Monoclonal antibody combination for detecting canine parainfluenza virus NP protein and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting canine parainfluenza virus NP protein and application thereof. The combination is composed of monoclonal antibodies 5H10 and 1H7, 5H10 is used as a coating antibody, and 1H7 is used for colloidal gold labeling, so that efficient and specific sandwich detection of CPIV / NP antigen can be realized. The variable region CDR sequences of the heavy chains and light chains of the two antibodies are clear (SEQ ID NO. 1-12). The application also provides a rapid detection test strip based on the antibody combination, which can be used for on-site screening, early diagnosis and epidemiological monitoring of canine parainfluenza virus, solves the problem that existing detection technologies lack high-specificity recognition tools, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody combination for detecting canine parainfluenza virus NP protein and its application. Background Technology

[0002] Canine parainfluenza virus (CPIV) is a single-stranded negative-sense RNA virus belonging to the Paramyxoviridae family. CPIV is prevalent year-round and globally, and is one of the most contagious respiratory pathogens, a major cause of infectious bronchitis—kennel cough. CPIV has a wide host range, with canines such as dogs, minks, foxes, and raccoon dogs being susceptible. The virus is easily transmitted through respiratory secretions and the air, commonly found in densely populated dog-keeping places such as kennels, pet shops, and veterinary hospitals. The higher the density of dogs, the faster the spread. Furthermore, when CPIV is co-infected with mycoplasma or Bordetella bronchiseptica, it often leads to more severe illness and even death. Therefore, the harm caused by canine parainfluenza virus infection should not be underestimated.

[0003] CPIV virus has a diameter between 80 and 200 nm, an envelope containing two types of spikes: a fusion protein (F) and a hemagglutinin-neuraminidase protein (HN). The envelope also contains nucleocapsid protein (NP), a major structural protein of the viral particle and considered an important target for antigen detection. Diagnosis of CPIV infection is usually based on a preliminary assessment of clinical symptoms combined with epidemiological data. However, the clinical symptoms of CPIV infection are very similar to those of other canine respiratory infectious diseases, thus requiring more precise laboratory testing methods to identify the pathogen. CPIV has only one serotype; therefore, serum neutralization tests and hemagglutination inhibition tests can be used for epidemiological investigations and retrospective diagnosis. Molecular biological methods such as PCR can more sensitively diagnose CPIV infection; however, routine immunization vaccines, such as canine quadrivalent live vaccines, may interfere with this detection, posing a certain risk of false positives.

[0004] In summary, although existing technologies such as serology and molecular biology are used for the detection of canine parainfluenza virus, there is still a lack of a simple, rapid, and highly specific on-site diagnostic tool. Summary of the Invention

[0005] This invention provides a highly specific and sensitive monoclonal antibody for detecting canine parainfluenza virus nucleoprotein (CPIV / NP) and its application, solving the technical problem of the lack of efficient and rapid detection tools for canine parainfluenza virus antigens in the prior art.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] In a first aspect, the present invention provides a monoclonal antibody combination for detecting canine parainfluenza virus NP protein, the monoclonal antibody combination comprising monoclonal antibody 5H10 and monoclonal antibody 1H7.

[0008] The heavy chain variable region of the monoclonal antibody 5H10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0009] The light chain variable region of the monoclonal antibody 5H10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0010] The heavy chain variable region of the monoclonal antibody 1H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0011] The light chain variable region of the monoclonal antibody 1H7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0012] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO. 14.

[0013] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO. 16.

[0014] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO.18.

[0015] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO. 20.

[0016] Secondly, the present invention provides an application of the above-described monoclonal antibody combination in the preparation of a tool for detecting canine parainfluenza virus NP protein.

[0017] In a further embodiment, the tool includes colloidal gold test strips, reagents, kits, and antibody chips.

[0018] In a further embodiment, the colloidal gold test strip uses monoclonal antibody 5H10 as the capture antibody and monoclonal antibody 1H7 as the labeling antibody.

[0019] In a further embodiment, the colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold 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 5H10, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 1H7.

[0021] Beneficial effects:

[0022] This application provides a monoclonal antibody ensemble for detecting canine parainfluenza virus nucleoprotein. The ensemble consists of monoclonal antibodies 5H10 and 1H7, and can efficiently and specifically recognize canine parainfluenza virus nucleoprotein. The amino acid sequences of the complementarity-determining regions (CDRs) of the heavy and light chains of each antibody in this ensemble are clearly defined: for monoclonal antibody 5H10, the heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs 1, 2, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs 4, 5, and 6, respectively; for monoclonal antibody 1H7, the heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs 7, 8, and 9, respectively, and the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs 10, 11, and 12, respectively. The defined CDR sequences ensure high specificity and high affinity of antibody binding, significantly reducing the risk of non-specific binding and cross-reactivity.

[0023] The monoclonal antibody against canine parainfluenza virus NP protein provided by this invention exhibits high specificity and good sensitivity. It can specifically recognize the nucleoprotein of canine parainfluenza virus and shows no cross-reactivity with antigens of other common canine pathogens such as canine distemper virus, canine coronavirus, canine respiratory coronavirus, and canine parvovirus, effectively avoiding misdiagnosis or missed diagnosis in clinical practice due to similar pathogen symptoms. This antibody can not only be used in the construction of colloidal gold immunochromatographic test strips for rapid, on-site detection of CPIV antigens in samples, but is also suitable for various detection platforms such as ELISA and immunofluorescence, with a wide range of applications. This invention solves the technical bottleneck of the lack of efficient, specific, and stable canine parainfluenza virus detection reagents in the prior art, significantly improving the early diagnostic efficiency and epidemiological monitoring capabilities of canine parainfluenza virus infection, and has significant clinical application value and industrialization prospects. Attached Figure Description

[0024] 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.

[0025] Figure 1 Results of protein identification by SDS-PAGE;

[0026] Figure 2 The results of the identification of CPIV / NP recombinant proteins;

[0027] Figure 3 Identification results for purified monoclonal antibodies;

[0028] Figure 4 This is a schematic diagram of colloidal gold assembly.

[0029] Figure 5 The results are from the specificity analysis of the test strips;

[0030] Figure 6 The results are from the sensitivity test of the test strips;

[0031] Figure 7 This is the result of antibody binding identification. Detailed Implementation

[0032] 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.

[0033] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0034] The detection method described in this application is not intended for disease diagnosis and treatment.

[0035] Immunological detection of antigens, such as rapid antigen test strips, is convenient to operate and enables rapid qualitative detection. It has been widely used for rapid pathogen detection and has shown significant advantages. Monoclonal antibodies against the canine parainfluenza virus (CPIV) NP protein are core tools for immunoassay and essential raw materials for developing highly specific and sensitive diagnostic reagents. They are of great significance for the rapid diagnosis, epidemiological surveillance, and scientific research of CPIV.

[0036] This invention utilizes hybridoma technology to obtain a monoclonal antibody that can be used for targeted detection of canine parainfluenza virus NP protein, and has been successfully applied to colloidal gold test strips. The antibody has been verified to have excellent detection sensitivity and specificity for canine parainfluenza virus NP recombinant protein, providing a raw material basis for the development of diagnostic reagents for canine parainfluenza infection.

[0037] The canine parainfluenza virus (NP) protein includes the natural NP protein and the recombinant NP protein.

[0038] The CPIV / NP recombinant protein is either a canine parainfluenza virus NP recombinant protein or a canine parainfluenza virus nucleocapsid recombinant protein.

[0039] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs that specifically recognize CPIV / NP proteins. This combined antibody pair can efficiently recognize CPIV / NP recombinant proteins, exhibiting good specificity and sensitivity. This invention applies the monoclonal antibody pair to an immunoassay platform, constructing a rapid test strip or test card based on colloidal gold immunochromatography technology. This test strip has high sensitivity to CPIV / NP recombinant proteins and exhibits no cross-reactivity with other proteins, solving the problem of false positives in PCR testing caused by canine quadrivalent live vaccine administration.

[0040] Example 1

[0041] 1. Expression of recombinant NP protein of canine parainfluenza virus

[0042] The CPIV / NP gene was synthesized by Qingke Biotechnology and cloned into the pET28a vector. Its nucleotide sequence is shown in SEQ ID NO. 21.

[0043]

[0044] The amino acid sequence is shown in SEQ ID NO.22:

[0045] MSSVLKAYERFTLTQELQDQSEEGTIPPTTLKPIIRVFILTSNNPELRSRLLLFCLRIVLSNGARDSHRFGALLTMFSLPSATMLNHVKLADQSPEADIERVEIDGFEEGSFRLIPNARSGMSRGEI NAYAALAEDLPDTLNHETPFVDSEVEGTAWDEIETFLDMCYSVLMQAWIVTCKCMTAPDQPAASIEKRLQKYRQQGRINPRYLLQPEARRIIQNVIRKGMVVRHFLTFELQLARAQSLVSNRYYAMVG DVGKYIENCGMGGFFLTLKYALGTRWPTLALAAFSGELTKLKSLMALYQTLGEQARYLALLESPHLMDFAAANYPLLYSYAMGIGYVLDVNMRNYAFSRSYMNKTYFQLGMETARKQQGAVDMRMAE DLGLTQAERTEMANTLAKLTTANRGADTRGGVNPFSSVTGTTQVPAAVTGDTFESYMVADRLRQRYADASTHDDEMPPLEEEEEDDTSAGPRTGPTLEQVALDIQNTAVGAPIHTDDLNAALGDLDI.

[0046] The recombinant plasmid pET28a-CPIV / NP was transformed into BL21(DE3) competent cells using standard methods and induced to express the gene. 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.

[0047] 2. Purification of recombinant NP protein of canine parainfluenza virus

[0048] 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 0.22 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 the target protein was collected and dialyzed overnight at 4°C with buffer A. The purification process was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C. Figure 1 In the diagram, M represents the protein marker, and R represents the purified CPIV / NP recombinant protein, which shows a distinct band between 55-70 kDa. The estimated molecular weight of the fusion protein is 61.4 kDa, indicating that this application has successfully prepared the CPIV / NP recombinant protein with high purity and good integrity, meeting the needs of downstream applications.

[0049] 3. Identification of recombinant protein of canine parainfluenza virus NP (CPIV / NP)

[0050] The purified CPIV / NP recombinant protein was coated onto an ELISA plate, and its reaction with CPIV-positive serum was identified by indirect ELISA. The positive serum was canine pentavalent hyperimmune serum, obtained by repeatedly immunizing healthy dogs with canine distemper, parainfluenza, infectious hepatitis, parvovirus, and coronavirus enteritis virus. The recombinant protein was first coated into 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, pH 7.4) and patted dry. CPIV / NP positive sera were diluted with PBS at gradients of 100, 1000, 10000, 100000, and 1000000. 50 μL of each diluted solution was added to each well of the antigen-coated microplate. PBS was used 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 plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to stop the reaction. The OD450nm value was measured using a microplate reader. The results are as follows: Figure 2 The purified CPIV / NP recombinant protein can specifically react with CPIV-positive serum and maintain good immunoreactivity over a wide range of serum dilution gradients. This recombinant protein has the correct antigen structure and good biological activity, meeting the needs of downstream applications.

[0051] 4. Screening for monoclonal antibodies against CPIV / NP recombinant proteins

[0052] 4.1 Mouse Immunization

[0053] Mice were immunized with high-purity CPIV / NP recombinant protein, and other recombinant proteins expressed by the pET28a vector were used as screening antigens for monoclonal antibody selection. Specifically, purified CPIV / NP recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. At weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. At week 6, mice were immunized by direct injection of insulin (5 μg / mouse) into the spleen. 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 CPIV / NP protein via intraperitoneal pulse, and the spleen was collected 3 days later for hybridoma cell preparation.

[0054] 4.2 Screening of hybridoma cells

[0055] 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 to the bottom of the well, clones positive for CPIV / NP recombinant protein were selected by indirect ELISA. Since the immunogen was a prokaryotic expression source of the pET28a vector containing a His tag, background components needed to be screened to identify specific cell lines targeting CPIV / NP protein. Positive cells were cloned to monoclonal status using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0056] Indirect ELISA method for screening positive clones:

[0057] Recombinant CPIV / NP proteins and other recombinant proteins of the pET28a vector (pET28a-HPV16 / E7, His tag, expression of which is described in patent 202510831708.X) 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), with 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 150 μL of 3% sucrose + 2% BSA, 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. 50 μL of cell culture supernatant was added, and the plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with the CPIV / NP recombinant protein but not with the control recombinant protein were selected for subsequent experiments.

[0058] Table 1: Screening Results of Monoclonal Antibodies

[0059]

[0060] 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.

[0061] 5. Purification of monoclonal antibodies

[0062] 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, and 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, and let it precipitate overnight at 2–8°C. 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 at 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 solution 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.

[0063] The purified antibody was diluted to 1 μg / ml with PBS, and the antibody-antigen binding affinity was detected using the indirect ELISA method described above. The results are shown below. Figure 3 As shown in Table 2, the binding ability of the purified monoclonal antibody to the antigen was detected by indirect ELISA. The monoclonal antibody showed strong reactivity to the CPIV / NP recombinant protein, but no significant reaction to the irrelevant antigen HPV16 / E7 recombinant protein, indicating that the obtained monoclonal antibody has good specificity and high affinity.

[0064] Table 2: Identification results of monoclonal antibodies

[0065]

[0066] 6. Preparation of test strips coated with different CPIV / NP monoclonal antibodies:

[0067] The selected CPIV / NP monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS at pH 7.4) were sprayed horizontally in a linear pattern using a scribing instrument, with a spray volume of 0.8 μL / cm per line, forming the detection line (T line). Goat anti-mouse IgG antibodies were then sprayed horizontally in a linear pattern at 6 mm intervals, diluted to a final concentration of 1 mg / mL with 0.01 M PBS at pH 7.4, and coated onto the nitrocellulose membrane at a volume of 0.8 μL / cm, forming the control line (C line).

[0068] 7. Colloidal gold pairing of CPIV / NP monoclonal antibodies

[0069] Preparation of antibody-colloidal gold labeled complex:

[0070] 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. To optimize the conjugation efficiency between the antibody and colloidal gold, 1 mL of colloidal gold solution was placed in a centrifuge tube, and 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL of 0.2M potassium carbonate solution were added respectively to adjust the pH environment of the colloidal gold solution. The optimal conjugation effect was found to be 5 μL. After mixing, 5 μg of the CPIV / 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 the mixture was incubated at room temperature 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 (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0071] 8. Screening of paired monoclonal antibodies

[0072] Nitrocellulose membranes streaked with different monoclonal antibodies against CPIV / NP were individually paired with different colloidal gold-labeled monoclonal antibodies. CPIV / NP protein was diluted to 20 ng / mL for detection, while HPV16 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection. Combinations that showed the strongest staining for CPIV / NP protein and did not react with the control protein were selected. Therefore, the optimal pairing for detecting CPIV / NP recombinant protein was determined to be 5H10 streaking and 1H7 gold labeling.

[0073] Table 3: Results of screening paired monoclonal antibodies using CPIV / NP recombinant protein

[0074]

[0075] - 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.

[0076] Table 3 shows the screening results using CPIV / NP recombinant protein diluted to a concentration of 20 ng / ml as a positive antigen. The detection results for HPV16 / E7 recombinant protein and blank dilution were negative and are not shown. The results showed that the 5H10 monoclonal antibody, applied with a membrane scratch and the 1H7 monoclonal antibody, produced the deepest staining for CPIV / NP recombinant protein, making it the optimal pairing. That is, the combination of monoclonal antibody anti-5H10 as the capture antibody and monoclonal antibody 1H7 as the labeling antibody can specifically recognize CPIV / NP recombinant protein.

[0077] 9. Preparation and Assembly of Colloidal Gold Test Strips

[0078] Preparation of gold-labeled pads: A 6mm x 300mm glass fiber membrane was treated with PBS containing 1% BSA and 1% Tween-20 at pH 7.4. The prepared colloidal gold-labeled antibody was then uniformly added to the glass fiber at a rate of 1200ul / strip. After air drying, the membrane was dried at 37℃ for 2 hours before use.

[0079] See Figure 4 , Figure 4This is a schematic diagram of the colloidal gold assembly. A 60mm x 300mm PVC backing plate is used as a support. Sample pads, gold-labeled pads (colloidal gold pads), nitrocellulose membranes, and absorbent paper are attached to the backing plate. The nitrocellulose membrane is coated with two lines and dried at 37℃ for 12 hours before use. The nitrocellulose membrane is coated with a detection line (monoclonal antibody 5H10) and a control line (goat anti-mouse IgG). The colloidal gold pad is coated with monoclonal antibody 1H7. The assembled plate is cut into 4mm strips using a strip cutter and wrapped with colloidal gold plastic casings. The sample pad is exposed at the sample application well of the plastic casing, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip is now assembled.

[0080] 10. Test strip specificity test

[0081] Recombinant protein samples: CPIV / NP recombinant protein, CDV / NP recombinant protein (Genbank ID: AFC40213.1), CCoV / NP recombinant protein (Genbank ID: UVT36859.1), CRCoV / NP (Genbank ID: ANA11064.1), and CPV / VP2 (Genbank ID: QYI48652.1) recombinant proteins were diluted to 1 μg / mL with sample diluent for detection. 80 μL of the diluted sample was added to the sample well of the test strip, and the results were evaluated within 20 minutes. A positive result was indicated by the appearance of clear red bands on both the T and C lines; a negative result was indicated by only the C line showing color; and an invalid result was indicated by no color development on the C line.

[0082] The sample dilution solution was 0.01 MPB + 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 detect CPIV / NP recombinant protein well, and there is no cross-reactivity with CDV / NP recombinant protein, CCoV / NP recombinant protein, CRCoV / NP, and CPV / VP2, indicating that the test strip has good specificity.

[0083] 11. Sensitivity test of test strips

[0084] The CPIV / NP recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.5 ng / mL before detection. Figure 6 The results are from the sensitivity test of the test strip. Figure 6The 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 (0.01 MPB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (BLK), did not show color development, indicating that the limit of detection for CPIV / NP recombinant protein on the test strip is 1 ng / mL.

[0085] 12. Monoclonal antibody binding activity

[0086] Based on the screening of potential paired antibodies using a double-antibody sandwich ELISA, the selected paired monoclonal antibodies and other murine-derived 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 the CPIV / NP recombinant protein. A murine CPV monoclonal antibody (Hytest, CAT#3PV16) 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 CPV mouse monoclonal antibody. Monoclonal antibodies 1H7 and 5H10 still showed strong signals at concentrations as low as 10 ng / mL.

[0087] 13. Variable region gene sequence of monoclonal antibodies

[0088] 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 two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction 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.

[0089] Gold-labeled monoclonal antibody 1H7:

[0090] The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 1H7 is shown in SEQ ID NO.20:

[0091] GATGTCCAGATAACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTCTAGCACATCTTATTTGGC TTCTGGAGTCCCACCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCGCTCACGTTCGGTGGTGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0092] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1H7 is shown in SEQ ID NO.16:

[0093] DVQITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWISSTSYLASGVPPRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGGGTKLEIKRTV.

[0094] Light chain CDR area annotation:

[0095] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO.10:

[0096] CDR-L1: TASSSVSSSYLH;

[0097] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO.11:

[0098] CDR-L2: STSYLAS;

[0099] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO. 12:

[0100] CDR-L3: HQYHRSPLT.

[0101] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO.19:

[0102] GAAGTGCAGCTGTTGGAGACTGGACCTGGCCTGGTGGCGCCCTCACTGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAAGCAGTCATGGTGTTCACTGGATTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGACTGGTGGAAACACAAATTA TAATTCGGCTCTCATGTCCAGACTGACCATCACCAAAGACGACTCCAGGAGCCAAGTTTTCTTAGAAGTGAACAGTCTACAAACTGATGACACAGCCATATATTATTGTGCCAGAGACCACTATGATTACGGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0103] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO.15:

[0104] EVQLLETGPGLVAPSLSLSITCTVSGFSLSSHGVHWIRQSPGKGLEWLGVIWTGGNTNYNSALMSRLTITKDDSRSQVFLEVNSLQTDDTAIYYCARDHYDYGFDYWGQGTTLTVSS.

[0105] Heavy chain CDR region annotation:

[0106] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO. 7:

[0107] CDR-H1: SHGVH;

[0108] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO. 8:

[0109] CDR-H2: VIWTGGNTNYNSALMS;

[0110] The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 1H7 is shown in SEQ ID NO. 9:

[0111] CDR-H3: DHYDYGFDY.

[0112] Scratch-resistant monoclonal antibody 5H10:

[0113] The nucleotide sequence encoding the variable region of the 5H10 light chain of the monoclonal antibody is shown in SEQ ID NO.18:

[0114] GACATTTGTGATGTCACAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTATCCTGCAGGGCCAGTCAGAGCATTGGCACAAATATAAACTGGTATCAGCAAAGAACAAGTGGTTCTCCAAGGCTTCTCATAAAGCGTGCTTCTGAGTCTGTC TCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGACTTTATTCTTAGCATCAACAGTTTGGAGTCTGAAGATATTGCAGATTACTACTGTCACAGAATAGTAGCTGGCCGCTCACGTTCGGTGCTGGGACAAAGCTGGAGCTGAAACGTACGGTG.

[0115] The amino acid sequence of the variable region of the 5H10 light chain of the monoclonal antibody is shown in SEQ ID NO.14:

[0116] DIVMSQSPAILSVSPGERVSLSCRASQSIGTNINWYQQRTSGSPRLLIKRASESVSGIPSRFSGSGSGTDFILSINSLESEDIADYYCQQNSSWPLTFGAGTKLELKRTV.

[0117] Light chain CDR area annotation:

[0118] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 4:

[0119] CDR-L1: RASQSIGTNIN;

[0120] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 5:

[0121] CDR-L2: RASESVS;

[0122] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 6:

[0123] CDR-L3: QQNSSWPLT.

[0124] Heavy chain variable region nucleotide sequence:

[0125] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO.17:

[0126] GAAGTGATGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCAGTTTCAGTGAAGATGGCATGTCTTGGATTCGCCAGACTCCAGACAAGAGGCTGGAATGGGTCGCAAGTATTACTAGTGGAGGTAGTTTCACC TACTATCGAGACAGTGTGAGGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTATACCTACAAATGAGCAGTCTGAAGTCTGAGGACACAGCCACGTATTACTGTGCAAGACAGGGATTACTGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0127] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO.13:

[0128] EVMLVESGGDLVKPGGSLKLSCAASGFSFSEDGMSWIRQTPDKRLEWVASITSGGSFTYYRDSVRGRFTISRDNAKNTLYLQMSSLKSEDTATYYCARQGLLFAYWGQGTLVTVSS.

[0129] Heavy chain CDR region annotation:

[0130] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 1:

[0131] CDR-H1: EDGMS;

[0132] The amino acid sequence of the complementarity-determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 2:

[0133] CDR-H2: SITSGGSFTYYRDSVRG;

[0134] The amino acid sequence of the complementarity-determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 3:

[0135] CDR-H3: QGLLFAY.

Claims

1. A monoclonal antibody combination for detecting canine parainfluenza virus NP protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 5H10 and monoclonal antibody 1H7. The heavy chain variable region of the monoclonal antibody 5H10 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 5H10 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 1H7 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 1H7 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 for detecting canine parainfluenza virus NP protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting canine parainfluenza virus NP protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting canine parainfluenza virus NP protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5H10 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting canine parainfluenza virus NP protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H7 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination of claim 1 in the preparation of a tool for detecting canine parainfluenza virus NP protein.

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

8. The application according to claim 7, characterized in that, The colloidal gold test strip uses monoclonal antibody 5H10 as the capture antibody and monoclonal antibody 1H7 as the labeling antibody.

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

10. The application according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 5H10, the control line is coated with goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 1H7.

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