A monoclonal antibody based on H5N8 subtype avian influenza virus NA and its application

Abstract

The present invention belongs to the biological field and relates to a monoclonal antibody based on the NA of the H5N8 subtype avian influenza virus and its application. The monoclonal antibody has good reactivity with MDCK cells infected with the NA8 subtype AIV, good broad spectrum, and good specificity, sensitivity, repeatability and accuracy. It can be used for rapid detection of antibodies specific to the NA8 subtype avian influenza.

Description

Technical Field

[0001] The present invention belongs to the biological field and relates to a monoclonal antibody based on H5N8 subtype avian influenza virus NA and an application thereof. Background Art

[0002] In 2010, the H5N8 subtype of influenza virus (AIV) was isolated from poultry in a live poultry market in Jiangsu, my country. In 2020 and 2021, complex reassortment between the H5N8 wild bird virus and native Chinese H5N6, H3N2, H4N6, and H6N6 domestic duck viruses, as well as the H9N2 wild bird virus, resulted in the H5N6 subtype of influenza virus. During the evolution and recombination of AIV, the H5N8 subtype, a highly pathogenic strain, acquired novel biological properties through genetic reassortment with other subtypes. It can cause a variety of symptoms in poultry, ranging from respiratory illness to systemic sepsis, and is also capable of infecting mammals. This poses a significant challenge to influenza virus prevention and control.

[0003] Vaccination is an important means of preventing infectious diseases. During the prevention and control of infectious diseases, vaccination has largely prevented the outbreak and spread of diseases. However, pathogen monitoring and differential diagnosis cannot be neglected in the prevention and control of infectious diseases. During AIV infection, virus isolation and antigen detection are important rapid diagnostic methods. Currently, the commonly used AIV detection methods are mainly hemagglutination inhibition tests. Identification of AIV pathogen subtypes mainly relies on NI tests and real-time reverse transcription polymerase chain reaction (rRT-PCR). These are cumbersome and time-consuming procedures, prone to cross-reactions, and unsuitable for testing large numbers of serum samples. Therefore, it is particularly important to establish an experimental method that can quickly, simply, and efficiently distinguish NA subtypes. Summary of the Invention

[0004] The present invention provides a monoclonal antibody based on H5N8 subtype avian influenza virus NA and application thereof. The monoclonal antibody can be used for rapid detection of NA8 subtype avian influenza-specific antibodies.

[0005] The technical solutions provided by the present invention are as follows:

[0006] A monoclonal antibody based on NA of H5N8 subtype avian influenza virus, comprising a heavy chain variable region and a light chain variable region;

[0007] The heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises V L CDR1, V L CDR2 and V L CDR3;

[0008] The V H The amino acid sequence of CDR1 is shown in SEQ ID NO. 3;

[0009] The V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 4;

[0010] The V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 5;

[0011] The V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 6;

[0012] The V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 7;

[0013] The V L The amino acid sequence of CDR3 is shown in SEQ ID NO. 8.

[0014] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2.

[0015] The present invention also provides a nucleotide molecule, which encodes the above-mentioned monoclonal antibody based on the NA of the H5N8 subtype avian influenza virus.

[0016] Furthermore, the sequence of the nucleotide molecule is selected from SEQ ID NO. 9 and SEQ ID NO. 10;

[0017] Sequence SEQ ID NO. 9 encodes the heavy chain variable region of the monoclonal antibody;

[0018] The sequence SEQ ID NO. 10 encodes the light chain variable region of the monoclonal antibody.

[0019] The present invention also provides an expression vector containing the above-mentioned nucleotide molecule.

[0020] The present invention also provides a host cell, which contains the above expression vector.

[0021] The present invention also provides the use of the above-mentioned monoclonal antibody based on H5N8 subtype avian influenza virus NA in the preparation of NA8 subtype avian influenza specific antibody detection reagent.

[0022] Furthermore, the detection reagent is an ELISA detection reagent.

[0023] Furthermore, the blocking solution in the detection reagent is 8-12% FBS, the blocking time is 100-140 minutes, and the TMB color development solution reaction time is 8-12 minutes.

[0024] Furthermore, the dilution concentration of the monoclonal antibody is 1:70 to 90 times.

[0025] The present invention prepares a monoclonal antibody of the NA8 subtype. After biological characteristics are identified, the antibody can be used as a blocking antibody for blocking ELISA. The blocking ELISA method is established using the antibody, and is optimized and validated for the detection of clinical serum samples. The technical route is as follows: Figure 1 shown.

[0026] The invention discloses an NA8 subtype monoclonal antibody hybridoma cell line N8 / 17.

[0027] Beneficial effects

[0028] To rapidly assess antibody levels following NA8 subtype avian influenza vaccination or the presence of antibodies to NA8 subtype avian influenza virus infection in infected chickens, a method for detecting NA8 subtype avian influenza-specific antibodies is essential. Currently, numerous methods exist for detecting avian influenza antibodies, including the hemagglutination inhibition test (HI), enzyme-linked immunosorbent assay (ELISA), agar diffusion assay (AGID), immunofluorescence assay, and colloidal gold immunochromatography. ELISA, with its high sensitivity, excellent reproducibility, high throughput, high efficiency, and ease of use, has made it a widely used method for antibody detection.

[0029] The present invention is based on hybridoma cells that can stably secrete NA8 subtype monoclonal antibodies to establish a blocking ELISA. The prepared monoclonal antibodies have good reactivity with MDCK cells infected with NA8 subtype AIV and good broad spectrum. In addition, WB verifies that the protein sample prepared by the monoclonal antibody and the prepared NA8 subtype AIV infected cells has a single band at the 55kDa position, which can provide detection for NA8 subtype AIV infected cells. The established NA8 subtype AIV blocking ELISA method has been optimized and verified. The method has good specificity, sensitivity, repeatability and accuracy. It can quickly detect NA8 subtype avian influenza-specific antibodies and distinguish NA8 subtype AIV infected samples, providing technical support for the rapid diagnosis of NA8 subtype AIV. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a technical route flow chart of the present invention.

[0031] Figure 2 This is the result of PAGE identification after dialysis and renaturation of the recombinant NA8 protein.

[0032] Figure 3 Results of ascites IFA specificity test using the N8 / 17 monoclonal antibody (100×). A: MDCK cells infected with H5N8 AIV; B: MDCK cells infected with H9N2 AIV; C: MDCK cells infected with H5N1 AIV; D: MDCK cells infected with H5N6 AIV; E: MDCK cells uninfected.

[0033] Figure 4 Results of broad-spectrum IFA testing of ascites with the N8 / 17 monoclonal antibody (100×). A: MDCK cells infected with CIXI (H5N8); B: MDCK cells infected with 22.3.4C4 (H3N8); C: MDCK cells infected with CZ (H5N8); D: MDCK cells infected with JD0707 (H5N8); E: MDCK cells infected with TC1209 (H5N8); F: MDCK cells uninfected.

[0034] Figure 5 The results of Western blot analysis of ascites fluid using the N8 / 17 monoclonal antibody. M: Protein marker; 1: Uninfected MDCK cells; 2: MDCK cells infected with CIXI(H5N8); 3: MDCK cells infected with JY(H5N8); 4: MDCK cells infected with YZ02(H5N8). DETAILED DESCRIPTION

[0035] Expression and purification of 1NA8 recombinant protein

[0036] 1.1 Construction of recombinant plasmid

[0037] The H5N8 AIV strain CIXI was amplified in 9-10-day-old SPF chicken embryos. Allantoic fluid was harvested and RNA was extracted. The cDNA was frozen and stored at -20°C using the PrimeScript™ RT reagent Kit instructions. Primers were designed based on the NA genomic sequence (Table 1), and the NA8 target gene was amplified.

[0038]

[0039] The expression vector pET-32a was double-digested using the designed NA8 primer restriction sites. The resulting vector fragment and the target gene fragment obtained above were excised and recovered using a gel recovery kit. The target gene and vector gene were then ligated using T4 DNA ligase to obtain a recombinant plasmid.

[0040] 1.2 Inducible expression of NA8 protein

[0041] The recombinant plasmid, once identified as correct, was transformed into competent E. coli BL21 (DE3). Successfully transformed competent strains were cultured. A portion of the culture medium was added to 25 mL of LB medium containing the corresponding resistance at a ratio of 1:50. The culture was shaken for approximately 2 hours. At this point, the medium became slightly turbid. The OD600nm reading was measured. A reading between 0.4 and 0.7 indicates that the strain is in the logarithmic growth phase. IPTG was then added for induction. For the initial induction attempt, the final IPTG concentration was 0.5 mM. The resulting culture medium was collected and centrifuged at 8000 rpm for 10 minutes at 16°C. The LB supernatant was discarded, and the cells were resuspended in 10 mL of PBS. The cells were then resuspended in 1 mL of PBS and transferred to a 1.5 mL EP tube. The cells were disrupted by sonication for approximately 5 minutes. Successfully disrupted cells appeared clear and transparent under light, clearly distinguishable from undisrupted cells. Centrifuge the disrupted cells at 13,000 rpm in a pre-cooled 4°C centrifuge for 10 minutes. Transfer the supernatant to a clean EP tube and resuspend the disrupted cells in 30 μL of PBS. This provides the supernatant and precipitate from the induced cells. Resuspend the precipitate in 30 μL of PBS. Add 30 μL of each sample to 10 μL of 4× Protein Loading Buffer and cook the sample in a 100°C metal bath for 10 minutes to prepare protein samples for SDS-PAGE analysis.

[0042] 1.3 Purification and refolding of recombinant NA8 protein

[0043] SDS-PAGE analysis revealed that the recombinant NA8 protein was present as inclusion bodies in the precipitate. The same method was used to induce expression in 800 mL of bacterial culture. The precipitate after bacterial lysis was dissolved in LE Buffer containing 8 M urea and collected for purification of the recombinant protein.

[0044] Column purification: Add 2 mL of Ni-NTA medium to a 10 mL purification column. According to the instructions, filter the 8M LE Buffer containing the recombinant protein through a filter membrane and add it to the purification column for purification.

[0045] Dialysis refolding: Add the eluate containing the recombinant protein to a dialysis bag. Place the dialysis bag in 2 L of PBS solution and dialyze overnight at 4°C. Prepare the dialyzed protein sample.

[0046] The purified protein was analyzed again by SDS-PAGE to check whether the protein band was single. The purified protein was detected by BCA kit to determine the corresponding protein concentration and then frozen at -70℃ for future use.

[0047] 2 Monoclonal Antibody Preparation

[0048] 2.1 Animal immunization

[0049] 5-6 week old BALB / c female mice were immunized subcutaneously in the back at a dose of 100 μg of the prepared NA8 recombinant protein per mouse. After three immunizations, serum was collected and evaluated for immune efficacy using ELISA and IFA. Mice with the highest immune titer were selected and immunized intraperitoneally with 100 μg of NA8 protein. Cell fusion was performed three days later.

[0050] 2.2 Fusion

[0051] 2.2.1 Preparation of feeder cells

[0052] ICR mice were sacrificed by cervical dislocation and sterilized in 70% alcohol for 10 minutes. The mice were mounted on a dissecting table, and the abdominal cavity was opened aseptically to expose the peritoneum. Using a 20 mL syringe with a 16-gauge needle, 15 mL of HAT medium was injected into the peritoneal cavity. Using forceps and an alcohol cotton ball, the mouse abdomen was gently massaged. The intraperitoneal medium was repeatedly aspirated until the syringe gradually turned yellow. Finally, the fluid was withdrawn and injected into a saline bottle containing HAT medium that had been cooled in ice water. The culture medium containing feeder cells was aliquoted into a 96-well cell culture plate, containing approximately 2 × 104 cells per well. After addition of the feeder cells, the plates were placed in a cell culture incubator.

[0053] 2.2.2 Preparation of splenocytes

[0054] Three days after booster immunization, BALB / c mice were eviscerated, blood was collected from their eyeballs, and positive serum was used. The mice were then sacrificed by cervical dislocation. The body surface was disinfected with 75% alcohol for 10 minutes, and the limbs were secured on a dissecting table. The abdominal cavity was opened aseptically, and the spleen was removed. The spleen was washed with DMEM, and surrounding connective tissue was carefully removed using forceps. The spleen was then transferred to another dish containing 5 mL of DMEM. The spleen was then triturated with a grinding rod to fully release the spleen cells. A spleen cell suspension was prepared and the cells were counted.

[0055] 2.2.3 Preparation of SP2 / 0 cells

[0056] Select SP2 / 0 cells in the logarithmic growth phase. When they grow to 75%, discard the supernatant, wash once with DMEM, and then gently blow the SP2 / 0 cells off the flask wall with 10 mL of DMEM medium and count the cells.

[0057] 2.2.4 Cell fusion

[0058] Prepared spleen cells and myeloma cells were mixed in a fusion tube at a ratio of 2:1 to 5:1. The tubes were centrifuged at 1000 rpm for 10 min at 4°C, the supernatant discarded, and the bottom of the tube gently rubbed with the palm of the hand to thoroughly mix the pelleted cells. 1 mL of preheated PEG was slowly added over 60 s in a 37°C water bath, with shaking. 30 mL of 37°C preheated DMEM was then added dropwise over 90 s, slowly then rapidly. The tubes were allowed to stand in a 37°C water bath for 10 min, and then centrifuged at 1000 rpm for 10 min at 4°C. The supernatant was discarded, and the cells were slowly resuspended in 30 mL of HAT medium and aliquoted into 96-well plates containing feeder cells. The 96-well plates were cultured in a 37°C 5% CO2 incubator. After 5 days of fusion, half of the medium was replaced with HAT medium, and after 10 days, the medium was replaced entirely with HT medium. When white cell clumps appeared at the bottom of the 96-well plate or the cells grew to 1 / 10 of the bottom of the well, the supernatant was aspirated for antibody detection.

[0059] 2.3 Screening of hybridoma cells

[0060] 2.3.1 Indirect ELISA method

[0061] When hybridoma cells grow more or the cell culture turns yellow, the culture medium is extracted and tested using the indirect ELISA method. The expressed protein was used as an antigen to coat the ELISA plate, which was coated overnight at 4°C. PBST solution was added to the ELISA plate and washed on a shaker at 700 rpm for 5 min. After washing, the liquid in the plate was discarded and the ELISA plate was patted dry on absorbent paper. The washing process was repeated three times. After washing, 5% skim milk was added for blocking. The plate was incubated at 37°C for 2 h at a volume of 200 μL / well. The washing operation was the same as above. 100 μL of the collected hybridoma cell supernatant was added to each well as the primary antibody. 1000-fold dilution of immune mouse serum was used as a positive control and SPF mouse negative serum was added as a negative control. The plate was incubated at 37°C for 1 h and washed as above. 100 μL of HRP enzyme-labeled secondary antibody diluted 1:8000 was added to each well, incubated at 37°C for 35 min, and washed as above. 100 μL of TMB colorimetric solution was added to each well in the dark for 10 min. Finally, 2M H2SO4 was added at 50 μL / well to terminate the color reaction. Measure OD 450nm Read the value and analyze the result. When P / N is greater than 2.1, it is judged as positive.

[0062] 2.3.2 Indirect immunofluorescence assay (IFA)

[0063] Culture MDCK cells in 96-well plates. Observe under a microscope until the MDCK cells have grown to 70%-80% of the 96-well plate. Discard the medium and wash the plate two to three times with preheated PBS. Dilute the CIXI virus allantoic fluid to 10⁻² using infection solution, add 70 μL to each well, and incubate in a constant temperature incubator for 24 hours. Wash the cell plates as above, then add 100 μL / well of 4% paraformaldehyde to each well and fix at room temperature for 15 min. Wash as above, then add 100 μL / well of permeabilization solution and permeabilize at room temperature for 10 min. Wash as above. Add 100 μL / well of 3% BSA blocking solution and block at 37°C for 2 h. Wash as above. Add 100 μL / well of hybridoma supernatant to the inoculated wells of the cell plates. Add 100 μL / well of positive serum as a positive control and incubate at 37°C for 2 h. Wash as above, pat dry on absorbent paper, and add 100 μL of a 1:2000 diluted fluorescent secondary antibody to each well in the dark for 1 h. Wash as above. Observe under a fluorescence microscope, using the negative wells as the standard, and adjust the field of view to the darkest setting. If a well shows specific bright green fluorescence, it is considered positive; otherwise, it is negative.

[0064] 2.4 Subcloning of hybridoma cells

[0065] Hybridoma wells were screened using an indirect ELISA method. After ELISA verification, positive wells were screened using IFA. For subcloning, feeder cells were plated onto a 96-well plate. Hybridoma cells of the desired subclone were blown down with preheated HT medium, and the cells were diluted and counted. 100 μL of HT medium containing hybridoma cells (approximately 100 hybridoma cells) was added to the first well of the first row of the 96-well plate. Then, dilutions were made from top to bottom using a pipette. All wells in the first column were filled with 200 μL of HT medium. Using a pipette, dilutions were made again from left to right to the last column. Finally, all wells were filled with 200 μL of HT medium and cultured. Cell status was observed daily, and hybridoma supernatant was tested when the cell culture medium turned yellow. This method was used for three consecutive subcloning cycles until the supernatant of the entire 96-well plate tested positive, and the strain was then identified.

[0066] 2.5 Preparation of monoclonal antibody ascites

[0067] Five healthy, multiparous BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin per mouse. One week later, hybridoma cells in the logarithmic growth phase were harvested and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the hybridoma cells were resuspended in prewarmed PBS and counted for later use. 5 × 105 hybridoma cells were present in the peritoneal cavity of each mouse. Approximately one week later, the mouse abdomen showed noticeable distension. Ascites was collected using a 16-gauge needle and centrifuged at 1500 rpm for 20 minutes to remove oil and other contaminants. The supernatant was aspirated and aliquoted into finger-shaped tubes. Finally, the supernatant was stored at -70°C until further use.

[0068] 2.6 Characterization of Monoclonal Antibody Ascites

[0069] 2.6.1 Identification of Monoclonal Antibody Ascites Subclasses

[0070] The subclass of the screened mAbs was identified using an indirect ELISA method with diluted ascites as the primary antibody and SPF mouse serum as the negative control. The N8 / 17 mAb strain was found to be of the IgM subclass.

[0071] 2.6.2 Results of indirect immunofluorescence testing of monoclonal antibody ascites

[0072] The method was the same as 2.3.2. The results showed that the prepared N8 / 17 mAb did not react with MDCK cells infected with non-NA8 subtype AIV, indicating that it has good specificity ( Figure 3 Broad-spectrum identification results showed that the monoclonal antibody had good reactivity with MDCK cells infected with 5 strains of NA8 subtype AIV ( Figure 4 ), indicating that it has a good broad spectrum.

[0073] 2.6.3 Monoclonal antibody ascites Western blot test results

[0074] CIXI, JY, and YZ02 were inoculated into MDCK cells at an MOI of 1, and protein samples were collected 24 hours later. The ascites fluid was used as the primary antibody, and WB results showed a target band at the expected position of 55 kDa ( Figure 5 ).

[0075] 3 Blocking ELISA Procedure

[0076] Use a matrix test to determine the optimal coating antigen concentration and blocking antibody dilution. Dilute the recombinant protein synthesized earlier as the antigen in the coating buffer, and coat the ELISA plate with 100 μL / well. Incubate overnight at 4°C. Add 200 μL of PBST to each well of the coated plate, shake at 700 rpm for 5 minutes, and pat dry on absorbent paper. Repeat three times. All subsequent steps require washing, using the same procedures as above. Add 5% skim milk (100 μL / well) and incubate at 37°C for 1 h. After washing, add diluted immune serum (100 μL / well) and incubate at 37°C for 1 h. After washing, dilute blocking monoclonal antibody and add it to the ELISA plate at 100 μL / well and incubate at 37°C for 1 h. After washing, dilute HRP enzyme-labeled antibody 8000-fold and add 100 μL / well, incubate at 37°C for 1 h. After washing, add TMB colorimetric solution and incubate at 37°C for 10 min. Finally, add 2 M sulfuric acid to terminate the color reaction, measure the OD450nm reading, and calculate the inhibition rate. Inhibition rate (I) = (1-OD450nm positive value P / negative value N) × 100%.

[0077] 4. Optimization of blocking ELISA detection conditions

[0078] 4.1 Determination of the optimal working concentrations of coating antigen and blocking mAb

[0079] The prokaryotic expressed NA8 protein was diluted with coating solution to four concentrations: 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0.625 μg / mL. 100 μL was used to coat the microplate. Array experiments were performed with immune serum diluted 1:2 and competing mAbs diluted 1:40, 1:80, 1:160, and 1:320. The antigen and mAb concentrations with the highest inhibition rates were selected. According to the test results (Table 2), the highest inhibition rate of 47.37% was achieved when the coating concentration of recombinant NA8 protein was 5 μg / mL and the N8 / 17 mAb dilution was 1:80.

[0080]

[0081] 4.2 Determination of serum dilution

[0082] The ELISA plate was coated with prokaryotic expressed NA8 protein at a concentration of 5 μg / mL, the competitive monoclonal antibody N8 / 17 was diluted at a concentration of 1:80, and the CIXI immune serum was diluted at 1:1, 1:2, and 1:4, respectively. According to the test results (Table 3), the highest inhibition rate of 53.41% was achieved when the serum to be tested was diluted at a 1:1 dilution.

[0083]

[0084] 4.3 Determination of the optimal blocking solution

[0085] Following the optimized experimental conditions, the optimal blocking solution was explored using various concentrations of skim milk (1%, 2%, 3%, and 5%), BSA (1%, 2%, 3%, and 5%), and FBS (1%, 2%, 5%, and 10%). The results (Table 4) showed that the highest inhibition rate, 53.62%, was achieved when the blocking solution contained 10% FBS.

[0086]

[0087] 4.4 Determination of the optimal sealing time

[0088] The optimal blocking time was determined according to the optimized experimental conditions. Blocking was performed at 37°C for 60, 90, and 120 minutes, and the inhibition rate was calculated. The results (Table 5) showed that the highest inhibition rate, 56.34%, was achieved when the blocking time was 120 minutes.

[0089]

[0090] 4.5 Determination of the optimal substrate action time

[0091] Following the optimized experimental conditions, the optimal substrate exposure time was determined. After adding TMB colorimetric solution, the cells were incubated at 37°C in the dark for 5, 10, and 15 minutes, and the inhibition rate was calculated. According to the experimental results (Table 6), the highest inhibition rate of 54.27% was achieved when the TMB exposure time was 10 minutes.

[0092]

[0093] 4.6 Blocking ELISA positive and negative judgment criteria

[0094] Twenty negative sera were tested under the optimal experimental conditions. The results (Table 7) showed that the mean inhibition rate for negative sera was 8.61% (X) with a standard deviation of 4.50% (SD). The sum of the mean inhibition rate and two times the standard deviation was the negative cutoff value, 17.61%, and the sum of the mean inhibition rate and three times the standard deviation was the positive cutoff value, 22.11%.

[0095]

[0096] 4.7 Blocking ELISA specificity test results

[0097] The established blocking ELISA assay was used to detect sera positive for H3N1, H3N2, H3N3, H4N6, H5N1, H5N2, H5N6, H7N9, and H9N2 subtypes, with SPF and CIXI sera used as controls. The results (Table 8) showed that the inhibition rates of sera positive for non-NA8 subtypes were all below the negative cutoff value (17.61%), demonstrating good specificity for sera positive for the NA8 subtype of AIV.

[0098]

[0099] 4.8 Blocking ELISA broad spectrum test results

[0100] The established blocking ELISA assay was used to detect NA8-positive sera, with SPF chicken serum used as a control. The results (Table 9) showed that the inhibition rate of the assay for all NA8-subtype immune sera tested was greater than the positive cutoff value (22.11%), and all were judged as positive, demonstrating that the established assay has a good broad spectrum for NA8-subtype AIV sera.

[0101]

[0102] 4.9 Blocking ELISA sensitivity test results

[0103] Positive sera from chickens with H5N8 and H3N8 subtypes were diluted two-fold to a HI titer of 20, and the inhibition rate was calculated (Table 10). Based on the cutoff value, the results showed that all sera with an HI titer of 23 or greater were considered positive. Even when the HI titer was less than 23, some sera were still considered positive, indicating that the blocking ELISA method has good sensitivity.

[0104]

[0105] 4.10 Blocking ELISA reproducibility test results

[0106] Three NA8-positive chicken sera and three non-NA8-positive sera were tested in replicates within the same batch, using the same protocol across different batches. The results (Table 11) showed that both the intra- and inter-assay coefficients of variation were less than 10%, demonstrating the good reproducibility of the established blocking ELISA method.

[0107]

[0108] 4.11 Clinical Sample Test Results

[0109] Blocking ELISA was performed on 30 samples with titers greater than 6 log2 as determined by the HI assay. The results (Table 12) showed that the inhibition rates for all 30 clinical samples were greater than 22.11%, indicating positive results. The inhibition rates for non-NA8 subtype sera were all less than 17.61%, indicating negative results. These results were 100% consistent with the HI assay results, demonstrating that the established blocking ELISA method is suitable for testing clinical samples.

[0110]

[0111] 5 The nucleotide or amino acid sequences involved in the present invention are as follows:

[0112] SEQ ID NO. 1 (amino acid sequence of the heavy chain variable region of N8 / 17-mAb)

[0113] QVQLQESGGDFVKPGGSLKLSCALSGFTLSTYGMSWVRQTPDKRLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQRTTVVPDYYTMDYWGQGTSVTVSS;

[0114] SEQ ID NO. 2 (amino acid sequence of the light chain variable region of N8 / 17-mAb)

[0115] DIVMTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSDLASGVPARFGGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPRTFGAGTKLELK;

[0116] SEQ ID NO. 3 (HCDR1 sequence of the heavy chain variable region of N8 / 17-mAb)

[0117] TYGMS;

[0118] SEQ ID NO. 4 (HCDR2 sequence of the heavy chain variable region of N8 / 17-mAb)

[0119] TISSGGTYTYYPDSVKG;

[0120] SEQ ID NO. 5 (HCDR3 sequence of the heavy chain variable region of N8 / 17-mAb)

[0121] QRTTVVPDYYTMDY;

[0122] SEQ ID NO. 6 (LCDR1 sequence of the light chain variable region of N8 / 17-mAb)

[0123] SASSSISSNYLH;

[0124] SEQ ID NO. 7 (LCDR2 sequence of the light chain variable region of N8 / 17-mAb)

[0125] RTSDLAS;

[0126] SEQ ID NO. 8 (LCDR3 sequence of the light chain variable region of N8 / 17-mAb)

[0127] QQGSSIPRT.

[0128] SEQ ID NO. 9 (Nucleotide sequence of the heavy chain variable region of N8 / 17-mAb)

[0129] CAAGTTCAGCTGCAGGAGTCTGGGGGAGACTTTGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCACTCTCTGGATTCACTCTCAGTACCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTACTTACACCTACTATCCAGA CAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACAGAGGACTACGGTAGTACCTGATTACTATACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG;

[0130] SEQ ID NO.10 (Nucleotide sequence of the light chain variable region of N8 / 17-mAb)

[0131] GATATTGTGATGACACAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCGATCTGGCTTCTGGAGTCCCAGCTCGCTTCGGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGTAGTATACCACGCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC。

Claims

1. A monoclonal antibody based on NA of H5N8 subtype avian influenza virus, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO. 3; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 4; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 5; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 6; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 7; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO.

8.

2. The monoclonal antibody based on H5N8 subtype avian influenza virus NA according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

3. A nucleotide molecule, characterized in that The nucleotide molecule encodes the monoclonal antibody based on H5N8 subtype avian influenza virus NA as claimed in claim 1 or 2.

4. The nucleotide molecule according to claim 3, wherein The sequence of the nucleotide molecule is selected from SEQ ID NO. 9 and SEQ ID NO. 10; Sequence SEQ ID NO. 9 encodes the heavy chain variable region of the monoclonal antibody; The sequence SEQ ID NO. 10 encodes the light chain variable region of the monoclonal antibody.

5. An expression vector, characterized in that The expression vector contains the nucleotide molecule according to claim 3 or 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 5.

7. Use of the monoclonal antibody based on NA of H5N8 subtype avian influenza virus as claimed in claim 1 or 2 in the preparation of a detection reagent for NA8 subtype avian influenza virus.

8. The use according to claim 7, characterized in that The detection reagent is an ELISA detection reagent.

9. The use according to claim 7, characterized in that The blocking solution in the detection reagent is 8-12% FBS, the blocking time is 100-140 minutes, and the TMB color development solution reaction time is 8-12 minutes.

10. The use according to claim 7, characterized in that The dilution concentration of the monoclonal antibody is 1:70 to 90 times.

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