CN111117970A - Monoclonal antibody for recognizing N6 subtype avian influenza virus neuraminidase protein and application thereof - Google Patents
Monoclonal antibody for recognizing N6 subtype avian influenza virus neuraminidase protein and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Abstract
The invention discloses a monoclonal antibody for identifying N6 subtype avian influenza virus neuraminidase protein and application thereof. The monoclonal antibody is secreted and generated by a hybridoma cell strain 3H7 with the preservation number of CGMCC No. 18900. Experiments prove that the monoclonal antibody 3H7 obtained by the invention can be specifically combined with the N6 subtype avian influenza virus, and can be combined with 15 strains of N6 subtype avian influenza viruses with different evolutionary branches, which shows that the 3H7 has good broad spectrum. The invention establishes an ELISA detection kit for N6 subtype avian influenza virus neuraminidase protein antibody blocking and a detection method thereof by utilizing the better broad spectrum and specificity of the monoclonal antibody. Experiments prove that the method has high sensitivity and good repeatability, can be used for detecting the level of N6 subtype avian influenza virus neuraminidase protein antibody in clinical serum samples, and has good development prospect.
Description
Technical Field
The invention relates to a hybridoma cell strain capable of secreting N6 subtype avian influenza virus neuraminidase protein monoclonal antibody, and also relates to the monoclonal antibody secreted by the hybridoma cell strain and application thereof. The invention belongs to the field of biotechnology.
Background
Avian Influenza (AI) is an acute infectious disease of birds caused by influenza a viruses of the orthomyxoviridae family, and is classified as a type a virulent infectious disease by the world animal health organization. Avian Influenza Virus (AIV) is a single negative strand segmented RNA virus, numerous in subtype, classified into 16 different HA subtypes and 9 different NA subtypes according to antigenic differences of its surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA), and further isolated in bat as influenza viruses of H17N10 and H18N11 subtypes.
NA is a type II glycoprotein encoded by fragment 6, and the NA proteins structurally form a heterotetramer, each NA protein monomer consisting of an amino-short cytoplasmic domain, a hydrophobic transmembrane domain, a stem domain and a globular head domain. The NA protein has neuraminidase activity, and can cut sialic acid on the surface of a host cell and the surface of a new virus particle in the later period of virus infection, so that the release and the diffusion of the new virus particle are promoted, and the accumulation of the new virus particle on the cell surface is prevented.
N6 is one of the main NA subtypes of the current domestic epidemic avian influenza virus, and the H5N6 and H6N6 subtypes are most commonly combined. The H5N6 subtype avian influenza virus not only seriously harms the poultry industry, but also can cross the interspecific barrier to infect human, and by 8 months and 8 days in 2019, 22 cases of people have infected the H5N6 subtype avian influenza virus. In addition, there are also reports of H6N6 subtype avian influenza virus infecting pigs across the interspecific barrier. Therefore, the enhancement of the monitoring of the avian influenza virus subtype N6 has important public health significance. At present, the NA subtype detection method recommended by WHO is a neuraminidase inhibition experiment, but the method has long experimental period, and the experimental reagent sodium arsenite is a highly toxic reagent, so the method is difficult to popularize and use clinically.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain capable of stably secreting a broad-spectrum N6 subtype avian influenza virus resistant neuraminidase protein monoclonal antibody.
The invention also aims to provide a monoclonal antibody secreted by the hybridoma cell strain and having broad-spectrum resistance to N6 subtype avian influenza virus neuraminidase protein.
The invention also aims to apply the monoclonal antibody to the diagnosis and treatment of the N6 subtype avian influenza.
The invention achieves the above purpose by the following technical scheme:
the invention firstly selects 3 strains of N6 subtype avian influenza viruses positioned in different evolutionary branches, immunizes BALB/c female mice of 6 weeks old with inactivated viruses at intervals of 14 days in sequence, and fuses spleens of the immunized mice with SP2/0 cells. The indirect immunofluorescence method is utilized to screen 1 monoclonal antibody hybridoma cell strain which can stably secrete N6 subtype avian influenza virus neuraminidase resistant protein with broad spectrum, the name is 3H7, the classification name is hybridoma cells, the cell strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address is the institute of microbiology, China academy of sciences, No. 3, of West Lu No.1 Hospital, on the sunward area of Beijing, the preservation number is CGMCC No.18900, and the preservation time is 12 months and 4 days in 2019.
Furthermore, the invention also provides a monoclonal antibody 3H7 secreted by the hybridoma cell strain and having broad spectrum resistance to N6 subtype avian influenza virus neuraminidase protein. The antibody was identified as having a heavy chain subclass of IgG2b and a light chain subclass of kappa chain. The monoclonal antibody 3H7 and the N6 subtype avian influenza virus are taken to carry out Western Blot detection, and the result that 3H7 can be specifically combined with the neuraminidase protein of the denatured N6 subtype avian influenza virus shows that 3H7 recognizes the linear epitope of the N6 protein. The ELISA titer of the hybridoma cell 3H7 supernatant was 1:6400, and the ELISA titer of the mouse ascites was 1: 409600. 15 strains of different clades of N6 subtype AIV are selected to infect DF-1 cells and are subjected to IFA detection, and the result that 3H7 can be combined with the 15 strains of viruses shows that 3H7 has good broad spectrum. The results of indirect ELISA tests using AIV subtype N1-N9 and Newcastle disease virus as coating antigens show that 3H7 can only bind to AIV subtype N6, and that 3H7 has good specificity.
Furthermore, the invention also provides the application of the monoclonal antibody in preparing a reagent for detecting the avian influenza virus subtype N6. And the application of the monoclonal antibody in preparing medicaments for treating the N6 subtype avian influenza.
A pharmaceutical combination comprising a monoclonal antibody according to the invention.
Furthermore, the invention establishes a blocking ELISA detection kit for detecting N6 subtype avian influenza virus neuraminidase protein antibody and a detection method thereof by utilizing better broad spectrum and specificity of 3H 7.
The blocking ELISA detection kit for detecting N6 subtype avian influenza virus neuraminidase protein antibody contains the monoclonal antibody.
Preferably, the kit further comprises an inactivated antigen A/chicken/Hunan/S3003/2009(H6N6), an enzyme label plate, a coating solution, a PBST solution, an HRP-labeled goat-anti-mouse secondary antibody, a TMB color development solution and a stop solution.
When the kit is used for detecting the N6 subtype avian influenza virus antibody, the method comprises the following steps:
(1) the HA titer is 28The inactivated antigen A/chicken/Hunan/S3003/2009(H6N6) is diluted by coating solution by 1:80 times and coated on an enzyme label plate, each hole is 100 mu L, and the temperature is kept overnight at 4 ℃;
(2) discarding the supernatant, washing the ELISA plate with PBST solution, washing with 200 μ L of each well for 3 times, adding 5% skimmed milk solution, sealing, incubating at 37 deg.C for 1h, and collecting the eluate;
(3) discarding the supernatant, washing the ELISA plate with PBST solution (200 μ L per well), washing for 3 times, adding the serum to be detected, the negative serum and the positive serum, incubating at 37 deg.C for 1h, and collecting the eluate after washing;
(4) discarding the supernatant, washing the ELISA plate with PBST solution (200 μ L per well), washing for 3 times, adding 1:12800 times diluted monoclonal antibody ascites 3H7 (100 μ L per well), and incubating at 37 deg.C for 1H;
(5) discarding the supernatant, washing the ELISA plate with PBST solution, washing 200 μ L per well for 3 times, adding HRP-labeled goat-anti-mouse secondary antibody diluted 1:3000 times, incubating at 37 deg.C for 1h, and collecting the eluate;
(6) discarding supernatant, washing the ELISA plate with PBST solution 200 μ L per well, washing for 3 times, adding TMB color development solution in dark place 100 μ L per well, developing for 15min, adding 50 μ L2M sulfuric acid to terminate color development, and reading OD on enzyme labeling instrument450And (5) nm value.
Experiments prove that the method has high sensitivity and good repeatability, can be used for detecting the level of N6 subtype avian influenza virus neuraminidase protein antibody in clinical serum samples, and has good development prospect.
Drawings
FIG. 1 is an indirect immunofluorescence assay of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H 7;
a: 3H 7; b: negative control
FIG. 2 is a Western Blot detection of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H 7;
1:3H 7: 2: negative control
FIG. 3 is a broad-spectrum assay of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody 3H 7;
A:DK/HuB/SP143/2014(H5N6)；B:DK/HuN/SE226/2017(H5N6)；
C:CK/HuN/S3003/2009(H6N6)；D:DK/HuN/S4270/2010(H6N6)；
E:DK/GX/S4111/2010(H6N6)；F:DK/HuN/S1351/2009(H6N6)；
G:DK/ZJ/S4354/2016(H11N6)；H:DK/GD/S4180/2017(H6N6)；
I:CK/GD/S1031/2018(H6N6)；J:DK/JX/S40199/2017(H6N6)；
K:DK/JX/S1129/2018(H6N6)；L:DK/JX/S1200/2018(H6N6)；
M:DK/GX/S40437/2017(H6N6)；N:DK/GX/S31293/2017(H6N6)；
CK/HuB/S1246/2018(H4N 6); p is negative control.
Detailed Description
In order to clearly explain the present invention, the present invention will be described in detail with reference to specific examples, but the present invention is not limited to the following examples.
Example 1 preparation and identification of N6 subtype avian influenza Virus neuraminidase protein monoclonal antibody
1.1 preparation of immunogens and immunization of animals
3 strains of avian influenza virus subtype N6 (CK/HuN/S3003/2009(H6N6), HuN3003 for short, DK/HuB/SP143/2014(H5N6), HuB143 for short, DK/HuN/SE226/2017(H5N6) for short, HuN226 for short) in different evolutionary branches are selected as immunogens, wherein amino acid homology between HuN226 and HuN3003 is 95%, amino acid homology between HuN3003 and HuB143 is 94.1%, and amino acid homology between HuN and HuB143 is 91.7%, viruses HuN, HuN3003 and HuB143 are firstly diluted by a 10-fold ratio, 9-11-day-old SPF chick embryos are inoculated, cultured in a 37 ℃ incubator for 48H, allantoic fluid is collected, β -propiolactone is added according to a ratio of 1:2000, the chick embryos are inoculated for detection of whether virus inactivation is sufficient, and are placed at-70 ℃ after virus inactivation is tested and aseptic storage.
8 female BALB/c mice, 6 weeks old, were selected and immunized sequentially with inactivated HuN226, HuN3003 and HuB143, 3 days prior to fusion, using HuN3003 for boosting, the specific immunization protocol was as follows (Table 1):
TABLE 1 immunization procedure for the preparation of N6 subtype avian influenza virus neuraminidase protein monoclonal antibody
1.2 preparation of hybridoma cell lines
The mouse spleen was collected aseptically, ground with the plunger of a 2.5mL syringe to disperse the splenocytes evenly, and then transferred to a sterile 50mL centrifuge tube. SP2/0 cells were blown down in serum-free DMEM and placed in centrifuge tubes containing splenocytes, gently mixed, centrifuged at 800rpm for 10min, and the supernatant was discarded. The centrifuge tube was then placed in a 37 ℃ water bath, 1mL of preheated PEG was slowly added dropwise over 45s while shaking the centrifuge tube to mix it evenly. After standing for 1min, slowly dropwise adding serum-free DMEM to 40mL, centrifuging at 800rpm for 10min, and discarding the supernatant. The fused cells were resuspended in 96-well plates at 100. mu.L per well using DMEM containing 1% HAT and 20% FBS preheated at 37 ℃ and then placed in a medium containing 5% CO2At 37 ℃ in a cell culture chamber.
1.3 screening of Positive hybridoma cells
And when the hybridoma cells in the cell holes grow to about half of the hybridoma cells, sucking cell supernatants for IFA detection, and determining positive holes. 293T cells were transfected with the eukaryotic expression plasmid pCAGGS-N6(HuN3003) the day before selection, cultured for 24h, the cell supernatant was discarded, washed 3 times with PBST solution, and then formalin-fixed at 100. mu.L per well for 30min at room temperature was added. After fixation, the cells were washed 3 times with PBST solution, then blocked with 5% skim milk solution at 37 ℃ for 2h or overnight at 4 ℃ in 200. mu.L/well, and washed 3 times as above. The fused hybridoma cell supernatant was pipetted 100. mu.L into 293T cell plates, and mouse positive and negative serum diluted 1:400 was used as positive and negative controls, incubated at 37 ℃ for 1h, and washed 3 times as above. Then 100. mu.L of a 1: 1000-fold diluted FITC-labeled goat anti-mouse fluorescent secondary antibody was added to each well, incubated at 37 ℃ for 1h, and washed 3 times as above, and the process was performed in a dark environment. Then, 1 hybridoma cell recognizing the N6 protein was finally selected and named 3H7, as observed by a fluorescence microscope. The cell strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, the preservation number is CGMCC No.18900, and the preservation time is 12 months and 4 days in 2019.
1.4 subcloning of Positive hybridoma cells 3H7
Firstly, the methodGently and uniformly blowing and beating cells in the 3H7 well of the positive hybridoma cells, counting the cells, diluting the cells to 10mL by using DMEM containing 20% FBS to contain about 100 cells, uniformly mixing the cells, adding the mixture into a 96-well plate, adding 100 mu L of the mixture into each well, then supplementing 100 mu L of DMEM culture solution containing 20% FBS into each well, placing the 96-well plate into a culture medium containing 5% CO2At 37 ℃ in a cell culture chamber. Observing the cell condition every day until cell colonies appear, marking single cell holes, and screening positive hybridoma when the cells grow to about half. Subcloning was repeated 3 times in this manner.
1.5 preparation of monoclonal antibody 3H7
Mice were injected intraperitoneally with Freund's incomplete adjuvant, 0.5mL each. After injection for 3d, hybridoma cells in a good cell state, 3H7, were blown down, centrifuged at 800rpm for 10min, the supernatant was discarded, the cells were resuspended in sterile PBS, centrifuged again, and the supernatant was discarded. Add sterile PBS to resuspend the cells, count the cells, dilute the cells to 5X 10 per 500. mu.L5And (4) cells. The cell dilutions were mixed well and then injected into the abdominal cavity of mice, 500 μ L each. Observing the state of the mouse every day within 7-10 days after cell injection, sucking ascites of the mouse by using a syringe when the abdomen of the mouse swells, centrifuging at 4500rpm for 10min after overnight standing at 4 ℃, collecting the supernatant of the abdomen, and placing the supernatant in a refrigerator at-70 ℃ for later use.
1.6 subclass identification of monoclonal antibody 3H7
The 3H7 hybridoma cells were cultured in serum-free medium, and the cell supernatants were collected and the subclasses of 3H7 were identified according to the antibody subclass identification kit instructions. The heavy chain subclass was determined to be IgG2b and the light chain subclass was determined to be kappa chain.
1.7 potency assay for monoclonal antibody 3H7
The N6 subtype AIV was formulated as 8 units antigen and coated on an ELISA plate overnight at 4 ℃. After washing 3 times with PBST, 200. mu.L of 5% skim milk solution was added for blocking, and incubated at 37 ℃ for 1 h. The supernatant or ascites of the 3H7 hybridoma cells was diluted in a gradient as a primary antibody and incubated at 37 ℃ for 1H. After washing, a 1:3000 fold dilution of HRP-labeled goat anti-mouse secondary antibody was added, 100. mu.L per well, and incubated at 37 ℃ for 1 h. Washing, adding TMB color developing solution for dark color development, with each well being 100 μ L and developing color of 15min after 50. mu.L of 2M H was added to each well2SO4The color development was stopped and the OD read on the microplate reader450And (5) nm value. Finally, the ELISA titer of the hybridoma cell supernatant is determined to be 1:6400, and the ELISA titer of the ascites is determined to be 1: 409600.
1.8 IFA detection of monoclonal antibody 3H7
IFA detection was performed on 3H7 cell supernatant using the method of 1.3, wherein the plasmid used for transfection was a constructed eukaryotic expression recombinant plasmid pCAGGS-NA (HuN 226). The primary antibody is cell supernatant, the secondary antibody is FITC-labeled goat-anti-mouse fluorescent secondary antibody diluted by 1:1000 times, and the result is recorded under a fluorescent microscope. As a result, 3H7 was found to be able to react with N6 protein HuN226 to emit green fluorescence (fig. 1).
1.9 Western Blot detection of monoclonal antibody 3H7
Mixing whole virus HuN3003 with 5 xSDS-PAGE loading Buffer, heating for denaturation, performing electrophoresis, and transferring membrane. After the membrane transfer, the membrane was blocked with a 5% skim milk solution and incubated at 37 ℃ for 2 h. Wash with PBST solution 5min each time. The supernatant of 3H7 cells was used as a primary antibody, and placed in a shaker at 37 ℃ for 1H, after washing the membrane by the above method, a 1: 1000-fold diluted infrared-labeled anti-mouse secondary antibody was added, and incubated at 37 ℃ for 1H in the dark. And washing the membrane by the same method, and then placing the membrane on an infrared membrane scanner for scanning the result. As a result, 3H7 was found to be able to specifically react with N6 protein HuN3003 (fig. 2).
1.10 specific detection of monoclonal antibody 3H7
N1-N9 subtype AIV and Newcastle disease virus are selected as antigens, and the viruses are prepared into 8 units of antigen coated enzyme label plates and are kept overnight at 4 ℃. After washing 3 times with PBST, 200. mu.L of 5% skim milk solution was added for blocking, and incubated at 37 ℃ for 1 h. 3H7 cell supernatant was used as a primary antibody and incubated at 37 ℃ for 1H. After washing, a 1:3000 fold dilution of HRP-labeled goat anti-mouse secondary antibody was added, 100. mu.L per well, and incubated at 37 ℃ for 1 h. Washing, adding TMB color developing solution for dark development, each well is 100 μ L, and after developing for 15min, each well is added with 50 μ L of 2M H2SO4The color development was stopped and the OD read on the microplate reader450And (5) nm value. The results show that 3H7 can only bind to AIV subtype N6 (table 2), indicating that 3H7 has good specificity.
TABLE 2 specific detection of neuraminidase protein monoclonal antibody 3H7 of subtype N6 avian influenza virus
1.11 broad Spectrum detection of monoclonal antibody 3H7
Firstly, DF-1 cells are cultured in a 96-well plate, when the cells grow to about 80 percent, 15 strains of N6 subtype avian influenza virus in different evolutionary branches infect the DF-1 cells, the cells are cultured in a cell culture box at 37 ℃ for 48 hours, then the cells are taken out, the supernatant is discarded, and the cells are washed for 3 times by PBST. mu.L of formalin fixative was added to each well, fixed at room temperature for 30min and the supernatant discarded, and washed 3 times with PBST. The primary antibody was hybridoma 3H7 culture supernatant, 100. mu.L/well, incubated at 37 ℃ for 1H, and then washed 3 times with PBST. The secondary antibody was a FITC-labeled goat-anti-mouse fluorescent secondary antibody diluted 1:1000 times at 100. mu.L per well, and the procedure was performed in the dark, and after incubation at 37 ℃ for 1h, washed 3 times with PBST, and the results were recorded under a fluorescence microscope. Cells infected with these 15 strains of virus were observed to fluoresce green under a fluorescence microscope (FIG. 3), indicating that 3H7 has a good broad spectrum.
Example 2 establishment of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method
2.1N 6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA operation method
(1) The HA titer is 28The inactivated antigen A/chicken/Hunan/S3003/2009(H6N6) was diluted 1: 80-fold with a coating solution (50mmol/L carbonate buffer (pH 9.6)) and then coated on an ELISA plate at 4 ℃ overnight at 100. mu.L per well.
(2) The supernatant was discarded, and the plate was washed with PBST solution (50mmol/L phosphate buffer pH 7.2 containing 0.05% Tween-20) at 200. mu.l/well, washed 3 times, and then blocked by adding PBST solution containing 5% w/w skim milk at 200. mu.l/well, and incubated at 37 ℃ for 1 h.
(3) Discarding the supernatant, washing the ELISA plate with PBST solution (200 μ L per well), washing for 3 times, adding the serum to be detected, the negative serum and the positive serum, incubating at 37 ℃ for 1h and 100 μ L per well.
(4) Discarding the supernatant, washing the ELISA plate with PBST solution at 200. mu.l per well for 3 times, adding 1:12800 times diluted monoclonal antibody ascites 3H7 at 100. mu.L per well, and incubating at 37 ℃ for 1H.
(5) The supernatant was discarded, and the microplate was washed with PBST solution (200. mu.l/well) for 3 times, and then a 1: 3000-fold diluted HRP-labeled goat-anti-mouse secondary antibody (100. mu.L/well) was added and incubated at 37 ℃ for 1 hour.
(6) Discarding supernatant, washing the ELISA plate with PBST solution 200 μ L per well, washing for 3 times, adding TMB color development solution in dark place 100 μ L per well, developing for 15min, adding 50 μ L2M sulfuric acid to terminate color development, and reading OD on enzyme labeling instrument450And (5) nm value.
2.2 determination of the threshold value
60 portions of the negative serum were diluted 1:160 times, and the inhibition rates of the 60 portions of the serum were measured by the established blocking ELISA method using SPF chicken serum as a negative control (Table 3), and the average inhibition rate was found to be 9.04% and the standard deviation was found to be 4.36%. The positive cutoff value was calculated to be 22.12% and the negative cutoff value was calculated to be 17.76% according to the cutoff determination criteria. And if the intermediate is judged to be suspected, and the result is judged to be negative if the intermediate is still less than the positive critical value after re-measurement.
TABLE 3 determination of the cut-off value of the N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method
2.3 specific detection of blocking ELISA method
N1-N9 subtype AIV serum and Newcastle disease virus serum are selected to carry out blocking ELISA method detection, SPF chicken serum is used as a negative control, and the inhibition rate results show that the inhibition rates of the other sera except the N6 subtype serum are all smaller than a negative critical value (Table 4), which indicates that the method has better specificity.
TABLE 4 specificity detection of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method
2.4 repetitive detection of blocking ELISA method
The average inhibition rate X and standard deviation SD were determined by repeating 5 serum samples 4 times on one microplate and 4 on 4 microplate, respectively, and the coefficient of variation CV (%) ═ SD × 100%/X was determined. Calculating the variation coefficient in the plate to be between 1.14 and 6.57 percent; the coefficient of variation between plates was between 2.22% and 4.98% (table 5), and both were less than 10%, indicating that the method has good reproducibility.
TABLE 5 repeated detection of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method
2.5 detection of sensitivity by blocking ELISA method
30 positive sera with clear background are selected for detection by a blocking ELISA method, and the inhibition rate is calculated to be between 27.83% and 75.31% (table 6), and the coincidence rate is 100%, which indicates that the method has good sensitivity.
TABLE 6 sensitivity detection of N6 subtype avian influenza virus neuraminidase protein antibody blocking ELISA detection method
The results show that the blocking ELISA method established by the invention has good specificity, repeatability and sensitivity, and can be used for detecting the N6 subtype avian influenza virus neuraminidase protein antibody level in clinical serum samples.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (8)
1. A monoclonal antibody hybridoma cell strain capable of stably secreting N6 subtype avian influenza virus resistant neuraminidase protein is named as 3H7, the cell strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the microbial research institute of China academy of sciences No. 3 of West Lu No.1 of Beijing Shang, the south China area, the preservation number is CGMCC No.18900, and the preservation time is 12 months and 4 days in 2019.
2. A monoclonal antibody secreted by the hybridoma cell line of claim 1 and having broad-spectrum resistance to N6 subtype avian influenza virus neuraminidase protein.
3. Use of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting avian influenza virus subtype N6.
4. A blocking ELISA detection kit for detecting N6 subtype avian influenza virus neuraminidase protein antibody, characterized in that the kit contains the monoclonal antibody of claim 2.
5. The ELISA detection kit of claim 4 further comprising inactivated antigen A/chicken/Hunan/S3003/2009(H6N6), an ELISA plate, a coating solution, a PBST solution, a goat anti-mouse secondary antibody labeled with HRP, a TMB color developing solution and a stop solution.
6. The ELISA detection kit of claim 5, wherein when the kit is used for detecting N6 subtype avian influenza virus neuraminidase protein antibody, the following steps are carried out:
(1) the HA titer is 28The inactivated antigen A/chicken/Hunan/S3003/2009(H6N6) is diluted by coating solution by 1:80 times and coated on an enzyme label plate, each hole is 100 mu L, and the temperature is kept overnight at 4 ℃;
(2) discarding the supernatant, washing the ELISA plate with PBST solution, washing with 200 μ L of each well for 3 times, adding 5% skimmed milk solution, sealing, incubating at 37 deg.C for 1h, and collecting the eluate;
(3) discarding the supernatant, washing the ELISA plate with PBST solution (200 μ L per well), washing for 3 times, adding the serum to be detected, the negative serum and the positive serum, incubating at 37 deg.C for 1h, and collecting the eluate after washing;
(4) discarding the supernatant, washing the ELISA plate with PBST solution (200 μ L per well), washing for 3 times, adding 1:12800 times diluted monoclonal antibody ascites 3H7 (100 μ L per well), and incubating at 37 deg.C for 1H;
(5) discarding the supernatant, washing the ELISA plate with PBST solution, washing 200 μ L per well for 3 times, adding HRP-labeled goat-anti-mouse secondary antibody diluted 1:3000 times, incubating at 37 deg.C for 1h, and collecting the eluate;
(6) discarding supernatant, washing the ELISA plate with PBST solution 200 μ L per well, washing for 3 times, adding TMB color development solution in dark place 100 μ L per well, developing for 15min, adding 50 μ L2M sulfuric acid to terminate color development, and reading OD on enzyme labeling instrument450And (5) nm value.
7. The use of the monoclonal antibody of claim 2 in the preparation of a medicament for the treatment of avian influenza subtype N6.
8. A pharmaceutical combination comprising the monoclonal antibody of claim 2.
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