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

Abstract

The invention 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 of the monoclonal antibody combination. The combination is composed of monoclonal antibodies 5H10 and 1H7, the 5H10 serves as a coating antibody, the 1H7 is used for colloidal gold labeling, and efficient and specific sandwich detection of CPIV / NP antigens can be achieved. The heavy chain and light chain variable region CDR sequences of the two antibodies are clear (SEQ ID NO. 1-12). The invention further provides a rapid detection test strip based on the antibody combination, the rapid detection test strip can be used for on-site screening, early diagnosis and epidemiological monitoring of the canine parainfluenza virus, the problem that an existing detection technology is lack of a high-specificity recognition tool is solved, and the rapid detection test strip has a good application prospect.

Description

TECHNICAL FIELD

[0001] 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. BACKGROUND

[0002] Canine parainfluenza virus (CPIV) belongs to the single-stranded negative-strand RNA virus of paramyxoviridae. CPIV is highly infectious and is one of the respiratory pathogens, and is also the main culprit of infectious bronchitis-dog kennel cough. CPIV has a wide range of host infection, and canines, minks, foxes and raccoon dogs are susceptible. The virus is easily transmitted through respiratory secretions and air, and is commonly found in kennels, pet stores, pet hospitals and other densely populated places for dogs. The higher the density of feeding, the faster the transmission. In addition, when CPIV is mixedly infected with mycoplasma or bronchial septic bobram bacteria, it often leads to more severe illness or even death, so the harm caused by CPIV infection cannot be underestimated.

[0003] The diameter of CPIV virus is between 80-200nm, has a capsule membrane, and two kinds of filopodia are distributed on the capsule membrane, which are fusion protein (F) and hemagglutinin-neuraminidase protein (HN). There is a nucleocapsid protein (NP) in the capsule membrane, which is the main structural protein of the virus particle and is considered to be an important target for antigen detection. The diagnosis of CPIV infection is usually based on the combination of clinical symptoms and epidemiological data for preliminary judgment, but the clinical symptoms of CPIV infection are very similar to those of other canine respiratory infectious diseases, so more accurate laboratory detection means are needed to identify the pathogen. CPIV has only one serotype, so serum neutralization test and hemagglutination inhibition test can be used for epidemiological investigation and retrospective diagnosis of the disease. Molecular biology methods such as PCR can more sensitively diagnose CPIV infection, but routine immunization vaccines such as canine quadruple live vaccine may interfere with such detection, and there is a certain risk of false positives.

[0004] In summary, although there are various methods such as serology and molecular biology in the prior art for detecting canine parainfluenza virus, there is still a lack of a simple, rapid and specific on-site diagnostic tool. SUMMARY

[0005] The application provides a monoclonal antibody for detecting canine parainfluenza virus nucleoprotein (CPIV / NP) and application thereof, which has high specificity and good sensitivity, and solves the technical problem that there is a lack of efficient and rapid detection tools for canine parainfluenza virus antigens in the prior art.

[0006] In order to achieve the above-mentioned purpose, the application provides the following technical solutions: In a first aspect, the present application provides a monoclonal antibody combination for detecting canine parainfluenza virus NP protein, the monoclonal antibody combination comprising monoclonal antibody 5H10 and monoclonal antibody 1H7, The heavy chain variable region of the monoclonal antibody 5H10 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 1-SEQ ID NO. 3, respectively. The light chain variable region of the monoclonal antibody 5H10 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4-SEQ ID NO. 6, respectively. The heavy chain variable region of the monoclonal antibody 1H7 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7-SEQ ID NO. 9, respectively. The light chain variable region of the monoclonal antibody 1H7 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 10-SEQ ID NO. 12, respectively.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] In a second aspect, the present application provides use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting canine parainfluenza virus NP protein.

[0012] In a further embodiment, the tool comprises colloidal gold detection test strips, reagents, kits, and antibody chips.

[0013] In a further embodiment, the colloidal gold detection test strip uses monoclonal antibody 5H10 as the capture antibody and monoclonal antibody 1H7 as the labeled antibody.

[0014] In a further embodiment, the colloidal gold detection test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad and an absorbent paper connected to the back plate.

[0015] In a further embodiment, the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated with monoclonal antibody 5H10, and the quality control line comprises goat anti-mouse IgG; and the colloidal gold pad is coated with monoclonal antibody 1H7.

[0016] Beneficial effects: The application provides a combination of monoclonal antibodies for detecting canine parainfluenza virus nucleoprotein, which comprises 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 chain and light chain variable regions of each antibody in the combination are as follows: the heavy chain CDR1, CDR2 and CDR3 of monoclonal antibody 5H10 are SEQ ID NO. 1, 2 and 3, respectively, and the light chain CDR1, CDR2 and CDR3 are SEQ ID NO. 4, 5 and 6, respectively; and the heavy chain CDR1, CDR2 and CDR3 of monoclonal antibody 1H7 are SEQ ID NO. 7, 8 and 9, respectively, and the light chain CDR1, CDR2 and CDR3 are SEQ ID NO. 10, 11 and 12, respectively. The specific CDR sequences ensure high specificity and high affinity of antibody binding, and significantly reduce the risk of non-specific binding and cross-reaction.

[0017] The canine parainfluenza virus NP protein monoclonal antibody provided by the application has high specificity and good sensitivity, can specifically recognize canine parainfluenza virus nucleoprotein, has no cross-reaction with antigens of other common canine pathogens such as canine distemper virus, canine coronavirus, canine respiratory coronavirus and canine parvovirus, and effectively avoids misdiagnosis or missed diagnosis caused by similar symptoms of pathogens in the clinic. The antibody can be used not only for the construction of colloidal gold immunochromatography test strips to realize rapid and on-site detection of CPIV antigens in samples, but also for various detection platforms such as ELISA and immunofluorescence, and has a wide range of applications. The application solves the technical bottleneck of the lack of efficient, specific and stable canine parainfluenza virus detection reagents in the prior art, significantly improves the early diagnosis efficiency and epidemiological monitoring ability of canine parainfluenza virus infection, and has important clinical application value and industrialization prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative effort based on these drawings.

[0019] Figure 1 Results of identification of proteins for SDS-PAGE; Figure 2 Results of identification of recombinant proteins of CPIV / NP; Figure 3 Results of identification of purified monoclonal antibodies; Figure 4 Schematic diagram of colloidal gold assembly; Figure 5 Results of specificity analysis of test strips; Figure 6 Results of sensitivity test of test strips; Figure 7 Results of antibody binding identification. DETAILED DESCRIPTION

[0020] The embodiments of the present application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application, and the present application can be implemented in many different forms, and is not limited to the specific examples disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0021] The present application provides these examples in order to make the present application thorough and complete, and fully express the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specified, the relative arrangement of components and steps, the composition of materials, numerical expressions and values set forth in these examples should be interpreted as merely exemplary, and not as limiting.

[0022] The detection method of the present application is not used for disease diagnosis and treatment.

[0023] The immunological detection of antigens, such as rapid antigen test strips, is convenient to operate, can quickly realize qualitative detection, has been widely used in rapid detection of pathogens, and has shown obvious advantages. The monoclonal antibody against canine parainfluenza virus NP protein is the core tool of immunological detection, is the core raw material for developing high-specificity and high-sensitivity detection reagents, and has important significance for rapid diagnosis, epidemiological monitoring and scientific research of CPIV.

[0024] The application obtains a monoclonal antibody for targeted detection of canine parainfluenza virus NP protein through a hybridoma technology, and successfully applies the monoclonal antibody to a colloidal gold detection test paper, and the canine parainfluenza virus NP recombinant protein has good detection sensitivity and specificity, which provides a raw material basis for development of a diagnostic reagent for canine parainfluenza virus infection.

[0025] The canine parainfluenza virus NP protein includes canine parainfluenza virus NP natural protein and canine parainfluenza virus NP recombinant protein.

[0026] The CPIV / NP recombinant protein is canine parainfluenza virus NP recombinant protein, or canine parainfluenza virus nucleocapsid recombinant protein.

[0027] The application screens and obtains a monoclonal antibody pair capable of specifically recognizing CPIV / NP protein through a hybridoma technology. The combined antibody can efficiently recognize CPIV / NP recombinant protein, and has good specificity and sensitivity. The monoclonal antibody pair is applied to an immunodetection platform, and a rapid detection test paper strip or test paper card based on colloidal gold immunochromatography technology is constructed. The test paper has high sensitivity to CPIV / NP recombinant protein, and has no cross reaction with other proteins, and solves the problem of false positive in PCR detection caused by inoculation of canine quadruple live vaccine.

[0028] Example 1 1. Canine parainfluenza virus NP recombinant protein expression The CPIV / NP gene is synthesized by GenScript Biotechnology Co., Ltd. and cloned into a pET28a vector, and the nucleotide sequence is shown in SEQ ID NO. 21:

[0029] The amino acid sequence is shown in SEQ ID NO. 22: MSSVLKAYERFTLTQELQDQSEEGTIPPTTLKPIIRVFILTSNNPELRSRLLLFCLRIVLSNGARDSHRFGALLTMFSLPSATMLNHVKLADQSPEADIERVEIDGFEEGSFRLIPNARSGMSRGEINAYAALAEDLPDTLNHETPFVDSEVEGTAWDEIETFLDMCYSVLMQAWIVTCKCMTAPDQPAASIEKRLQKYRQQGRINPRYLLQPEARRIIQNVIRKGMVVRHFLTFELQLARAQSLVSNRYYAMVGDVGKYIENCGMGGFFLTLKYALGTRWPTLALAAFSGELTKLKSLMALYQTLGEQARYLALLESPHLMDFAAANYPLLYSYAMGIGYVLDVNMRNYAFSRSYMNKTYFQLGMETARKQQGAVDMRMAEDLGLTQAERTEMANTLAKLTTANRGADTRGGVNPFSSVTGTTQVPAAVTGDTFESYMVADRLRQRYADASTHDDEMPPLEEEEEDDTSAGPRTGPTLEQVALDIQNTAVGAPIHTDDLNAALGDLDI.

[0030] The recombinant plasmid pET28a-CPIV / NP was transformed into BL21 (DE3) competent cells according to a conventional method and induced for expression, specifically: the transformed bacteria were spread on LB agar plates (containing 50 μg / mL kanamycin) and incubated at 37°C overnight. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C, 220 rpm overnight. The culture was inoculated into LB medium (containing 50 μg / mL kanamycin) at a volume of 1% of the total volume of the medium, and incubated at 37°C, 220 rpm for about 3 hours, until the OD 600 was 0.6-0.9. IPTG was added at a final concentration of 0.1 mM, and the bacteria were induced at 30°C, 200 rpm for 4 hours, and then collected.

[0031] 2. Purification of canine parainfluenza virus NP recombinant protein The expressed recombinant protein has a histidine tag, and a protein purification instrument, HisTrap TMHP affinity chromatography column for purification. A buffer is 50 mM PB, 300 mM NaCl, pH 8.0, B buffer is 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0, the chromatography column with A buffer equilibration, then the fermentation bacteria 8000 rpm centrifugation 10 min, the precipitate was resuspended with A liquid, in ice water ultrasonic broken 30 min, interval 5 seconds ultrasonic 5 seconds, 12000 rpm centrifugation 30 min, the supernatant was filtered with jiet biology 0.22 micron filter after loading to chromatography column, the chromatography column with A buffer washing, followed by B buffer gradient elution. The collection of target protein elution peak with A buffer dialysis at 4 ℃ overnight, the purified protein by SDS-PAGE electrophoresis observation. The purified protein electrophoresis results as shown in Figure 1 The protein concentration was determined by ultramicro spectrophotometer and stored at -20 ℃. Figure 1 In the figure, M: protein Marker, R is: purified CPIV / NP recombinant protein, there is a clear band between 55-70 kDa, wherein the fusion protein estimated molecular weight: 61.4 kDa, indicating that the application has successfully prepared CPIV / NP recombinant protein, high purity, good integrity, meet the needs of downstream use.

[0032] 3. Identification of canine parainfluenza virus NP (CPIV / NP) recombinant protein The purified CPIV / NP recombinant protein was coated on the enzyme-labeled plate, and indirect ELISA method was used to identify its reaction with CPIV positive serum. The positive serum was canine pentavalent hyperimmune serum, which was obtained from healthy dogs immunized with canine distemper, parainfluenza, infectious hepatitis, parvovirus and coronavirus enteritis virus. First, the recombinant protein was coated in the microplate well (coating buffer: carbonate buffer, 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate were dissolved in 1 L of pure water, pH 9.6), the coating concentration was 1 μg / mL, 50 μL / well, 4°C overnight. The next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C for 2 hours, then washed with PBST (0.05% Tween-20 in PBS, pH 7.4) once, and dried. The CPIV / NP positive serum was diluted with PBS at a gradient of 100 times, 1000 times, 10000 times, 100000 times, and 1000000 times, and 50 μL was added to the microplate well coated with the antigen. PBS was used as a negative control, and the reaction was carried out at 37°C for 30 min. The liquid in the well was shaken out, and the plate was washed with PBST 4 times, and dried. Then 50 μL / well of HRP-labeled rabbit anti-dog IgG secondary antibody (Solebo, diluted 5000 times with PBS) was added, and the reaction was carried out at 37°C for 30 min. The plate was washed 4 times again, and dried. Then 50 μL / well of TMB color developing liquid was added, and the color was developed at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) was added to terminate the reaction, and the OD450nm value was measured by an enzyme-labeled instrument. The results are shown in Figure 2 The purified CPIV / NP recombinant protein can specifically react with CPIV positive serum, and still maintains good immunoreactivity under a wide serum dilution gradient. The recombinant protein has correct antigen structure and good biological activity, which meets the requirements of downstream use.

[0033] 4. Screening of monoclonal antibody of CPIV / NP recombinant protein 4.1. Mouse immunization The highly purified CPIV / NP recombinant protein was used to immunize mice, and other recombinant proteins expressed by pET28a vector were used as anti-screening antigens for monoclonal antibody screening. Specifically, the purified CPIV / NP recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected into 6-week-old female BALB / c mice at a dose of 30 μg per mouse. At 2 weeks and 4 weeks, the mice were given booster immunization by subcutaneous injection of the same dose mixed with an equal volume of Freund's incomplete adjuvant. At 6 weeks, the mice were immunized by directly injecting the spleen with insulin needles at a dose of 5 μg per mouse. Seven days after the last immunization, the mouse serum was detected for antibody titer. The mouse with higher titer was selected for intraperitoneal boost with 20 μg of recombinant CPIV / NP protein, and the mouse spleen was taken 3 days later for preparation of hybridoma cells.

[0034] 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 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.

[0035] Indirect ELISA method for screening positive clones: 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.

[0036] Table 1: Screening results of monoclonal antibodies

[0037] 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.

[0038] 5. Purification of monoclonal antibodies The ascites is centrifuged at 12000 r / min for 10 minutes, 1 ml of supernatant is taken, 4 ml of acetic acid-sodium acetate buffer (0.06M, pH 4.5) is added, mixed uniformly, then 10 μl of n-octanoic acid is slowly added while stirring, after adding, continue stirring for 30 minutes, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the supernatant. The supernatant is filtered with defatted cotton, saturated ammonium sulfate is added at a ratio of 50% (V / V) of the final volume, stirring while adding, after adding, continue stirring for 30 minutes, precipitate overnight at 2-8°C, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the precipitate. The precipitate is completely dissolved with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filtered through a 0.22 um filter, the filtered sample is pumped into a Protein L purification column equilibrated with binding buffer at low speed through a peristaltic pump, connected to a protein purification instrument, washed with 5-10 column volumes of binding buffer until the ultraviolet absorption peak is washed flat, then eluted with elution buffer (0.1M glycine, pH 2.7), collect the elution peak, the collected sample is neutralized with 1M Tris-HCl, pH 9, loaded into a dialysis bag (MW: 8000-14000), dialyzed in 20 mM PBS pH 7.4 solution at 2-8°C for 16 hours. The liquid in the dialysis bag is transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes, the supernatant is the purified monoclonal antibody.

[0039] The purified antibody is diluted to 1 ug / ml with PBS, and the antibody binding capacity is detected by the above indirect ELISA method, and the results are shown in Figure 3 and Table 2, the purified monoclonal antibody is detected by indirect ELISA, the monoclonal antibody shows strong reactivity on CPIV / NP recombinant protein, and has no significant reaction on irrelevant antigen HPV16 / E7 recombinant protein, indicating that the obtained monoclonal antibody has good specificity and high affinity.

[0040] Table 2: Identification results of monoclonal antibodies

[0041] 6. Preparation of test strips coated with different monoclonal antibodies of CPIV / NP The selected CPIV / NP monoclonal antibodies were respectively drawn on the nitrocellulose membrane with different specifications of 20 mm x 300 mm, using a membrane drawing instrument to spray the diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS with pH 7.4) in a horizontal line, with a spraying amount of 0.8 uL / cm, to form a test line (T line). With an interval of 6 mm, the goat anti-mouse IgG antibody was sprayed in a horizontal line with the same spraying amount, diluted to a final concentration of 1 mg / mL with 0.01 M PBS with pH 7.4, coated on the nitrocellulose membrane with an amount of 0.8 uL / cm, to form a quality control line (C line).

[0042] 7. Gold colloid pairing of CPIV / NP monoclonal antibody Preparation of antibody-gold colloid labeled complex: Antibody labeling: The colloidal gold solution was prepared by the trisodium citrate reduction method. Specifically, 100 mL of 0.01% chloroauric acid solution was heated and boiled, then 1 mL of 1% trisodium citrate solution was quickly added until the solution color was wine red. Boiling was continued for 5 minutes, and after the colloidal gold particles were stable, the solution was cooled to room temperature for standby. To optimize the coupling efficiency of the antibody and colloidal gold, 1 mL of colloidal gold solution was taken in a centrifuge tube, and 1 uL, 2 uL, 3 uL, 4 uL, 5 uL, and 6 uL of 0.2 M potassium carbonate solution were added to adjust the pH environment of the colloidal gold solution in a gradient. Finally, the coupling effect was optimal when 5 uL was selected. After mixing, 5 ug of the CPIV / NP monoclonal antibody to be labeled was added, and after quick mixing, it was incubated at room temperature for 10 minutes. Then, 10 uL of 10% (w / v) bovine serum albumin (BSA) was added to block the non-specific binding sites, and incubation was continued at room temperature for 10 minutes. Then, 10 uL of 10% (w / v) polyethylene glycol 20000 (PEG20000) was added to enhance the stability of the label, and after mixing, it was centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The lower precipitate was resuspended with 1 / 10 volume of resuspension solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4), to obtain the antibody-gold colloid labeled complex, which was stored at 4°C in the dark for standby.

[0043] 8. Screening of paired monoclonal antibodies The nitrocellulose membrane with different CPIV / NP monoclonal antibodies drawn was respectively paired with different colloidal gold labeled monoclonal antibodies. The CPIV / NP protein was diluted to 20 ng / mL for detection, and the HPV16 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection, and a combination that showed a deeper color development for CPIV / NP protein and did not react with the control protein was selected. Therefore, the combination of 5H10 drawn membrane and 1H7 labeled gold was selected as the best pair for detecting CPIV / NP recombinant protein.

[0044] Table 3: Results of screening paired monoclonal antibodies with CPIV / NP recombinant protein

[0045] - represents negative, i.e. no color development; + / ++ / +++ represents positive, i.e. color development, the more "+" signs, the darker color development, i.e. the stronger positive reaction.

[0046] Table 3 is the screening result using CPIV / NP recombinant protein diluted to a concentration of 20 ng / ml as a positive antigen. The detection results of HPV16 / E7 recombinant protein and blank diluent were negative, and no display was performed. The results showed that the 5H10 monoclonal antibody membrane combination and the 1H7 monoclonal antibody label had the deepest color development for the CPIV / NP recombinant protein, which was used as the optimal paired combination. That is, the combination of monoclonal antibody anti-5H10 as a capture antibody and monoclonal antibody 1H7 as a label antibody can specifically recognize the CPIV / NP recombinant protein.

[0047] 9. Preparation and assembly of colloidal gold detection test paper Preparation of gold label pad: 6mm X 300mm glass fiber membrane was used, which was treated with PBS containing 1% BSA and 1% Tween-20, pH 7.4. The prepared colloidal gold labeled antibody was uniformly added to the glass fiber at 1200ul / strip, naturally air-dried, and then placed in a 37°C oven for 2h for standby.

[0048] See Figure 4 , Figure 4 is a schematic diagram of colloidal gold assembly. A 60mm X 300mm PVC backboard was used as support, on which a sample pad, a gold label pad (colloidal gold pad), a nitrocellulose membrane and a water absorption paper were respectively pasted. The nitrocellulose membrane was coated with two lines, and was dried at 37°C for 12h for standby. Among them, the nitrocellulose membrane was coated with a detection line (monoclonal antibody 5H10 membrane) and a quality control line (goat anti-mouse IgG), the colloidal gold pad was coated with monoclonal antibody 1H7, and the assembled large plate was cut into 4mm naked strips with a cutting machine, and was wrapped with a colloidal gold plastic card shell. The sample pad was exposed to the sample hole position of the plastic card shell, the quality control line and the detection line were exposed to the result observation hole position, and the colloidal gold test paper card was assembled.

[0049] 10. Specificity test of test strip 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), CPV / VP2 (Genbank ID: QYI48652.1) recombinant protein were diluted to 1 ug / mL with sample diluent for detection. 80 uL of the above diluted samples were added to the sample wells of the test strip card, and the results were determined within 20 min. If clear red bands appeared on both T and C lines, it was judged as positive; only C line developed color was negative; if C line did not develop color, it was judged as invalid.

[0050] The sample diluent was 0.01 M PB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4. The results are shown in Figure 5 , Figure 5 The specificity analysis results of the test strip are shown in the table. The test strip card can well detect CPIV / NP recombinant protein, and has no cross reaction with CDV / NP recombinant protein, CCoV / NP recombinant protein, CRCoV / NP, and CPV / VP2, indicating that the test strip card has good specificity.

[0051] 11. Sensitivity test of test strip CPIV / NP recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.5 ng / mL for detection. Figure 6 The sensitivity test results of the test strip are shown in the table. Figure 6 The results show that the colloidal gold test strip still has weak color development at a recombinant protein concentration of 1 ng / mL, while the blank diluent, i.e. sample diluent (0.01 M PB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (BLK) does not develop color, indicating that the minimum detection limit of the test strip card for CPIV / NP recombinant protein is 1 ng / mL.

[0052] 12. Monoclonal antibody binding activity On the basis of screening potential pairing antibodies by double antibody sandwich ELISA, the pairing monoclonal antibodies and other irrelevant mouse antibodies were gradient diluted (concentrations were 10 ug / mL, 1 ug / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL and 0.1 ng / mL) according to the aforementioned indirect ELISA method to evaluate their binding activity with CPIV / NP recombinant protein. CPV mouse monoclonal antibody (Hytest, CAT#3PV16) was used as a negative control in the experiment to exclude the influence of non-specific binding. The results are shown in Figure 7 .Figure 7 The "Ctrl" represents the negative control CPV mouse monoclonal antibody, and the monoclonal antibodies 1H7 and 5H10 still showed strong signals at a concentration as low as 10 ng / mL.

[0053] 13. Variable region gene sequence of the monoclonal antibody Total RNA of the hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers; mouse antibody variable region universal primers were designed, and the VH and VL genes were amplified by 2 rounds of PCR, and an Age1 and Bsiw1 enzyme cutting site was introduced in the primer of the 3rd round of PCR. The PCR product was cut and purified, and then ligated to the pUC19 vector, transformed into the TOP10 strain, and cultured at 37°C for 14 h. Single colonies were picked for sequencing, and the gene sequences of the light and heavy chains of the monoclonal antibody were obtained.

[0054] Monoclonal antibody 1H7: The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1H7 is shown as SEQ ID NO. 20: GATGTCCAGATAACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAACGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTCTAGCACATCTTATTTGGCTTCTGGAGTCCCACCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCGCTCACGTTCGGTGGTGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0055] The amino acid sequence of the variable region of the light chain of the monoclonal antibody 1H7 is shown as SEQ ID NO. 16: DVQITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWISSTSYLASGVPPRFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGGGTKLEIKRTV.

[0056] Light chain CDR region annotation: The amino acid sequence of the complementarity determining region CDR-L1 of the light chain variable region of the monoclonal antibody 1H7 is shown as SEQ ID NO. 10: CDR-L1: TASSSVSSSYLH; The amino acid sequence of the complementarity determining region CDR-L2 of the light chain variable region of the monoclonal antibody 1H7 is shown as SEQ ID NO. 11: CDR-L2: STSYLAS; The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of the monoclonal antibody 1H7 is shown as SEQ ID NO. 12: CDR-L3: HQYHRSPLT.

[0057] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H7 is shown as SEQ ID NO. 19: GAAGTGCAGCTGTTGGAGACTGGACCTGGCCTGGTGGCGCCCTCACTGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAAGCAGTCATGGTGTTCACTGGATTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGACTGGTGGAAACACAAATTATAATTCGGCTCTCATGTCCAGACTGACCATCACCAAAGACGACTCCAGGAGCCAAGTTTTCTTAGAAGTGAACAGTCTACAAACTGATGACACAGCCATATATTATTGTGCCAGAGACCACTATGATTACGGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0058] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H7 is shown as SEQ ID NO. 15: EVQLLETGPGLVAPSLSLSITCTVSGFSLSSHGVHWIRQSPGKGLEWLGVIWTGGNTNYNSALMSRLTITKDDSRSQVFLEVNSLQTDDTAIYYCARDHYDYGFDYWGQGTTLTVSS.

[0059] Heavy chain CDR region annotation: The amino acid sequence of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 1H7 is shown as SEQ ID NO. 7: CDR-H1 : SHGVH; The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 1H7 are set forth in SEQ ID NO. 8, respectively: CDR-H2: VIWTGGNTNYNSALMS; The amino acid sequences of the complementarity determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 1H7 are set forth in SEQ ID NO. 9, respectively: CDR-H3: DHYDYGFDY.

[0060] Striated membrane monoclonal antibody 5H10: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5H10 is set forth in SEQ ID NO. 18: GACATTGTGATGTCACAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTATCCTGCAGGGCCAGTCAGAGCATTGGCACAAATATAAACTGGTATCAGCAAAGAACAAGTGGTTCTCCAAGGCTTCTCATAAAGCGTGCTTCTGAGTCTGTCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGACTTTATTCTTAGCATCAACAGTTTGGAGTCTGAAGATATTGCAGATTACTACTGTCAACAGAATAGTAGCTGGCCGCTCACGTTCGGTGCTGGGACAAAGCTGGAGCTGAAACGTACGGTG.

[0061] The amino acid sequence of the light chain variable region of the monoclonal antibody 5H10 is set forth in SEQ ID NO. 14: DIVMSQSPAILSVSPGERVSLSCRASQSIGTNINWYQQRTSGSPRLLIKRASESVSGIPSRFSGSGSGTDFILSINSLESEDIADYYCQQNSSWPLTFGAGTKLELKRTV.

[0062] Light chain CDR region labeling: The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of monoclonal antibody 5H10 are set forth in SEQ ID NO. 4, respectively: CDR-L1 : RASQSIGTNIN; 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: CDR-L2: RASESVS; 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: CDR-L3: QQNSSWPLT.

[0063] The heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 17: GAAGTGATGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCAGTTTCAGTGAAGATGGCATGTCTTGGATTCGCCAGACTCCAGACAAGAGGCTGGAATGGGTCGCAAGTATTACTAGTGGAGGTAGTTTCACCTACTATCGAGACAGTGTGAGGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTATACCTACAAATGAGCAGTCTGAAGTCTGAGGACACAGCCACGTATTACTGTGCAAGACAGGGATTACTGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0064] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5H10 is shown in SEQ ID NO. 13: EVMLVESGGDLVKPGGSLKLSCAASGFSFSEDGMSWIRQTPDKRLEWVASITSGGSFTYYRDSVRGRFTISRDNAKNTLYLQMSSLKSEDTATYYCARQGLLFAYWGQGTLVTVSS.

[0065] Heavy chain CDR region annotation: 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: CDR-H1: EDGMS; The amino acid sequence of the complementarity determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 5H10 is set forth in SEQ ID NO. 2: CDR-H2: SITSGGSFTYYRDSVRG; The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 5H10 is set forth in SEQ ID NO. 3: 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, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. 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. 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. 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.

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 according to 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 includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 1H7.

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