Monoclonal antibody for detecting Newcastle disease virus, antibody chip and application thereof

Abstract

The invention discloses a monoclonal antibody for detecting Newcastle disease virus, an antibody chip and application thereof, the Newcastle disease virus is adopted to prepare hybridoma cells, the heavy chain variable region sequence of the obtained monoclonal antibody is shown as SEQ ID NO: 7, and the light chain variable region sequence is shown as SEQ ID NO: 8. The antibody has high specificity and sensitivity to the Newcastle disease virus, and can be used for diagnosing the condition of the poultry Newcastle disease and preventing and controlling the propagation of the Newcastle disease. The antibody chip containing the antibody can simultaneously detect a plurality of Newcastle disease virus samples, realizes rapid, flexible and convenient detection of the to-be-detected samples, improves the detection efficiency, and expands the application range of the product.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a monoclonal antibody for detecting Newcastle disease virus, an antibody chip and applications thereof. Background Art

[0002] Newcastle disease (ND) is an acute infectious disease of poultry caused by the Newcastle disease virus (NDV). It is one of the most detrimental infectious diseases to the poultry industry (e.g., chickens, ducks, geese, pigeons, and quail). NDV belongs to the genus Paramyxovirus and has a single serotype, but strains vary significantly in virulence and pathogenicity to poultry. Conventional hemagglutination inhibition tests (HI) are not easily able to detect differences in antigenic structure. Furthermore, hemagglutination (HA) and hemagglutination inhibition (HI) methods have significant drawbacks, such as poor sensitivity, large errors, and small sample sizes. These methods lack reliability and require significant manpower to test large numbers of samples.

[0003] Monoclonal antibodies have the specificity to recognize a single antigenic determinant. Using monoclonal antibodies can detect differences in surface antigen structure between different NDV strains, with the advantages of rapidity, sensitivity, and high specificity.

[0004] Therefore, the development of monoclonal antibodies to detect Newcastle disease virus is of great significance for the prevention and control of Newcastle disease. Summary of the Invention

[0005] In order to accurately, quickly and timely diagnose and treat Newcastle disease and prevent the spread of Newcastle disease, and reduce the harm of Newcastle disease to the poultry industry, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof for detecting Newcastle disease virus, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises the heavy chain variable region (VH) complementarity determining regions 1-3 (CDR1-3) whose amino acid sequences are respectively as shown in SEQ ID NOs: 1-3, or a sequence having 90% or more homology to the sequences shown in SEQ ID NOs: 1-3, and the light chain variable region (VL) complementarity determining regions 1-3 (CDR1-3) whose amino acid sequences are respectively as shown in SEQ ID NOs: 4-6, and a sequence having 90% or more homology to the sequences shown in SEQ ID NOs: 4-6.

[0007] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises heavy chain variable region (VH) complementarity determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, and light chain variable region (VL) complementarity determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NOs: 4-6, respectively.

[0008] Furthermore, the amino acid sequences of SEQ ID NOs: 1-6 are as follows:

[0009] GYSFTGDT (SEQ ID NO: 1)

[0010] INPFNGGT (SEQ ID NO: 2)

[0011] ARNDYDGYGMDY (SEQ ID NO: 3)

[0012] QNINVW (SEQ ID NO: 4)

[0013] KAS (SEQ ID NO: 5)

[0014] QQGQSYPWT (SEQ ID NO: 6).

[0015] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) as shown in SEQ ID NO: 7 or a sequence having more than 90% homology to the sequence shown in SEQ ID NO: 7, and a light chain variable region (VL) as shown in SEQ ID NO: 8 or a sequence having more than 90% homology to the sequence shown in SEQ ID NO: 8.

[0016] Furthermore, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having a sequence as shown in SEQ ID NO: 7, and a light chain variable region (VL) having a sequence as shown in SEQ ID NO: 8.

[0017] More preferably, the amino acid sequences of SEQ ID NOs: 7-8 are as follows:

[0018] EVQLQQSGPEMVKPGASMKISCKASGYSFTGDTMNWVKQSHGKNLEWIGLINPFNGGTRYNQKFKGKATLTVDKSSSTAYMELLSLTFEDSAVYYCARNDYDGYGMDYWGQGTSVTVSS(SEQ ID NO:7)

[0019] DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEIK(SEQ ID NO:8)

[0020] Preferably, the above 90% homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0021] Preferably, the monoclonal antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, dAb, single-chain antibody, mouse antibody, humanized antibody, chimeric antibody or multispecific antibody, and any polypeptide comprising an antibody binding domain or a homologous antibody binding domain. The antibody binding domain may include a complete heavy chain and / or light chain CDR, a complete antibody heavy chain and / or light chain variable region, a complete full-length heavy chain and / or light chain, or a single, two, three, four, five or six CDRs from the antibody. A single-chain antibody comprises one heavy chain variable region and one light chain variable region.

[0022] Preferably, the monoclonal antibody further comprises a light chain constant region and a heavy chain constant region. Optionally, the type of the monoclonal antibody is independently selected from IgM, IgE, IgA, IgD or IgG. It is understood that the type of antibody can be converted by the constant region sequence.

[0023] Preferably, the monoclonal antibody or antigen-binding fragment thereof specifically binds to NDV.

[0024] In a second aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0025] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule described in the second aspect.

[0026] Preferably, the vector is a cloning vector or an expression vector. The vector can be a plasmid, a cosmid or a phage.

[0027] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the vector described in the third aspect.

[0028] Preferably, the host cells include, but are not limited to, prokaryotic cells (such as E. coli cells) and eukaryotic cells (such as yeast cells, insect cells, plant cells and animal cells). The cells of the present invention may also be cell lines, such as 293T cells.

[0029] Furthermore, the animal cells are mammalian cells, such as mouse cells.

[0030] In a fifth aspect, the present invention provides a hybridoma cell capable of producing the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0031] Preferably, the hybridoma cells are formed by fusion of sp2 / 0 cells and non-human animal spleen cells.

[0032] Preferably, the non-human animal is a non-human mammal, more preferably a rodent.

[0033] Preferably, the non-human animal is immunized with NDV.

[0034] In one embodiment of the present invention, the non-human animal is a rat or a mouse.

[0035] In a sixth aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof described in the first aspect, the method comprising culturing the host cell described in the fourth aspect or the hybridoma cell described in the fifth aspect to obtain an antibody or antigen-binding fragment thereof that specifically binds to NDV.

[0036] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0037] In an eighth aspect, the present invention provides a kit for detecting Newcastle disease virus, wherein the kit comprises the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0038] In a ninth aspect, the present invention provides an antibody chip, comprising the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0039] In a tenth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the host cell described in the fourth aspect, the hybridoma cell described in the fifth aspect, the pharmaceutical composition described in the seventh aspect, or the antibody chip described in the ninth aspect in the preparation of a product for detecting Newcastle disease virus.

[0040] Preferably, the product comprises poultry meat, poultry blood, poultry tissue fluid, etc. By detecting the NDV level in the product, the health status of poultry animals can be assessed and the spread of Newcastle disease can be prevented and controlled.

[0041] Beneficial effects of the present invention:

[0042] 1. The monoclonal antibody of the present invention can specifically bind to Newcastle disease virus, diagnose Newcastle disease in poultry, and prevent and control the spread of Newcastle disease.

[0043] 2. The antibody chip of the present invention can detect multiple Newcastle disease virus samples simultaneously, achieving rapid, flexible and convenient detection of samples to be tested, improving detection efficiency and expanding the application range of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Shown are the results of immune titer determination after immunization of mice in Example 1;

[0045] Figure 2 Shown are the results of fluorescence staining of monoclonal antibody detection, where A. Chicken embryo fibroblasts; B. Negative control; C. Positive control;

[0046] Figure 3 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V1;

[0047] Figure 4 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V2;

[0048] Figure 5 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V3;

[0049] Figure 6 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V4;

[0050] Figure 7 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V5;

[0051] Figure 8 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V6;

[0052] Figure 9 Shown is the detection sensitivity of Anti-A-ND-13 to vaccine V7;

[0053] Figure 10 Shown is the specificity of Anti-A-ND-13 for vaccine V4 detection;

[0054] Figure 11 Shown is the sensitivity curve of Anti-A-ND-13 for vaccine V4 detection. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be further described below in conjunction with the embodiments and drawings, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely illustrative and do not limit the scope of the present invention.

[0056] The sources of some experimental materials and experimental methods involved in Example 3 of this application are as follows:

[0057] The positive control was Bio-IgG: goat anti-mouse IgG 200 μg / mL + 50% glycerol, Solarbio,

[0058] Negative control: Buffer: 40% glycerol;

[0059] Antibodies: REV; FAdV-1; ALV; Anti-A-ND whole virus 3; Anti-A-ND whole virus 13; EDS Mab.

[0060] Live virus vaccines: The storage concentration is 50 doses / 100 μL. V1: Newcastle disease and infectious bronchitis combination vaccine (LaSota + H52), Chengdu Tianbang; V2: Newcastle disease and infectious bronchitis combination vaccine (LaSota + H52), Fujian Shengwei 064; V3: Newcastle disease and infectious bronchitis combination vaccine (LaSota + H52), Fujian Shengwei; V4: Newcastle disease and infectious bronchitis combination vaccine (HB1 + H120); V5: Infectious bronchitis (H120); V6: Fowlpox live vaccine (quail attenuated strain); V7: Newcastle disease live vaccine (V4 / HB92).

[0061] Reagents: Streptavidin phycoerythrin (SAPE); Secondary antibody: PE-conjugated goat anti-mouse IgG, ab97024, abcam; 5% milk; PBST; ddH2O

[0062] Microarrayer (Arrayjet, Roslin, UK); three-dimensional (3D) modified aldehyde-based slides (ProteomicsEraMedical Co., Beijing, China); GenePix 4300A microarray scanner (Molecular Devices, LLC, San Jose, CA, USA); GenePix Pro7 software (Molecular Devices, USA)

[0063] Example 1 Preparation of Newcastle disease monoclonal antibodies

[0064] 1.1 Animal Immunization: Four SPF-grade BALB / c female mice were selected and numbered 1, 2, 3, and 4. 60 μg of ND whole virus protein was injected subcutaneously per mouse. Two weeks later, 30 μg of antigen protein was injected per mouse via the same route for a booster immunization. Repeat this three times. After each booster immunization, orbital blood samples were collected at 9-day intervals and plated with ND whole virus. The titer of the immunized mice was determined by ELISA.

[0065] 1.2 Immunoassay: Use "ND whole virus", 2μg / mL, 4℃ overnight coating; 2% milk, 37℃ blocking for 2h; serum is diluted 2-fold starting from 200 times, blank control is 1×PBS, negative control is negative serum 200-fold dilution. Figure 1 The results shown were obtained by selecting shock #1 mice for cell fusion experiments.

[0066] 1.3 Cell Fusion: Before fusion, intraperitoneally challenge mouse #1 with 50 μg of the immunogen. Gently pipette healthy sp2 / 0 cells from the wall of the culture flask and transfer them to a 50 mL centrifuge tube. Eyeballs were removed to collect blood, and the mice were then sacrificed by cervical dislocation and soaked in 75% alcohol for 5 minutes. A small amount of serum-free IMDM was poured into a dish and a cell strainer and syringe plunger were placed into the dish. The spleen was removed from the mouse using scissors and forceps and placed on the cell strainer. Gently crush the spleen thoroughly with the syringe plunger and transfer the crushed cells to the sp2 / 0 centrifuge tube. Centrifuge at 1500 rad / min for 5 minutes. The thymus was removed from the mouse using scissors and forceps and crushed. The crushed thymic cells were transferred to a 15 mL centrifuge tube. 2 mL of HAT and 1 mL of HT were added and placed in an incubator until needed. Discard the supernatant from the centrifuged cells and gently and carefully homogenize the cells with serum-free IMDM. Centrifuge at 1500 rad / min for 5 minutes. Discard the supernatant of the centrifuged cells as much as possible. Tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, slowly add 1 mL of PEG in about 1 minute, and let it stand in warm water for 1 minute. Then slowly add 2 mL of serum-free IMDM within 2 minutes, and then slowly add 8 mL of serum-free IMDM within 2 minutes. Centrifuge at 1000 rad / min for 5 minutes. Discard the supernatant, add 10 mL of serum, carefully blow the cells evenly, and pour in the thymocytes prepared earlier. Add sterilized semi-solid culture medium to make the volume up to 50 mL and mix thoroughly. Then pour evenly into 30 cell culture dishes. Place the cell culture dishes in a humidified box and then culture them in an incubator.

[0067] 1.4 Screening of monoclonal cells: First, thymocytes were plated at 100 μL / well, and then 10 plates × 93 monoclonal cells were selected and cultured in 96-well cell culture plates.

[0068] 1.4.1 Monoclonal Cell Screening 1: Discard all the supernatants of monoclonal cells in the 96-well culture plate and add 20% newborn calf IMDM culture medium (containing HT) at a volume of 200 μL / well.

[0069] 1.4.2 Monoclonal Cell Screening 2: Use the "ND Whole Virus" plate to coat the plate and use the ELISA method to screen the selected clones. The specific process is as follows:

[0070] Dilute the "ND whole virus" with coating solution to a final concentration of 2 μg / mL, 100 μL / well, incubate at 4°C overnight, and wash three times with washing solution; block with blocking solution (2% skim milk powder), 200 μL / well, incubate at 37°C for 2 hours, and then wash three times with washing solution; add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times in PBS), all at 100 μL / well, and incubate at 37°C for 1 hour , then washed three times with washing solution; added secondary antibody diluted 20,000 times with PBS, 100 μL / well, incubated at 37°C for 1 hour, removed and washed three times with washing solution; added 100 μL / well of color development solution, and the color development time was about 10 minutes; added 50 μL of stop solution to each well to terminate; measured the absorbance value at dual wavelengths (450, 630), and the results are shown in Table 1. In descending order of OD value, 48 positive hybridoma cell lines with OD values ​​greater than the negative control were selected, and the data were recorded and saved.

[0071] Table 1 Screening of hybridoma cells

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Note: F 12 As negative control, G 12 As blank control, H 12 As a positive control.

[0078] 1.4.3 Monoclonal Cell Screening 3: The 48 positive cell lines were again coated with "ND Whole Virus" plates and screened for the third time using the ELISA method. The results are shown in Table 2. Fifteen positive hybridoma cell lines were obtained, numbered 3, 13, 15, 18, 25, 28, 29, 31, 33, 34, 36, 37, 43, 45, and 47.

[0079] Table 2 Hybridoma cell 3 screening results

[0080]

[0081]

[0082] 1.5 Identification of monoclonal cell subtypes: The 15 positive cell lines screened were coated with "subtype coating antibody" diluted with coating solution to a final concentration of 2 μg / mL, 100 μL / well, 4°C, overnight, and then washed 3 times with washing solution; blocking solution, 200 μL / well, incubated at 37°C for 2 hours, and then washed 3 times with washing solution; primary antibody (cell culture supernatant) and negative control (SP2 / 0 culture supernatant) were added at 100 μL / well, incubated at 37°C for 1 hour, and then washed 3 times with washing solution; PBS was used to dilute each type of subtype secondary antibody, 100 μL / well, added to appropriate wells, incubated at 37°C for 1 hour, removed and washed 3 times with washing solution; 100 μL / well of color development solution was added, and the color development time was about 10 minutes; 50 μL of stop solution was added to each well to stop; the absorbance value was measured at wavelengths (450, 630), and the results are shown in Tables 3 and 4. The data were recorded and saved. In descending order of OD values, 10 IgG-positive hybridoma cell lines were screened and numbered 3, 13, 18, 25, 28, 31, 34, 36, 37 and 43.

[0083] Table 3 Monoclonal cell subtype identification results (1)

[0084]

[0085]

[0086] Table 4 Monoclonal cell subtype identification results (2)

[0087] serial number 33 34 36 37 43 45 47 - M 0.223 0.095 0.135 0.104 0.106 0.293 0.337 0.153 G1 0.079 0.096 0.142 0.516 0.389 0.056 0.093 0.086 G2a 0.069 0.093 0.093 0.049 0.05 0.028 0.04 0.022 G2b 0.085 0.081 0.5 0.085 0.062 0.057 0.101 0.045 G3 0.085 0.414 0.079 0.095 0.067 0.052 0.084 0.072 A 0.096 0.093 0.106 0.073 0.067 0.04 0.048 0.062 κ 0.128 0.163 0.197 0.139 0.116 0.156 0.187 0.066 λ 0.102 0.086 0.112 0.099 0.097 0.147 0.113 0.063

[0088] The 10 IgG-positive hybridoma cell lines screened were screened again, and according to the results in Table 5, the hybridoma cell line numbered 13 was selected for mass culture and then frozen.

[0089] Table 5 Monoclonal cell subtype identification results (3)

[0090]

[0091]

[0092] 1.6 Detection of monoclonal antibodies

[0093] 1.6.1 Sample processing and inoculation:

[0094] 1.6.1.1 Hybridoma cell line No. 13 was cultured, the cell fluid was centrifuged to obtain the supernatant, and the ND monoclonal antibody was isolated and purified.

[0095] 1.6.1.2 Take well-grown chicken embryo fibroblasts from 96 wells and inoculate Newcastle disease virus (La Sota strain) at 10 EID50 / 0.1 mL per well. Incubate at 37°C, 5% CO2 for 5 days.

[0096] 1.6.2 Fluorescent staining

[0097] 1.6.2.1 Discard the cell culture medium and gently wash the cell surface once with approximately 0.5 mL of PBS (pH 7.2) per well. Discard as much PBS as possible, then add 0.3 mL of cold methanol to each well and fix at room temperature for 10-20 minutes. Discard the methanol and allow to air dry.

[0098] 1.6.2.2 Gently wash the cell surface once with PBS, then add 0.1 mL of ND monoclonal antibody diluted 1000-fold with PBS to each well and incubate at 37°C for 1 hour.

[0099] 1.6.2.3 Discard the liquid in the wells and wash 3 to 4 times with PBS.

[0100] 1.6.2.4 Discard the washing solution as much as possible and add 0.1 mL of fluorescently labeled goat anti-mouse IgG diluted appropriately with PBS to each well and incubate at 37°C for 1 hour.

[0101] 1.6.2.5 Discard the liquid in the wells and wash 3 to 4 times with PBS.

[0102] 1.6.3 Observation

[0103] Observe under an inverted fluorescence microscope using green excitation light (wavelength 555 nm). Figure 2 As shown, the positive cell wells showed red fluorescence and had complete cell morphology; the uninfected cells were not stained and the field of view was dark.

[0104] Example 2 Antibody Sequence Determination

[0105] Hybridoma cells numbered 13 were lysed to extract total RNA. The RNA was reverse transcribed into cDNA using RACE technology. The heavy and light chain variable region sequences were amplified by PCR. The target fragments were ligated to a vector using ligase, and the ligated product was transformed into competent E. coli cells. Single clones were then selected for sequencing. The sequencing results were analyzed and annotated. The amino acid sequences of the heavy chain variable region (SEQ ID NO: 7) and light chain variable region (SEQ ID NO: 8) of the monoclonal antibody (Anti-A-ND 13) were obtained. The specific sequences are as follows:

[0106] EVQLQQSGPEMVKPGASMKISCKASGYSFTGDTMNWVKQSHGKNLEWIGLINPFNGGTRYNQKFKGKATLTVDKSSSTAYMELLSLTFEDSAVYYCARNDYDGYGMDYWGQGTSVTVSS(SEQ ID NO:7)

[0107] DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGGGTKLEIK(SEQ ID NO:8)

[0108] Example 3 Antibody chip experiment

[0109] 3.1 Chip Design: Design the chip according to the composition in Table 6. Each chip consists of 2 × 7 blocks, each block arranged in a 6 × 5 array. Each array includes six antibodies, two positive controls, and two negative controls. Each sample is spotted in triplicate. The initial vaccine concentration is 50 aliquots, and a fivefold dilution method is used. For example, a 1:2 dilution yields 25 aliquots.

[0110] Table 6 Chip structure

[0111] chip Vaccine Vx Vaccine Vn 1 1:2 (25 birds) 1:2 (25 birds) 2 1:10 (5 birds) 1:10 (5 birds) 3 1:50 (1 bird) 1:50 (1 bird) 4 1:250 (1 / 5 bird) 1:250 (1 / 5 bird) 5 1:1250 (1 / 25 pigeon) 1:1250 (1 / 25 pigeon) 6 1:6250 (1 / 125 birds) 1:6250 (1 / 125 birds) 7 0 0

[0112] The array was designed according to the composition in Table 7.

[0113] Table 7 Array structure

[0114] Column / Row 1 2 3 4 5 6 1 Bio-IgG Bio-IgG Bio-IgG Buffer Buffer Buffer 2 REV REV REV FAdV-1 FAdV-1 FAdV-1 3 ALV ALV ALV Buffer Buffer Buffer 4 Anti-A-ND13 Anti-A-ND13 Anti-A-ND13 EDSMab EDSMab EDSMab 5 Buffer Buffer Buffer Bio-IgG Bio-IgG Bio-IgG

[0115] Note: Anti-A-ND 3 is derived from hybridoma cell number 3, and Anti-A-ND 13 is derived from hybridoma cell number 13.

[0116] 3.2 Chip preparation: Prepare the sample for spotting with gene chip spotting solution (or 50% DMSO). Purified antibodies REV, ALV, EDS Mab, FAdV-1, and Anti-A-ND whole virus 13 were diluted to 500 μg / mL, 500 μg / mL, 250 μg / mL, 250 μg / mL, and 250 μg / mL, respectively, and then spotted onto the microarray. Samples were spotted onto a three-dimensional (3D) modified aldehyde-based glass slide using a microarrayer. The slides were then fixed in a humidified chamber at 37°C for at least 12 hours. After fixation, the microarrays were washed with a wash solution (e.g., 0.2% SDS) and blocked in a blocking solution (e.g., 0.15-0.3% NaBH4 solution prepared in 1× PBS and 25% ethanol) for 5 minutes. The microarrays were then rinsed with water and dried with nitrogen air or by centrifugation. After printing, the microarrays were inspected for accuracy, vacuum-sealed, and stored at -20°C until use.

[0117] 3.3 Preparation of biotin-labeled detection antibody: dilute the test antibody with 5% milk, mix well, add 1 μL of 20 mg / mL biotin labeling reagent (dissolved in DMSO), and shake at room temperature for 1 hour; and block the antibody chip at room temperature for 1 hour (PBST with 5% milk); while reacting, turn the Bio-Spin6 column upside down to mix the gel and remove bubbles; then remove the bottom of the column and place it in a 2 mL centrifuge tube, remove the column cover, use gravity to remove bubbles in the gel, and remove the effluent; put the column back into the 2 mL centrifuge tube and centrifuge at 1000g for 2 minutes (use a centrifuge with a hanging basket, otherwise the gel bed will easily produce a slope); add 500 μL of 1× PBS, centrifuge at 1000g for 1 minute, remove the effluent, repeat four times, and centrifuge for the last time for 2 minutes; add 100 μL of sample to the center of the gel bed and centrifuge at 1000g for 4 minutes; the effluent is the labeled sample (small molecules are trapped on the gel), and then add 400 μL milk, vortex, centrifuge.

[0118] 3.4 Sample testing: First, the vaccine with an initial concentration of 50 copies was diluted fivefold (e.g., 1:2 dilution is 25 copies), and then the antibody chip was fenced, 100 μL / well 5% milk (1000 rpm, 10 min) was added and placed on a shaker (12 rpm / medium speed). After blocking at room temperature for 1 hour, the blocking solution was sucked out from the lower left corner with a vacuum pump, and the vaccine diluent (serum was centrifuged at 10,000 rpm for 5 minutes before use, and the supernatant was taken) was slowly added to the antibody chip from the lower left corner, 100 μL / well, incubated at room temperature for 1.5 hours or at 4°C overnight, and then washed 3 times with 0.05% PBST (600 μL / time), each time for 5 minutes; Det (5% milk dilution) was added: 100 μL of DetAb was mixed The complex was slowly added to the array and incubated at room temperature for 1 hour. The array was then washed three times with PBST (100 μL / well), each for 5 minutes. Fluorescent dye was added: SAPE (diluted in 5% milk): 100 μL of 2 μg / mL SAPE was slowly added to the array and incubated at room temperature in the dark for 30 minutes. Washes were performed in the dark: three times with 0.05% PBST (100 μL / well), each for 5 minutes, followed by two rinses with filtered ddH2O, each for 2 minutes. The array was air-dried and scanned using a GenePix 4300A microarray scanner (Molecular Devices, LLC, San Jose, CA, USA) using a 532 nm PMT600. Median fluorescence intensity (MFI) was extracted using GenePix Pro7 software (Molecular Devices, USA).

[0119] like Figure 3 As shown in the results, Anti-A-ND-13 could detect NDV in vaccine V1 with a sensitivity of up to 1 / 5 of the feathers, while the other four antibodies could not detect NDV in V1.

[0120] like Figure 4 As shown in the results, Anti-A-ND-13 could detect NDV in vaccine V2 with a sensitivity of 1 / 125 dose, while the other four antibodies could not detect NDV in V1.

[0121] like Figure 5 As shown in the results, Anti-A-ND-13 could detect NDV in vaccine V3 with a sensitivity of 1 / 25 dose, while the other four antibodies could not detect NDV in V1.

[0122] like Figure 6 As shown in the results, Anti-A-ND-13 could detect NDV in vaccine V4 with a sensitivity of 1 / 25 feather, while the other four antibodies could not detect NDV in V1.

[0123] like Figure 7 As shown, since the virus in V5 is infectious bronchitis virus (IBV), the five antibodies cannot detect the virus in vaccine V5.

[0124] like Figure 8 As shown, since the virus in V6 is poxvirus (PPV), the five antibodies cannot detect the virus in vaccine V6.

[0125] like Figure 9 As shown in the figure, Anti-A-ND-13 can detect NDV in vaccine V7 with a sensitivity of 1 / 125 dose, while the other four antibodies cannot detect NDV in V1.

[0126] Figure 3-Figure 9 The results showed that Anti-A-ND-13 has high specificity and sensitivity to NDV. Figure 6 The clarity and maximum dilution of the V4 vaccine were the best. The test results of V4 were further analyzed. Figure 10 and Figure 11 .

[0127] Depend on Figure 10 It can be seen that compared with Anti-A-ND-3, Anti-A-ND-13 has better specificity for NDV, and the MFI difference between the two is huge.

[0128] have Figure 11 It can be seen that the detection sensitivity of Anti-A-ND-13 for NDV in vaccine V4 reaches 1 / 25 dose.

[0129] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof for detecting Newcastle disease virus, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises heavy chain variable region complementary determining regions 1-3 as shown in SEQ ID NOs: 1-3, or sequences having more than 90% homology to the sequences shown in SEQ ID NOs: 1-3, respectively, and light chain variable region complementary determining regions 1-3 as shown in SEQ ID NOs: 4-6, or sequences having more than 90% homology to the sequences shown in SEQ ID NOs: 4-6, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 7 or a sequence having more than 90% homology to the sequence shown in SEQ ID NO: 7, and a light chain variable region as shown in SEQ ID NO: 8 or a sequence having more than 90% homology to the sequence shown in SEQ ID NO:

8.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The monoclonal antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, dAb, single-chain antibody, mouse antibody, humanized antibody, chimeric antibody or multispecific antibody.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A carrier, characterized in that: The vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that: The host cell comprises the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A hybridoma cell, characterized in that: The hybridoma cell can produce the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

9. An antibody chip, characterized in that: The antibody chip comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

10. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the hybridoma cell according to claim 7, the pharmaceutical composition according to claim 8, or the antibody chip according to claim 9 in the preparation of a product for detecting Newcastle disease virus.

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