in the family Paramyxoviridae. It has only one serotype. This virus exhibits a strong tropism for the skin, respiratory system, digestive system, and central and peripheral nervous system, and can cause a highly fatal infectious disease in susceptible hosts.

[0003] CDV has a wide host range, primarily infecting canids (such as dogs, wolves, and foxes) and mustelids (such as ferrets, badgers, and minks). Furthermore, studies have shown that CDV can interact with human signaling lymphocyte activation molecular receptor (hSLAM) and human Nectin-4 receptor, and can infect human cell lines in vitro. This suggests that CDV has the potential to spread from dogs to humans, but currently there is no clear evidence that it can infect individual humans. Therefore, effective detection and monitoring of CDV is of great importance.

[0004] CDV is an enveloped virus with hemagglutinin protein (H) and fusion protein (F) embedded on its envelope surface. These two proteins work together to participate in the recognition and membrane fusion of the virus with the host cell. Inside the virus, the nucleocapsid protein (NP) binds tightly to the viral RNA and, together with the large protein (L) and phosphoprotein (P), forms the ribonucleoprotein complex (RNP) with transcriptional and replication activities. Among these, the NP protein is not only the most abundant structural protein in the virus but also exhibits high conservation among different strains, providing the structural basis for viral replication and transcription, and is an important target protein for viral antigen detection.

[0005] Canine distemper is one of the most common and deadliest viral infectious diseases in dogs, with the greatest demand for clinical diagnosis. Canine distemper virus (CDV) is considered the main pathogen causing canine distemper, and there is currently no specific treatment; prevention and control mainly rely on vaccination. Although vaccination plays an important role in preventing canine distemper, the risk of immunization failure and cross-species transmission remains due to the continuous mutation of the virus and the expansion of its host range. Therefore, in addition to vaccine prevention, establishing early rapid diagnosis is of great significance in blocking the spread of the disease.

[0006] Currently, CDV detection methods mainly include virus isolation, RT-PCR, and ELISA. While these methods are relatively accurate, they are highly dependent on specialized instruments and technicians, and their complex procedures make them difficult to implement at the grassroots level. Therefore, developing simple, rapid, and readily applicable on-site detection technologies is of paramount importance.

[0007] Colloidal gold immunochromatographic test strips have become an important tool for rapid on-site screening at the grassroots level due to their advantages such as ease of operation, intuitive result interpretation, and no need for specialized equipment. However, the performance of existing colloidal gold detection systems is largely limited by the antibody raw materials. Currently, most commercially available or publicly patented CDV antibodies target non-conserved epitopes of the H protein, making them susceptible to viral mutations, and they generally suffer from low sensitivity and a high risk of cross-reactivity.

[0008] In contrast, CDV-NP proteins are highly conserved and possess good immunogenicity, making them an ideal target for developing highly sensitive detection antibodies. However, current research on CDV-NP antibodies is relatively limited, and information regarding antibody sequences and their pairing relationships in published literature and patents remains insufficient, which to some extent restricts the development and application of highly sensitive and specific on-site detection systems. Summary of the Invention

[0009] The purpose of this invention is to address the deficiencies in existing technologies by providing a high-affinity monoclonal antibody combination targeting CDV-NP and its applications. The antibodies possess well-defined heavy and light chain variable region sequences, and antibody combinations with good pairing relationships are obtained through optimized screening. Verification has shown that this antibody exhibits good detection sensitivity and specificity against recombinant CDV-NP protein, providing a reliable raw material basis for the development of rapid diagnostic reagents for CDV infection.

[0010] To achieve the above objectives, the main technical solutions adopted by the present invention include: A monoclonal antibody assemblies against canine distemper virus nucleocapsid protein, the monoclonal antibody assemblies comprising monoclonal antibody 1C8 and monoclonal antibody 1G8, wherein the heavy chain variable region of monoclonal antibody 1C8 includes three complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, the amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.3; The light chain variable region of the monoclonal antibody 1C8 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.6. The heavy chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.9. The light chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.12.

[0011] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO. 14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.16.

[0012] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.18.

[0013] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.20.

[0014] Secondly, the application of the monoclonal antibody combination provided in this application in the preparation of tools for recognizing nucleocapsid proteins of canine distemper virus.

[0015] In a further embodiment, the tool includes colloidal gold test strips, reagents, kits, and antibody chips.

[0016] The tool is used to detect canine distemper virus nucleocapsid protein in samples in vitro.

[0017] The monoclonal antibody combination can be used to detect various samples, including but not limited to conjunctival secretions, nasal secretions, saliva, and nasal / oral / pharyngeal swabs, and the detection is not used for the diagnosis of diseases.

[0018] In a further embodiment, the colloidal gold test strip uses monoclonal antibody 1C8 as the capture antibody and monoclonal antibody 1G8 as the labeling antibody.

[0019] In a further embodiment, the colloidal gold test strip includes a nitrocellulose membrane, a gold-labeled conjugate pad, a sample pad, and absorbent paper attached to a backing plate.

[0020] In a further embodiment, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 1C8, the control line is coated with goat anti-mouse IgG, and the gold-labeled binding pad is coated with monoclonal antibody 1G8.

[0021] Beneficial effects: This application provides a monoclonal antibody combination against canine distemper virus nucleocapsid protein, consisting of monoclonal antibody 1C8 and monoclonal antibody 1G8, which can specifically recognize canine distemper virus nucleocapsid protein. The complementarity-determining region (CDR) sequences of the heavy and light chain variable regions of 1C8 and 1G8 are clearly defined (as shown in SEQ ID NO.1-12, respectively), ensuring the high affinity and specific binding ability of the antibody combination. Screening and verification have shown no cross-reactivity with unrelated antigens such as canine parainfluenza virus nucleocapsid protein (CPIV-NP), canine coronavirus nucleocapsid protein (CCoV-NP), and canine parvovirus VP2 protein (CPV-VP2). The specificity of detection is guaranteed at the sequence level, solving the technical pain points of existing antibodies being susceptible to viral mutations and having a high risk of cross-reactivity. This antibody combination can be used to prepare various detection tools for recognizing CDV-NP, including colloidal gold test strips, reagents, kits, and antibody chips. Among them, the colloidal gold test strip constructed based on this combination, using 1C8 as the capture antibody and 1G8 as the labeling antibody, has the advantages of simple operation, intuitive result interpretation, and no need for professional instruments and technicians. It can be widely used for rapid screening at the grassroots level, solving the problems of complex operation and difficulty in grassroots promotion of existing detection methods such as RT-PCR and ELISA, and greatly improving the convenience and popularity of CDV detection. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the SDS-PAGE results of CDV-NP protein purification. Figure 2 The results of the identification of CDV-NP recombinant protein; Figure 3 The image shows the identification results of the purified monoclonal antibody; Figure 4 This is a schematic diagram of colloidal gold assembly; Figure 5 This is a graph showing the specificity analysis results of the test strip; Figure 6 This is a graph showing the sensitivity test results of the test strips; Figure 7 This is a graph showing the results of a monoclonal antibody binding activity assay. Detailed Implementation

[0024] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0025] The detection method described in this application does not involve the diagnosis of diseases.

[0026] Canine distemper virus nucleocapsid proteins include natural canine distemper virus nucleocapsid proteins and recombinant canine distemper virus nucleocapsid proteins.

[0027] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs capable of specifically recognizing the CDV-NP protein. These antibody pairs efficiently recognize the CDV-NP recombinant protein, exhibiting good specificity and sensitivity. This invention applies these monoclonal antibody pairs to an immunoassay platform, constructing rapid test strips or test cards based on colloidal gold immunochromatography technology. These test strips demonstrate high sensitivity to the CDV-NP recombinant protein and show no cross-reactivity with other proteins, making them suitable for rapid, on-site detection.

[0028] Example 1 1. Expression of CDV-NP recombinant protein The CDV NP gene was synthesized by Qingke Biotechnology and cloned into the pET28a vector.

[0029] The nucleotide sequence of the CDV-NP gene is shown in SEQ ID NO.21:

[0030] The amino acid sequence is shown in SEQ ID NO.22: .

[0031] The recombinant plasmid pET28a-CDV-NP was transformed into BL21(DE3) competent cells and induced to express the gene using standard methods. Specifically, the transformed bacteria were plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours until OD500 reached. 600 The concentration was 0.6-0.9, and the final concentration was 0.1 mM IPTG. The cells were collected after induction at 30℃ and 200 rpm for 4 hours.

[0032] 2. Purification of CDV-NP recombinant protein Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TMPurification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of 0.5 M imidazole was collected. The SDS-PAGE electrophoresis results of the purified protein are shown below. Figure 1 .

[0033] See Figure 1 M stands for protein marker, 1 represents before cell induction, 2 represents after cell induction, 3 represents 50mM imidazole elution buffer, and 4 represents 500mM imidazole elution buffer (the estimated molecular weight of recombinant CDV-NP protein is 62.0kDa).

[0034] The results are as follows Figure 1 As shown, there is a distinct main band between 55-70 kDa, consistent with the expected size, and CDV-NP with high purity was obtained after Ni column affinity chromatography, which can be used for subsequent experiments.

[0035] 3. Indirect ELISA to identify the activity of CDV-NP recombinant protein The purified CDV-NP recombinant protein was coated onto an ELISA plate, and its reactivity with CDV-positive serum was detected using an indirect ELISA method. The positive serum was a canine pentavalent hyperimmune serum obtained from healthy dogs repeatedly immunized with canine distemper virus, canine parainfluenza virus, canine adenovirus, canine parvovirus, and canine coronavirus. The recombinant protein was first coated onto microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20) and patted dry. CDV-positive serum was diluted with PBS at gradients of 100, 1000, 10000, 100000, and 1000000. 50 μL of each diluted solution was added to each well of a microplate coated with the antigen. Simultaneously, serum from healthy dogs, verified to be free of CDV-specific antibodies, was diluted at the same fraction as a negative control. The reaction was carried out at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL / well of HRP-labeled rabbit anti-dog IgG secondary antibody (Solepro, diluted 5000 times with PBS) was added. The reaction was carried out at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer was added, and the plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to terminate the reaction. The OD450nm value was measured using a microplate reader. Results are as follows: Figure 2 . Figure 2 The purified CDV-NP recombinant protein showed a significant positive reaction with CDV-positive serum, indicating that the recombinant protein has the correct antigenic structure and good biological activity, which can meet the requirements of downstream experiments.

[0036] 4. Screening for monoclonal antibodies against CDV-NP recombinant protein 4.1 Mouse Immunization Mice were immunized with high-purity CDV-NP recombinant protein. CPIV-NP recombinant protein expressed via the pET28a vector (pET28a-CPIV-NP, His tag, recombinant protein expression details are found in patent 202511500450.1) was used as a reverse screening antigen for monoclonal antibody selection. Six-week-old female BALB / c mice were immunized subcutaneously at multiple sites with a mixture of CDV-NP recombinant protein and an equal volume of Freund's complete adjuvant (total volume 200 μL), at a dose of 20 μg / mouse. At weeks 4 and 8, booster immunizations were administered subcutaneously at multiple sites with a mixture of the above dose and an equal volume of Freund's incomplete adjuvant. At week 12, mice were immunized by direct injection of insulin into the spleen at a dose of 5 μg / mouse. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg of recombinant CDV-NP protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0037] 4.2 Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway up the bottom of the well, clones that reacted positively with the CDV-NP recombinant protein were selected by indirect ELISA. Since the immunogen is a prokaryotically expressed recombinant protein containing a His tag, background components needed to be screened to identify specific cell lines targeting the CDV-NP protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0038] 4.3 Screening of positive clones using indirect ELISA.

[0039] To efficiently screen for CDV-NP-specific monoclonal antibodies, recombinant CDV-NP protein expressed in *E. coli* was used for detection. Simultaneously, recombinant CPIV-NP protein was used as a reverse screening antigen to remove cross-reactivity with other canine respiratory virus NP proteins and non-specific immune background. Specifically, recombinant CDV-NP protein and other recombinant proteins of the pET28a vector (pET28a-CPIV-NP, His tag) were coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6), at a coating concentration of 1 μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. Add 50 μL of cell culture supernatant, and simultaneously use another mouse monoclonal antibody diluted to 1 μg / mL as a negative control. Incubate at 37℃ for 30 min. Discard the liquid from the wells, wash the plate 4 times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37℃ for 30 min, wash the plate 4 more times, blot dry, and add 50 μL / well of TMB chromogenic solution for color development at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction, and measure the OD450nm value using a microplate reader. Select positive cell lines that react with CDV-NP recombinant protein but not with the control recombinant protein for subsequent experiments. The results are shown in Table 1. In Table 1, the other mouse monoclonal antibody is HaiTai Bio's CPV-VP2 monoclonal antibody, and its clone number is 5G7 on the microplate. The other clone numbers are also indicated by the well numbers on the microplate.

[0040] Table 1: Screening Results of Monoclonal Antibodies

[0041] After the selected hybridoma cell lines were expanded and cultured, 0.2 mL (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0042] 5. Purification and Identification of Monoclonal Antibodies Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 mL of the supernatant, add 4 mL of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μL of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8 °C, and collect the supernatant. Filter the supernatant through defatted cotton, add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, incubate the precipitate overnight at 2–8 °C, and centrifuge at 12000 rpm for 30 minutes at 2–8 °C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl (pH 9) and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS (pH 7.4) for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0043] The purified antibody was diluted to 1 μg / mL, and another mouse monoclonal antibody (HaiTai Biotechnology CPV-VP2 monoclonal antibody: 5G7) was used as a negative control at the same dilution. The binding activity of the antibody to CDV-NP recombinant protein and the control antigen CPIV-NP recombinant protein was detected using an indirect ELISA method. Results are shown below. Figure 3 The selected monoclonal antibodies showed strong reactivity to the CDV-NP recombinant protein, but no significant response to the irrelevant antigen CPIV-NP recombinant protein, indicating that the obtained monoclonal antibodies have good specificity and high affinity.

[0044] 6. Preparation of test strips coated with different monoclonal antibodies of CDV-NP.

[0045] The 25 selected CDV-NP monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1 mg / mL with 0.01 M PBS + 1% BSA + 0.5% Trehalose, pH 7.8) were sprayed horizontally in a linear pattern using a scribing instrument, with each line containing 0.8 μL / cm, forming the detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01 M PBS (pH 7.4), were then sprayed horizontally in a linear pattern at 6 mm intervals, forming the control line (C line).

[0046] 7. Colloidal gold pairing of CDV-NP monoclonal antibodies Preparation of antibody-colloidal gold labeled complex.

[0047] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. The optimal conjugation effect was found to be 4 μL. After mixing, 5 μg of the CDV-NP monoclonal antibody to be labeled was added, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and incubation at room temperature was continued for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.05M Tris + 0.5% N100 + 1% BSA + 2% Sucrose + 0.01% PC300, pH 8) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0048] 8. Screening of paired monoclonal antibodies Nitrocellulose membranes streaked with different monoclonal antibodies against CDV-NP were paired with different colloidal gold-labeled monoclonal antibodies. CDV-NP protein was diluted to 20 ng / mL for detection, and CPIV-NP recombinant protein was diluted to 20 ng / mL as a negative antigen for detection. The screening results are shown in Table 2. Combinations that showed deep color development for CDV-NP protein and did not react with the control protein were selected. Therefore, the 1C8 streaking and 1G8 gold-labeled combination was chosen as the optimal pairing for detecting CDV-NP recombinant protein.

[0049] Table 2: Results of screening paired monoclonal antibodies using CDV-NP recombinant protein

[0050] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0051] Table 2 shows the screening results using CDV-NP recombinant protein diluted to a concentration of 20 ng / mL as a positive antigen. Results for both the CDV-NP recombinant protein and the blank dilution were negative and are not presented. The results showed that the combination of 1C8 monoclonal antibody scratching and 1G8 monoclonal antibody labeling resulted in the deepest staining of CDV-NP recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-1C8 as the capture antibody and monoclonal antibody 1G8 as the labeling antibody can specifically recognize CDV-NP recombinant protein.

[0052] 9. Preparation and Assembly of Colloidal Gold Test Strips Preparation of gold-labeled pads: Using a 6mm×300mm glass fiber membrane, the prepared colloidal gold-labeled antibody was evenly dropped onto the glass fiber at a rate of 1200μL / strip, allowed to air dry naturally, and then dried at 37℃ for 2 hours for later use. See Figure 4 , Figure 4 This is a schematic diagram of the colloidal gold assembly. A 60mm × 300mm PVC backing plate is used as a support, on which a sample pad, a gold pad (gold-labeled binding pad), an NC membrane, and an absorbent pad are attached. The nitrocellulose membrane is coated with two lines: a detection line (T line, monoclonal antibody 1C8 streaking) and a control line (C line, goat anti-mouse IgG). The gold-labeled binding pad is coated with monoclonal antibody 1G8 and dried at 37℃ for 12 hours before use. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings, exposing the sample pad at the sample application well of the plastic casing, and exposing the control and detection lines at the result observation wells. The colloidal gold test strip is now assembled.

[0053] 10. Test strip specificity test Recombinant protein samples: CDV-NP recombinant protein, CPIV-NP recombinant protein, CCoV-NP (Genbank ID:UVT36859.1) recombinant protein, CPV-VP2 (Genbank ID:QYI48652.1) recombinant protein, and CRCoV-NP (Genbank ID:ANA11064.1) recombinant protein were diluted to 1 μg / mL with sample dilution buffer for detection. Add 80 μL of the diluted sample to the sample well of the test strip. Simultaneously, add another 80 μL of the diluent to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid.

[0054] The sample dilution solution was 0.01M PBS + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4. Results are shown below. Figure 5 , Figure 5 The results of the specificity analysis of the test strip show that the test strip can specifically detect CDV-NP recombinant protein, and no cross-reactivity was observed with CPIV-NP, CCoV-NP, CPV-VP2, and CRCoV-NP recombinant proteins, indicating that the test strip has good specificity.

[0055] 11. Sensitivity test of test strips The CDV-NP recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL before detection. Figure 6 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 1 ng / mL, while the blank dilution, i.e. the sample dilution (0.01M PBS + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (0 ng / mL), showed no color development, indicating that the test strip's limit of detection for CDV-NP recombinant protein is 1 ng / mL.

[0056] 12. Monoclonal antibody binding activity Based on the screening of potential paired antibodies using colloidal gold, the selected paired monoclonal antibodies and other murine unrelated monoclonal antibodies were serially diluted (concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, respectively) using the aforementioned indirect ELISA method to evaluate their binding activity with CDV-NP recombinant protein. CPV-VP2 murine monoclonal antibody (HaiTai Biotechnology CPV-VP2 monoclonal antibody: 5G7) was used as a negative control to exclude the influence of non-specific binding. Results are shown below. Figure 7. Figure 7 In the text, “Ctrl” represents the negative control mouse monoclonal antibody. Monoclonal antibody 1C8 and monoclonal antibody 1G8 still showed strong signals at concentrations as low as 10 ng / mL.

[0057] 13. Gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by PCR. The PCR products were then purified by gel excision and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0058] membrane-scraped monoclonal antibody 1C8 sequence Light chain variable region nucleotide sequence: The nucleotide sequence encoding the variable region of the 1C8 light chain of the monoclonal antibody is shown in SEQ ID NO.18: gatgttgtgatgacccagactccactcactttgtcgcttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttacatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtg tctcaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctcagacgttcggtggaggcaccaagctggaaatcaaa.

[0059] Light chain variable region amino acid sequence: The amino acid sequence of the variable region of the 1C8 light chain of the monoclonal antibody is shown in SEQ ID NO.14; DVVMTQTPLTLSLTIGQPASISCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSQLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0060] Light chain CDR area annotation: The amino acid sequence of CDR-L1 of the monoclonal antibody 1C8 is shown in SEQ ID NO.4: CDR-L1: KSSQSLLHSDGKTYLN; The amino acid sequence of CDR-L2 of the monoclonal antibody 1C8 is shown in SEQ ID NO.5: CDR-L2: LVSQLDS; The amino acid sequence of CDR-L3 of the monoclonal antibody 1C8 is shown in SEQ ID NO. 6: CDR-L3: WQGTHFPQT.

[0061] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.17: gaggtgcacctggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctatggcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaaccattagtactgatggtactttcacc tactatccagacattgtaaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgaccagtctgaggtctgaggacacagccatttattactgtgcaagatatggtaactactttgactactggggccaaggcaccactctcacagtctcctca.

[0062] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.13; EVHLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISTDGTFTYYPDIVKGRFTISRDNAKNTLYLQMTSLRSEDTAIYYCARYGNYFDYWGQGTTLTVSS.

[0063] Heavy chain CDR region annotation: The amino acid sequence of CDR-H1 of the monoclonal antibody 1C8 is shown in SEQ ID NO.1: CDR-H1: SYGMS; The amino acid sequence of CDR-H2 of the monoclonal antibody 1C8 is shown in SEQ ID NO.2: CDR-H2: TISTDGTFTYYPDIVKG; The amino acid sequence of CDR-H3 of the monoclonal antibody 1C8 is shown in SEQ ID NO.3: CDR-H3: YGNYFDY.

[0064] The 1G8 sequence of the gold monoclonal antibody.

[0065] Light chain variable region nucleotide sequence: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1G8 is shown in SEQ ID NO.20.

[0066] caaattgttctcacccagtctccagcaatcatgtctgcatctctaggggaggagatcaccctaacctgcagtgccagctcgaatgcaggttacatgcactggtaccaacagaagtcaggcacttctcccaaactcttgatttataggacatccaacctg gcttctggagtcccttctcgcttcagtggcagtgggtctgggaccttttattctctcacaatcagcagtgtggaggctgaagatgctgccgattattattgccatcagtggagtcgttatccatggacgttcggtggaggcaccaagctggaaatccaa.

[0067] Light chain variable region amino acid sequence: The amino acid sequence of the variable region of the light chain of the monoclonal antibody 1G8 is shown in SEQ ID NO.16: QIVLTQSPAIMSASLGEEITLTCSASSNAGYMHWYQQKSGTSPKLLIYRTSNLASGVPSR FSGSGSGTFYSLTISSVEAEDAADYYCHQWSRYPWTTFGGGTKLEIQ.

[0068] Light chain CDR area annotation: The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody 1G8 is shown in SEQ ID NO.10: CDR-L1: SASSNAGYMH; The amino acid sequence of the light chain variable region CDR-L2 of the monoclonal antibody 1G8 is shown in SEQ ID NO.11: CDR-L2: RTSNLAS; The amino acid sequence of the light chain variable region CDR-L3 of the monoclonal antibody 1G8 is shown in SEQ ID NO.12: CDR-L3: HQWSRYPWT.

[0069] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.19: gaggtgcacctggtggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtgactatggcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaaccattagtactgatggtactttcacc tactatccagacagtttaaaggggcgcttcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgaccagtctgaggtctgaggacacagccatttattactgtgcaagatatggtaactactttgactactggggccgaggcaccactctcacagtctcctca.

[0070] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.15; EVHLVESGGDLVKPGGSLKLSCAASGFTFSDYGMSWVRQTPDKRLEWVATISTDGTFTYYPDSLKGRFTISSRDNAKNTLYLQMTSLRSEDTAIYYCARYGNYFDYWGRGTTLTVSS.

[0071] Heavy chain CDR region annotation: The amino acid sequence of the heavy chain variable region CDR-H1 of the monoclonal antibody 1G8 is shown in SEQ ID NO.7: CDR-H1: DYGMS; The amino acid sequence of the heavy chain variable region CDR-H2 of the monoclonal antibody 1G8 is shown in SEQ ID NO. 8: CDR-H2: TISTDGTFTYYPDSLKG; The amino acid sequence of the heavy chain variable region CDR-H3 of the monoclonal antibody 1G8 is shown in SEQ ID NO. 9: CDR-H3: YGNYFDY.

Claims

1. A monoclonal antibody combination against canine distemper virus nucleocapsid protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 1C8 and monoclonal antibody 1G8. The heavy chain variable region of monoclonal antibody 1C8 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 1C8 includes three complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5 and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 1G8 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody combination against canine distemper virus nucleocapsid protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.

14. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.

16.

3. The monoclonal antibody combination against canine distemper virus nucleocapsid protein according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1C8 is shown in SEQ ID NO.

18.

4. The monoclonal antibody combination against canine distemper virus nucleocapsid protein according to claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1G8 is shown in SEQ ID NO.

20.

5. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for recognizing the nucleocapsid protein of canine distemper virus.

6. The application according to claim 5, characterized in that, The tools include colloidal gold test strips, reagents, kits, and antibody chips.

7. The application according to claim 6, characterized in that, The colloidal gold test strip uses monoclonal antibody 1C8 as the capture antibody and monoclonal antibody 1G8 as the labeling antibody.

8. The application according to claim 7, characterized in that, The colloidal gold test strip includes a nitrocellulose membrane, a gold-labeled conjugate pad, a sample pad, and absorbent paper attached to a backing plate.

9. The application according to claim 8, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 1C8, the control line is coated with goat anti-mouse IgG, and the gold-labeled binding pad is coated with monoclonal antibody 1G8.

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