Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof
阅读说明：本技术 一种新城疫病毒hn蛋白单克隆抗体1g4及其应用 (Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof ) 是由 钱晶 王永山 欧阳伟 王晓丽 马孙婷 夏兴霞 王晶宇 诸玉梅 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种新城疫病毒HN蛋白单克隆抗体1G4及其应用,属于生物技术领域。本发明从建立的分泌抗新城疫病毒单克隆抗体杂交瘤细胞库中筛选出一株杂交瘤细胞1G4株,用其制备的细胞上清和诱生小鼠腹水的单克隆抗体的病毒中和效价分别为2<Sup>15</Sup>和10<Sup>8</Sup>,该单克隆抗体识别的抗原表位是针对病毒HN蛋白的。另外,本发明检测方法用于检测动物血清中新城疫病毒血凝抑制抗体的效价,具有敏感性强、特异性高、稳定性好,适用于高通量检测血清样本,为养殖户科学评估新城疫疫苗免疫效果提供技术手段。(The invention discloses a Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof, belonging to the technical field of biology. The invention screens out a hybridoma cell strain 1G4 from an established hybridoma cell bank secreting monoclonal antibody against Newcastle disease virus, and the virus neutralization titer of cell supernatant prepared by the hybridoma cell strain and the virus neutralization titer of the monoclonal antibody inducing ascites of mice are respectively 2 15 And 10 8 The epitope recognized by the monoclonal antibody is directed against the virus HN protein. In addition, the detection method is used for detecting the titer of the Newcastle disease virus hemagglutination inhibition antibody in the serum of the animal and has sensitivityThe kit has the advantages of strong specificity and good stability, is suitable for high-throughput detection of serum samples, and provides a technical means for farmers to scientifically evaluate the immune effect of the newcastle disease vaccine.)
1. A Newcastle disease virus HN protein monoclonal antibody 1G4, which is characterized in that: the monoclonal antibody is secreted by a monoclonal antibody hybridoma cell 1G4 strain secreting high-neutralization-activity Newcastle disease virus, and the hybridoma cell 1G4 strain is preserved in the China center for type culture Collection on 7-9/2019 with the address: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019146, classification naming: a monoclonal antibody hybridoma cell strain 1G4 secreting high neutralizing activity Newcastle disease virus.
2. The newcastle disease virus HN protein monoclonal antibody 1G4 according to claim 1, wherein: the epitope recognized by the monoclonal antibody is on the HN protein of the Newcastle disease virus.
3. The use of the monoclonal antibody 1G4 for the HN protein of Newcastle disease virus of claim 1 or 2.
4. Use according to claim 3, characterized in that: the Newcastle disease virus HN protein monoclonal antibody 1G4 is applied to preparation of a Newcastle disease virus antibody diagnostic detection reagent.
5. A kit for diagnosing and detecting newcastle disease virus antibody, which is prepared by using the newcastle disease virus HN protein monoclonal antibody 1G4 as claimed in claim 1 or 2.
6. The newcastle disease virus HN protein monoclonal antibody 1G4 according to claim 1 or 2, which is an enzyme-labeled monoclonal antibody obtained by labeling with horseradish peroxidase.
7. A Newcastle disease virus specific antibody competition ELISA detection method which is established by using the enzyme-labeled monoclonal antibody of claim 6 and is not used for the purpose of disease diagnosis.
Technical Field
The invention discloses a Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof, belonging to the technical field of biology.
Background
Newcastle Disease (ND) is an acute, virulent and highly contagious infectious disease caused by poultry infected by a virulent strain of Newcastle Disease Virus (NDV), and is one of animal epidemics of a type of animal epidemic disease which is preferably prevented in the national middle and long term animal epidemic prevention and control program (2012-2020) of our country.
In recent years, the disease presents new epidemic characteristics, such as atypical newcastle disease, NDV new gene (subtype) appearance, host range expansion, virulence enhancement and the like, so that the newcastle disease prevention and control are complicated. Currently, typical newcastle disease is well controlled in production practice, but local areas are seriously polluted by virus, and the epidemic situation is continuously endemic (Ding Zhuang, et al, new characteristics of newcastle disease epidemiology and goose newcastle disease prevention and control strategy, Chinese veterinary statement, 2015,35(1): 38-40.).
NDV is avian paramyxovirus type I of paramyxovirus of Paramyxoviridae, the genome is nonsegmented single-stranded negative-strand RNA, the genome structural pattern is 3 '-NP-P-M-F-HN-L-5', and six structural proteins are sequentially coded: nucleocapsid Protein (NP), phosphoprotein (P), Matrix protein (M), Fusion protein (F), hemagglutinin-Neuraminidase protein (HN), and macromolecular protein (L) as well as two non-structural proteins (V and W proteins) (Wangzha, et al, N.C.. recent research on Newcastle disease virus molecular biology has progressed [ J ] animal medicine, 2002,23(2): 33-36.). Among them, HN protein is the main protective antigen, and hemagglutination test and hemagglutination inhibition test involved in the conventional newcastle disease detection technology are both based on the biological activity (i.e. ability to agglutinate erythrocytes) of HN protein (in fei, et al. However, the above-mentioned tests are often affected by various factors, such as poor sensitivity, large error, small amount of sample detected each time, lack of reliability, and require a lot of labor in the detection of a large amount of samples (Dasworden et al., hemagglutination inhibition assay influencing factor analysis [ J ] zootechnics and veterinarians, 2013,45(9):108- & 109.). ELISA has the advantages of strong specificity, high sensitivity, good stability and the like, is suitable for detecting large-scale samples, and the antibody detection by using the method is very important for monitoring Newcastle disease.
The current ELISA methods for detecting antibodies against Newcastle disease virus are indirect ELISA and competitive ELISA (also called blocking ELISA), but the methods also have some limitations (the detection method involves poor specificity, sensitivity and stability of reagents). However, indirect ELISA requires an additional incubation step with secondary antibodies, and thus competitive ELISA methods are more advantageous in terms of timeliness. For example, Zhang diazepam invented a competitive ELISA kit for detecting newcastle disease antibody, including ELISA plate coated with inactivated newcastle disease virus standard hemagglutination inhibition antigen, monoclonal antibody of pigeon newcastle disease virus marked by Horse Radish Peroxidase (HRP) (Zhang diazepam, etc.. Pigeon newcastle disease virus monoclonal antibody and its application in preparation of diagnosis and detection kit [ P ]. publication No. CN108918875A, published No. 2018-11-30.), however, the competitive ELISA kit only aims at detection of chicken and pigeon newcastle disease virus antibody, and the monoclonal antibody only describes ELISA antibody titer, and does not indicate whether the monoclonal antibody has virus neutralization activity or hemagglutination inhibition activity. ELISA titer, which simply reflects the binding ability of the virus to the antibody; different from ELISA titer, the neutralizing activity or hemagglutination inhibiting activity of the antibody can directly reflect the titer of the antibody neutralizing virus or the titer of the hemagglutinin HN protein, and has obvious biological activity. The child break discloses a newcastle disease virus antibody detection blocking ELISA kit, which is characterized by comprising: the kit comprises an ELISA plate coated with newcastle disease virus inactivated antigen, newcastle disease virus positive control serum, newcastle disease virus negative control serum and a newcastle disease virus NP protein monoclonal antibody (Zhu Qin Yun, etc.. newcastle disease virus antibody detection blocking ELISA kit [ P ] publication number: CN106596933A, published: 2017-04-26.), however, an enzyme-labeled antibody in the kit is directed at newcastle disease virus NP protein, and the antibody does not have neutralizing activity and hemagglutination inhibition activity, so that the level of the detected antibody cannot represent the level of a neutralizing antibody or hemagglutination inhibition antibody of newcastle disease virus, and an ELISA detection method based on HN protein antibody is required to reflect the real biological activity of a sample to be detected.
Therefore, the invention selects specific antibodies (especially monoclonal antibodies aiming at the HN protein of the newcastle disease virus) and recombinant specific antigens (such as the extracellular domain of the recombinant HN protein) to establish a newcastle disease virus antibody competition ELISA detection method for high-throughput detection of the titer of the antibody for hemagglutination inhibition of the newcastle disease virus in animal serum, is expected to replace the traditional hemagglutination inhibition test, avoids result deviation and provides a technical means for farmers to scientifically evaluate the immune effect of the newcastle disease vaccine.
Disclosure of Invention
Technical problem
The invention aims to solve the problems of missing of a HN protein monoclonal antibody with real biological activity capable of reflecting a sample to be detected, omission and false detection phenomena and the like in the prior art, and provides a Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof.
Technical scheme
In order to achieve the purpose, the method is realized by the following technical scheme:
a Newcastle disease virus HN protein monoclonal antibody 1G4, which is characterized in that: the monoclonal antibody is secreted by a monoclonal antibody hybridoma cell 1G4 strain secreting high-neutralization-activity Newcastle disease virus, and the hybridoma cell 1G4 strain is preserved in the China center for type culture Collection on 7-9/2019 with the address: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019146, classification naming: a monoclonal antibody hybridoma cell strain 1G4 secreting high neutralizing activity Newcastle disease virus. The epitope recognized by the monoclonal antibody is on the HN protein of the Newcastle disease virus.
The Newcastle disease virus HN protein monoclonal antibody 1G4 can be applied to preparation of a Newcastle disease virus antibody diagnostic detection reagent.
The Newcastle disease virus HN protein monoclonal antibody 1G4 can be used for preparing a Newcastle disease virus antibody diagnosis and detection kit.
The Newcastle disease virus HN protein monoclonal antibody 1G4 is an enzyme-labeled monoclonal antibody obtained by horseradish peroxidase labeling. The enzyme-labeled monoclonal antibody can be used for establishing a Newcastle disease virus specific antibody competition ELISA detection method which does not aim at diagnosing diseases.
Advantageous effects
The invention screens out a hybridoma cell strain 1G4 from an established hybridoma cell bank secreting monoclonal antibody against Newcastle disease virus, and the virus neutralization titer of cell supernatant prepared by the hybridoma cell strain and the virus neutralization titer of the monoclonal antibody inducing ascites of mice are respectively 215And 108The epitope recognized by the monoclonal antibody is directed against the virus HN protein.
The invention has the following characteristics and advantages:
1. the invention screens out a hybridoma cell strain 1G4 from an established hybridoma cell bank secreting monoclonal antibody against newcastle disease virus, the identified epitope is directed at the HN protein of the newcastle disease virus, and the virus neutralization titers of the cell supernatant prepared by the hybridoma cell bank and the monoclonal antibody inducing ascites of mice are respectively 215And 108The virus has high neutralization activity and wide range of anti-NDV strains.
2. The envelope antigen used by the Newcastle disease virus specific antibody competition ELISA detection method is prokaryotic expression recombinant HN protein (extracellular domain), and non-purified inactivated virus or standard hemagglutination inhibition antigen, so that virus culture, purification and inactivation steps are avoided, and the cost of biological materials is greatly reduced. In addition, the detection method can be used for evaluating the hemagglutination inhibition antibody of poultry such as chicken, duck, goose, pigeon and the like after Newcastle disease immunization, and has wide applicability.
3. The invention establishes a competition ELISA detection method for a specific antibody of the Newcastle disease virus by an enzyme-linked immunosorbent assay (ELISA) technology based on the monoclonal antibody, and aims to solve the problems of artificial errors, complex operation and the like in the existing hemagglutination inhibition test of the Newcastle disease virus.
Biological preservation
Hybridoma cell line 1G4 was deposited with the chinese type culture collection on 7/9/2019 at address: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019146, classification naming: a monoclonal antibody hybridoma cell strain 1G4 secreting high neutralizing activity Newcastle disease virus.
Detailed Description
Establishment of monoclonal antibody hybridoma cell strain
1. Preparation of antigens
Inoculating 9-day-old SPF chick embryos (purchased from Nanjing Tianbang Biotechnology Co., Ltd.) to a Newcastle disease attenuated LaSota vaccine strain (purchased from Beijing Meiliya Viton laboratory animal technology Co., Ltd.), collecting allantoic fluid after 72 hours, centrifuging at 8000r/min for 30 minutes, taking supernatant, purifying by adopting a PEG precipitation and discontinuous sucrose density gradient method, determining the virus concentration by adopting a BCA protein quantitative detection kit (product of Seimenfiel science Co., Ltd.), and storing at-70 ℃ for later use.
2. Animal immunization
Female BALB/C mice (purchased from the university of Yangzhou, comparative medicine laboratory center) at the age of 8 weeks were immunized with purified NDV immunizing antigen by intraperitoneal injection at 50. mu.g/mouse. The antigen was emulsified with an equal volume of Freund's complete adjuvant (Sigma Co.) and then re-immunized with Freund's complete adjuvant (Sigma Co.) such as floral antigen every 14 daysThe disease is epidemic for 2 times. Collecting blood after 3 rd immunization at 6 th day, measuring serum antibody titer by indirect ELISA method, selecting antibody titer > 106The mice of (3) are re-boosted with purified NDV antigen 3 days prior to fusion.
3. Cell fusion
Adopting PEG cell fusion method, mixing well grown SP2/0 myeloma cell (purchased from ATCC cell bank) and immunized BALB/C mouse spleen cell at a ratio of 1:5, centrifuging at 1000r/min for 10min, discarding supernatant, tapping the tube bottom with palm to make the cells loose and uniform, preheating in 40 deg.C water bath, adding 50% PEG preheated to 40 deg.C with 1mL suction tube within 45 s4000(Sigma Co., Ltd.) 1mL of the medium was gently shaken while adding the medium, 15mL of fetal calf serum-free RPMI-1640 medium preheated to 37 ℃ was added over 90 seconds, the mixture was left to stand at room temperature for 10 minutes, centrifuged at 1000r/min for 10 minutes, the supernatant was discarded, resuspended in 10% Fetal Calf Serum (FCS) (Seimer Feishi technology Co., Ltd.) and HAT (Sigma Co., Ltd.) in RPMI-1640 medium, and the resulting suspension was dispensed onto a 96-well plate containing feeder cells and placed in a 5% CO-free plate2Culturing in an incubator. After 3 days, the cells were supplemented with RPMI-1640 medium containing HAT (Sigma) and 10% FCS (Seimer Feishal technologies), and after 5 days, the cells were cultured in RPMI-1640 medium containing HT (Sigma) and 10% FCS (Seimer Feishal technologies), and after 10 days, the cells were cultured in RPMI-1640 medium containing 10% FCS (Seimer Feishal technologies), and when the fused cells had grown to 1/5 of the bottom area of the well of 96-well plate, the supernatant was collected and subjected to antibody detection.
4. Screening of hybridoma cell lines
Determining the coating concentration of the purified NDV antigen according to a square matrix method, performing double-ratio dilution on the purified NDV antigen by using a carbonate buffer solution (pH9.6) with the coating solution being 0.05mol/L, coating an ELISA plate by using the amount of the NDV antigen diluted to be 500 times, coating 100 mu L/hole at 4 ℃ overnight, washing 3 times by using PBST, each time for 5 minutes, and finally drying by beating for the last time; blocking each well with PBST containing 10% calf serum, 200 μ L/well, standing at 37 deg.C for 2 hr, washing with PBST for 3 times, each time for 5 min, and drying at the last time; supernatant of cells 12 days after fusion, 1:1000 diluted immune mouse positive serum and 1:1000 diluted mouse negative serumAdding into corresponding hole, 100 μ L/hole, acting at 37 deg.C for 1 hr, washing with PBST for 3 times, each time for 5 min, and drying at last; adding 1:5000 diluted Horse Radish Peroxidase (HRP) -labeled goat anti-mouse IgG (Shanghai Biyuntian biotechnology Co., Ltd.), 100 μ L/well, standing at 37 deg.C for 1 hr, washing with PBST for 3 times (5 min each time), and drying by patting for the last time; adding TMB substrate, 100 mu L/hole, and developing for 10 minutes at room temperature in a dark place; the reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay450nmValues were zeroed for blank control, P is the value of each well, N is the OD of the negative reference serum450nmThe value is that when the OD450nm value of the negative reference serum is less than or equal to 0.1, the OD of the positive reference serum450nmValue and OD of negative reference serum450nmThe ratio of the values is more than or equal to 2.1, namely that the detection holes with the P/N more than or equal to 2.1 are judged to be positive under the premise that negative and positive controls are established, the detection is carried out once after every 2 days, and the hybridoma cells with positive detection results of the two times are cloned.
5. Cloning of hybridoma cells
Viable cells in positive wells were stained and counted with trypan blue, diluted to 100 cells/15 mL in RPMI-1640 medium containing 10% FCS (Seimer Feishell technology Co., Ltd.), the diluted cell suspension was added to a 96-well cell culture plate, 0.15mL in each well, 37 ℃ and 5% CO2, and after 4 days, formation of clonal cells was observed under a microscope, only single clonal growth wells were recorded, and after 8 days, cell supernatants were collected and subjected to ELISA detection in time. Selecting positive monoclonal cells, cloning for more than 3 times until all cell wells are positive and each well detects OD450nmThe values are closer. And (3) performing expanded culture on the cloned NDV specific monoclonal antibody hybridoma cell strain, and freezing and storing. After 20 times of cell fusion, screening, cloning and identification, a hybridoma cell bank (128 strain) which stably secretes the NDV specific monoclonal antibody is established.
6. Expression and purification of Newcastle disease virus HN protein extracellular domain
Selecting extracellular structure according to HN protein sequence information in Newcastle disease attenuated LaSota vaccine strain (purchased from Nanjing Tianbang Biotech Co., Ltd.)The domain (amino acids 46-577) is optimized by Escherichia coli codon, corresponding nucleic acid sequence (synthesized by general biological system (Anhui) Limited) is synthesized by artificial synthesis, and Nhe I and EcoR I enzyme cutting sites are respectively introduced at two ends of the sequence. Then, the artificially synthesized sequence and prokaryotic expression plasmid pET28a (+) (product of Beijing Solebao technology Co., Ltd.) are subjected to Nhe I and EcoR I double enzyme digestion respectively, after being identified by agarose gel electrophoresis, gel recovery kit (product of Tiangen Biochemical technology Co., Ltd.) is adopted to recover fragments respectively, and under the connection action of T4 ligase (product of Beijing technology Co., Ltd.), the fragments are incubated at 4 ℃ for 16 hours and transformed into E.coli Rosetta competence (product of Beijing technology Co., Ltd.) to obtain the strain capable of expressing HN protein of Newcastle disease virus. The strain was cultured to OD600nmWhen the value is about 1.0, IPTG (product of Baori doctor's technology (Beijing) Co., Ltd.) with a final concentration of 1mmol/L is added, induced expression is carried out at 37 ℃ for 6 hours, the thalli are collected and purified by a Ni-NTA affinity chromatography column (product of GE Co., Ltd.), purified recombinant protein is collected, the concentration of the purified recombinant protein is measured by a BCA protein quantitative determination kit (product of Saimer Feishell science Co., Ltd.), and the recombinant protein is stored at-70 ℃ for later use.
7. Screening of Newcastle disease virus HN protein (extracellular domain) monoclonal antibody hybridoma cell strain
Determining the coating concentration of the purified HN protein (ectodomain) according to a matrix method, diluting the purified HN protein (ectodomain) by a double ratio by using a coating solution which is 0.05mol/L of a carbonate buffer solution with pH of 9.6, diluting to 0.1 mu g/hole, coating overnight at 4 ℃, washing for 3 times by PBST (Poly-p-Phenylene Benzobisoxazole) (PBST), each time for 5 minutes, and finally drying by beating; blocking each well with PBST containing 10% calf serum, 200 μ L/well, standing at 37 deg.C for 2 hr, washing with PBST for 3 times, each time for 5 min, and drying at the last time; adding 128 cell supernatants (1:1000 dilution) stably secreting NDV specific monoclonal antibody into corresponding holes, performing action at 37 ℃ for 1 hour, washing 3 times with PBST, each time for 5 minutes, and finally drying by beating; adding 100 μ L/well of Horse Radish Peroxidase (HRP) -labeled goat anti-mouse IgG (Shanghai Biyuntian Biotech Co., Ltd.) diluted at 1:5000Standing at 37 deg.C for 1 hr, washing with PBST for 3 times, 5 min each time, and drying at the last time; adding TMB substrate, 100 μ L/hole, and developing in dark at room temperature for 10 min; the reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay450nmValue, selecting OD450nmThe NDV specific monoclonal antibody hybridoma cell strain corresponding to the highest value. The result of repeating the above experiment for 3 times shows that a monoclonal antibody hybridoma cell 1G4 strain against HN protein of newcastle disease virus is finally obtained, and the hybridoma cell 1G4 strain is deposited in the chinese type culture collection on 7/9/2019, with the address: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019146, classification naming: a monoclonal antibody hybridoma cell strain 1G4 secreting high neutralizing activity Newcastle disease virus.
8. Preparation of ascites
Injecting sterilized liquid paraffin into BALB/C mice (purchased from the university of Yangzhou, comparative medicine experiment center) with the age of 8-10 weeks, 0.5 mL/mouse, injecting hybridoma cell strain into the abdominal cavity of the mice after 7 days, wherein each hybridoma cell strain is 0.2mL (containing 5 multiplied by 10)6Individual hybridoma cells), after 7-10 days, taking ascites of a mouse with obviously swollen abdomen, centrifuging for 10 minutes at 3000r/min, collecting supernatant, subpackaging and storing at-20 ℃ for later use.
Biological Properties of monoclonal antibody 1G4
1. Chromosome analysis of hybridoma cell lines
The hybridoma cells were chromosome-counted by giemsa staining. Respectively culturing SP2/0 myeloma cell and positive hybridoma cell, growing to logarithmic phase, adding colchicine into cell bottle to make its final concentration be 0.1 μ g/ml, and placing into cell culture box for further culture for 4 hr. The cells were blown up and mixed with 5mL of 0.075mol/L KCI hypotonic solution pre-warmed at 37 ℃, placed in a 37 ℃ incubator for 30 minutes, added with L mL of newly prepared fixative (methanol: glacial acetic acid 3:1) while dropping, and centrifuged at 1000r/min for 10 minutes. The cell pellet was left by discarding the supernatant, the cells were blown up with 5mL of the fixative, acted at 37 ℃ for 30 minutes, centrifuged at 1000r/min for 10 minutes, and the above operation was repeated once. The cell sediment is suspended and mixed evenly by lmL fixing solution, 1 drop of the suspension is absorbed by a dropper, dropped on a pre-frozen glass slide, laid on the glass slide and dried naturally. Staining for 10min with newly prepared giemsa staining solution, washing with tap water, air drying, and observing under microscope. The number of chromosomes of the hybridoma cell line is 94, the number of chromosomes of myeloma cells is 54-64, and the number of chromosomes of mouse spleen cells is 40, so that the obtained hybridoma cell line 1G4 is proved to be the result of fusion of the two cells.
2. Characterization of the specificity of monoclonal antibodies
Experiments were performed in 24-well cell culture plates. Respectively inoculating a Newcastle disease virus LaSota strain, an infectious bursal disease virus B87 strain, an H9N2 subtype avian influenza virus NJ02 strain and an infectious bronchitis virus M41 strain (the strains are purchased from Nanjing Tianbang Biotechnology Co., Ltd.) to primary Chick Embryo Fibroblast (CEF) cells, after culturing for 72 hours, removing a cell culture solution by suction, washing for 2 times by using a serum-free culture solution, then adding-20 ℃ precooled absolute ethyl alcohol 1 mL/hole into a cell culture hole, fixing for 30 minutes at 4 ℃, washing for 3 times by using PBS, and patting to dry; adding culture supernatant of hybridoma cell 1G4, incubating at 200 μ L/well for 1 hr at 37 deg.C, washing with PBS for 3 times, and drying; a200-fold dilution of FITC-labeled goat anti-mouse IgG antibody (product of Wuhan Dr. Debioengineering, Ltd.) was added thereto, and the mixture was incubated at 200. mu.L/well for 1 hour at 37 ℃ and washed 5 times with PBS, and then observed under a fluorescence microscope. Under a fluorescence microscope, the monoclonal antibody 1G4 can only react with CEF cells infected by a Newcastle disease virus strain to generate fluorescence, but does not have fluorescence with CEF cells infected by other pathogens, and the specificity of the monoclonal antibody on NDV is proved.
3. Determination of monoclonal antibody type
The subclass of the monoclonal antibody secreted by hybridoma cell strain 1G4 was determined using a monoclonal antibody subclass identification kit (SeimearFeishell scientific Co., Ltd.), and the result showed that the subclass of the monoclonal antibody was IgG2a K.
4. Stability assay for monoclonal antibodies
Continuously culturing and subculturing the obtained hybridoma cell strain 1G4 for 50 times, freezing and storing by liquid nitrogen and recovering, and continuously detecting antibody titer in hybridoma cell culture supernatant to 10 by indirect ELISA method6It is proved that the hybridoma cell line 1G4 can continuously and stably secrete anti-Newcastle disease virus HN protein monoclonal antibody.
5. Potency assay for monoclonal antibodies
5.1 ELISA Titer assay
The indirect ELISA method using the Newcastle disease virus strain as the envelope antigen (see the detailed implementation mode of the invention, (one) establishment of monoclonal antibody hybridoma cell strain, 4. screening of hybridoma cell strain section contents) measures the titer of hybridoma cell culture supernatant and mouse ascites, and the result shows that the ELISA titer (i.e. reaction titer) of hybridoma cell 1G4 culture supernatant is 105Ascites reaction titer is 109。
5.2 Virus neutralization Titers assay
Chicken embryo fibroblasts (DF-1 cells, purchased from ATCC cell bank) were digested by the method of fixing virus to dilute the antibody, and then seeded into 96-well cell plates. The cell culture supernatant and mouse ascites of the monoclonal antibody serially diluted 2 times (cell supernatant)/10 times (mouse ascites) were separately mixed with an equal volume of 200TCID50Mixing the NDV suspension, acting at 37 deg.C for 1 hr, inoculating 0.1ml of the disease-antibody suspension per well into the 96-well cell plate, setting NDV and normal DF-1 cell control, standing at 37 deg.C and 5% CO2The cell culture supernatant of monoclonal antibody 1G4 was incubated in an incubator and the neutralization titer was observed to be 215The ascites neutralization potency of the mice is 108。
5.3 hemagglutination inhibition potency assay
Hemagglutination inhibition potency assay (refer to national standard of the people's republic of China GB/T16550-2008 New castle disease diagnosis technology, release date: 2008-12-31, implementation date: 2009-05-01, release organization: State administration of quality supervision and inspection and quarantine of the people's republic of China, China Committee of standardization management) was performed on cell culture supernatant of monoclonal antibody 1G4 and ascites of mice. The results showed that the hemagglutination inhibition titers of the cell culture supernatant and the ascites fluid of the mouse of the monoclonal antibody 1G4 were 2 respectively16And 227。
(III) establishment of Newcastle disease virus specific antibody competition ELISA detection method
1. Preparation of coating antigen
The envelope antigen (Newcastle disease virus HN protein) is prepared according to the specific embodiment of the invention (see the specific embodiment of the invention, (I) establishment of a monoclonal antibody hybridoma cell strain, 6. expression and purification of the extracellular domain of the Newcastle disease virus HN protein section content), the purified recombinant protein is collected, the concentration of the purified recombinant protein is measured to be 1.5mg/mL by a BCA protein quantitative detection kit (product of Saimer Feishell science and technology company), and the envelope antigen is stored at-70 ℃ for later use.
2. Preparation of enzyme-labeled antibody
The harvested monoclonal antibody 1G4 (the preparation method is shown in the detailed implementation mode of the invention, the establishment of the monoclonal antibody hybridoma cell strain, 8. the section content of ascites preparation) is acted by saturated ammonium sulfate for 2 hours, then the monoclonal antibody is centrifuged at 3000r/min for 15 minutes, and 2mL PBS solution is used for resuspension and precipitation; then, monoclonal antibody purification was performed using an antibody purification kit (product of Saimer Feishell science and technology), and the purified monoclonal antibody 1G4 was collected and stored at-70 ℃ for later use, at a concentration of 5.7mg/mL as determined by a BCA protein quantitative determination kit (product of Saimer Feishell science and technology).
The purified monoclonal antibody 1G4 is labeled by Horse Radish Peroxidase (HRP), and the specific steps are as follows: weighing 5mg of horseradish peroxidase (HRP, a product of Sigma Co.) and dissolving in 0.5mL of distilled water, adding freshly prepared 0.06mol/L NaIO40.5mL of aqueous solution, mixing, standing at 4 deg.C for 30 min, adding 0.16mol/L of ethylene glycol aqueous solution 0.5mL, standing at room temperature for 30 min, adding purified monoclonal antibody 1G41mL, mixing, placing into dialysis bag, slowly stirring in 0.05mol/L of carbonate buffer (pH 9.5), dialyzing for 6 hr, sucking out the liquid, adding NaBH40.2mL of the aqueous solution is acted at 4 ℃ for 2 hours, after the equivalent volume of saturated ammonium sulfate is added for 2 hours, the solution is centrifuged at 12000r/min for 15 minutes, the precipitate is dissolved in 2mL of PBS, the equivalent volume of glycerol is added, and the solution is preserved at minus 20 ℃ for standby.
3. Preparation of coating liquid, sealing liquid, diluent, washing liquid, developing liquid, stopping liquid and standard substance
The coating solution is 0.05mol/L carbonate buffer solution (pH 9.5), the confining solution is 10% calf serum, the diluent is 0.02mol/L phosphate buffer solution (pH 7.4), the washing solution is 0.02mol/L phosphate buffer solution (pH 7.4) containing 0.5% Tween-20, the developing solution is TMB substrate developing solution (product of Tiangen Biochemical technology (Beijing) Co., Ltd.), and the stop solution is 2mol/L sulfuric acid aqueous solution. The negative standard substance is negative serum of a Newcastle disease hemagglutination inhibition test standard, the positive standard substance is positive serum of the Newcastle disease hemagglutination inhibition test standard (purchased from Haerbinidae biotechnology, Inc.), and the standard substance is properly diluted by a diluent.
4. Establishment of detection method
The detection steps of the Newcastle disease virus specific antibody competition ELISA detection method are as follows:
(1) antigen coating: coating the purified HN protein of the recombinant Newcastle disease virus on a 96-well enzyme label plate (product of Corning, China) by 100 ng/well by using a coating solution, and acting for 1 hour at 37 ℃;
(2) washing: washing with a washing solution, adding 200 mu L of the washing solution into the ELISA plate in the step (1), washing for 3 times with shaking for 5 minutes each time, and spin-drying;
(3) and (3) sealing: adding 200 mu L of sealing liquid into the ELISA plate in the step (2), and sealing for 1 hour at 37 ℃;
(4) washing: repeating the step (2);
(5) adding an antibody: diluting enzyme-labeled monoclonal antibody at a ratio of 1:2000, diluting standard serum at different dilution times, respectively adding 50 μ L (total 100 μ L) into enzyme-labeled plate, and reacting at 37 deg.C for 1 hr (negative and positive standard substances are required);
(6) washing: repeating the step (2);
(7) color development: adding 50 mu L of TMB color development liquid into each hole, and developing for 10 minutes in a dark place at room temperature;
(8) and (4) terminating: adding 50 mu L of stop solution into each hole, and reading the value within 5 minutes;
(9) and (3) computer detection: setting enzyme-labeling Instrument OD450nmWavelength, reading a numerical value;
(10) and (4) judging a result: competitive inhibition ratio (%) (competitive antibody OD)450nmSample antibody OD450nm) Competitive antibody OD450nm100% by weight, according to different dilution timesSeveral negative and positive standards were tested for the rate of competitive inhibition by this assay. If the competitive inhibition rate is more than or equal to 20%, judging the sample to be positive; and if the competitive inhibition rate is less than 20%, judging the sample to be negative.
5. Detection and compliance comparison of clinical samples
300 parts of chicken serum, 180 parts of duck serum, 120 parts of goose serum, 50 parts of pigeon serum and 18 parts of wild bird serum are collected by the established detection method, and the coincidence rate is compared by adopting a traditional hemagglutination inhibition test (refer to national standard of the people's republic of China GB/T16550-2008 New castle disease diagnosis technology, release date: 2008-12-31, implementation date: 2009-05-01, release organization: State administration of quality supervision and quarantine of the people's republic of China, State Committee of standardization management). The detection results show in table 1, and the coincidence rate of the results of the detection method established by the invention for detecting the serum of the chicken, the duck, the goose, the pigeon and the wild bird and the hemagglutination inhibition test results is 83.3-94.3%, which shows that the detection method has higher specificity, improves the positive detection rate in clinical samples, avoids the phenomena of omission, false detection and the like, and is expected to replace the traditional detection method.
TABLE 1 comparison of detection and compliance rates of clinical samples
The invention screens out a hybridoma cell strain 1G4 from an established hybridoma cell bank secreting monoclonal antibody against Newcastle disease virus, and the virus neutralization titer of cell supernatant prepared by the hybridoma cell strain and the virus neutralization titer of the monoclonal antibody inducing ascites of mice are respectively 215And 108The epitope recognized by the monoclonal antibody is directed against the virus HN protein. In addition, the invention establishes a specific antibody competition ELISA detection method for the Newcastle disease virus based on the monoclonal antibody through an enzyme-linked immunosorbent assay technology to solve the problems of artificial errors, complex operation and the like existing in the existing Newcastle disease virus hemagglutination inhibition testThe kit has high performance and good stability, is suitable for high-throughput detection of serum samples, improves the positive detection rate of clinical samples, and avoids the phenomena of missed detection, false detection and the like.