Anti-influenza A virus NP protein antibody pair and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to an anti-influenza A virus NP protein antibody pair and application thereof. The antibody pair comprises two monoclonal antibodies; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region; one monoclonal antibody in the monoclonal antibody pair is named as a 10G10 antibody, and the other monoclonal antibody is named as a 11F8 antibody. The invention also discloses a nucleic acid molecule for coding the monoclonal antibody and a hybridoma cell strain for secreting the monoclonal antibody. In addition, the invention also discloses a kit for detecting influenza virus protein, which comprises the monoclonal antibody pair, the 10G10 antibody is used as a capture antibody, and the 11F8 antibody is used as a labeled antibody. The antibody for resisting the influenza A virus NP protein provided by the invention has relatively good specificity on the influenza virus protein.

Description

Technical Field

[0001] The present application relates to the technical field of biomedicine, and in particular to an antibody pair against influenza A virus NP protein and applications thereof. Background Art

[0002] Influenza refers to a contagious disease caused by influenza viruses (Influenza viruses, Flu, referred to as influenza viruses). Influenza viruses are mainly transmitted through droplets in the air, contact between susceptible and infected persons, or contact with contaminated objects. They are characterized by rapid mutation, strong infectivity, rapid transmission, short incubation period, and high incidence. According to the characteristics of the nucleoprotein (NP) and matrix protein (M) of influenza viruses, influenza viruses are divided into four types: A, B, C, and D. Among them, the types that are pathogenic to humans are mainly concentrated in influenza A viruses, which have the strongest viral toxicity and are easy to mutate; followed by influenza B viruses. Therefore, the detection and research of influenza A and influenza B viruses has great clinical significance.

[0003] At present, there are three main methods for influenza virus detection. Method 1: Isolation and culture method, which is considered the gold standard for influenza virus detection; however, due to the complex operation and long detection time (the detection cycle takes about 14 days), it is difficult to carry out in clinical practice. Method 2: Nucleic acid detection method, although this method has high sensitivity, it is costly, the experimental time takes 4-6 hours, and the experimental operation is highly professional, so its application in the field is limited. Method 3: Rapid antigen detection method, colloidal gold immunochromatography is commonly used to assist in the diagnosis of influenza A virus and influenza B virus infection. This is the most commonly used detection method on the market. The entire detection process does not require any instruments, and the operation is simple and fast (only 10-15 minutes). It is particularly suitable for self-examination screening of suspected patients, hospital laboratories, special departments (blood transfusion, preoperative, emergency department, etc.), disease control systems (epidemic prevention stations), entry-exit quarantine and other places.

[0004] The influenza virus NP protein is very conservative and has a high content in influenza viruses. It is the target protein in immunochromatographic detection. Although the immunochromatographic reagents in the related art can detect early infection of influenza viruses, they are not ideal. Summary of the invention

[0005] The present application provides an antibody pair against influenza A virus NP protein and its application. The antibody pair against influenza A virus NP protein provided in the present application is a monoclonal antibody targeting influenza A virus NP protein with higher sensitivity, which can effectively improve the quality of influenza virus chromatography detection reagents.

[0006] Continuously improving the sensitivity and specificity of influenza virus detection through technological iteration is an important research and development direction in the diagnostic reagent industry. Antibodies against influenza virus NP protein are the main biologically active raw materials in chromatographic detection reagents and have an important impact on the sensitivity and specificity of the reagents.

[0007] In a first aspect, the present application provides an antibody pair against influenza A virus NP protein, using the following technical solution:

[0008] An antibody pair against influenza A virus NP protein, the antibody pair comprising two monoclonal antibodies; the monoclonal antibodies comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3;

[0009] One monoclonal antibody in the antibody pair is named 10G10 antibody, and the sequence information of the 10G10 antibody is as follows:

[0010] The heavy chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 1, or an amino acid sequence comprising the above sequence;

[0011] The heavy chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, or an amino acid sequence comprising the above sequence;

[0012] The heavy chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 3, or an amino acid sequence comprising the above sequence;

[0013] The light chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 4, or an amino acid sequence comprising the above sequence;

[0014] The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 5, or an amino acid sequence comprising the above sequence;

[0015] The light chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 6, or an amino acid sequence comprising the above sequence;

[0016] The other monoclonal antibody in the antibody pair is named 11F8 antibody, and the sequence information of the 11F8 antibody is as follows:

[0017] The heavy chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 7, or an amino acid sequence comprising the above sequence;

[0018] The heavy chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 8, or an amino acid sequence comprising the above sequence;

[0019] The heavy chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 9, or an amino acid sequence comprising the above sequence;

[0020] The light chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 10, or an amino acid sequence comprising the above sequence;

[0021] The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence comprising the above sequence;

[0022] The light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 12, or an amino acid sequence comprising the above sequence.

[0023] The heavy chain sequence of the 10G10 antibody includes the amino acid sequence shown in SEQ ID NO:13.

[0024] The light chain sequence of the 10G10 antibody includes the amino acid sequence shown in SEQ ID NO:15.

[0025] The heavy chain sequence of the 11F8 antibody includes the amino acid sequence shown in SEQ ID NO:17.

[0026] The light chain sequence of the 11F8 antibody includes the amino acid sequence shown in SEQ ID NO:19.

[0027] In a second aspect, the present application provides a nucleic acid molecule, using the following technical solution:

[0028] A nucleic acid molecule, which encodes the amino acid sequence of the monoclonal antibody.

[0029] The nucleic acid molecule encoding the heavy chain sequence of the 10G10 antibody includes the nucleotide sequence shown in SEQ ID NO: 14. The nucleic acid molecule encoding the light chain sequence of the 10G10 antibody includes the nucleotide sequence shown in SEQ ID NO: 16.

[0030] The nucleic acid molecule encoding the heavy chain sequence of the 11F8 antibody includes the nucleotide sequence shown in SEQ ID NO: 18; the nucleic acid molecule encoding the light chain sequence of the 11F8 antibody includes the nucleotide sequence shown in SEQ ID NO: 20.

[0031] In a third aspect, the present application provides a hybridoma cell line, which adopts the following technical solution:

[0032] A hybridoma cell strain secretes the above monoclonal antibody.

[0033] In a fourth aspect, the present application provides a kit for detecting influenza virus protein, which adopts the following technical solution:

[0034] A kit for detecting influenza virus protein, comprising the above-mentioned antibody pair against influenza A virus NP protein.

[0035] Optionally, the 10G10 antibody is used as a capture antibody, and the 11F8 antibody is used as a labeling antibody.

[0036] In a fifth aspect, the present application provides use of the above-mentioned antibody pair, the above-mentioned nucleic acid molecule or the above-mentioned kit in the preparation of a reagent for detecting influenza virus protein.

[0037] In summary, this application has the following beneficial effects:

[0038] The present application provides an antibody pair against influenza A virus NP protein, respectively named 10G10 antibody and 11F8 antibody. Among them, 10G10 antibody is used as a capture antibody, and 11F8 antibody is used as a labeling antibody, which has good specificity and sensitivity to influenza virus protein. DETAILED DESCRIPTION

[0039] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0040] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0042] The present application is further described in detail below in conjunction with the embodiments and test results.

[0043] Example

[0044] Example 1

[0045] This example provides a preparation process and screening process of hybridoma cell lines. After screening, a total of 8 hybridoma cell lines were obtained in this example.

[0046] The above process specifically includes the following steps:

[0047] 1. Animal Immunization

[0048] The immunized subjects were 8-week-old BALB / C female mice. The immunogen was the influenza A virus NP protein expressed by Escherichia coli, with 498 amino acids, a molecular weight of 55 kDa, and an amino acid sequence as shown in SEQ ID NO: 21.

[0049] The immunization process specifically includes the following steps:

[0050] (1) Preparation of immunogens: The influenza virus protein recombinantly expressed by Escherichia coli was mixed with Freund's complete adjuvant (Cat. No. F5881, Sigma) in equal volumes to prepare the first immunogen; the influenza virus protein recombinantly expressed by Escherichia coli was mixed with Freund's incomplete adjuvant (Cat. No. F5506, Sigma) in equal volumes to prepare the second immunogen; the influenza virus protein recombinantly expressed by Escherichia coli was mixed with physiological saline in equal volumes to prepare the third immunogen.

[0051] (2) Immune process:

[0052] First immunization: 100 μg of the first immunogen was injected subcutaneously at 3-4 points on the back of BALB / C female mice;

[0053] Two weeks later, the second immunization was performed by injecting 100 μg of the second immunogen subcutaneously at 3-4 points on the back of BALB / C female mice;

[0054] Two weeks later, the third immunization was performed by injecting 100 μg of the second immunogen subcutaneously at 3-4 points on the back of BALB / C female mice;

[0055] Two weeks later, the fourth immunization-shock immunization was performed by intraperitoneal injection of 100 μg of the third immunogen into BALB / C female mice;

[0056] The fifth immunization was performed 24 hours later by injecting 50 μg of the third immunogen into the tail vein of BALB / C female mice.

[0057] 2. Cell Fusion

[0058] Cell fusion began on the third day after the fifth immunization of BALB / C female mice.

[0059] The process of cell fusion specifically includes the following steps:

[0060] (1) Preparation of spleen cell suspension: On the third day after the fifth immunization of BALB / C female mice, the eyeballs of the BALB / c female mice were removed and blood was collected, and the serum of the BALB / c female mice was separated as a positive control for antibody detection; at the same time, the BALB / c female mice were killed by cervical dislocation, and the spleens were removed to prepare spleen cell suspension;

[0061] Preparation of myeloma cell suspension: Resuscitate myeloma cells (ATCC, catalog number BNCC100908) two weeks in advance (to ensure that myeloma cells are in the logarithmic growth phase when used) to prepare myeloma cell suspension;

[0062] Preparation of feeder layer cells: One day before cell fusion, blank BALB / c female mouse peritoneal macrophages and spleen cells were taken and added to 96-well plates for culture to obtain a cell plate containing feeder layer cells (cell concentration was 1×10 4 / well) to prepare feeder layer cells.

[0063] (2) Cell fusion process:

[0064] Polyethylene glycol (PEG) was used to mediate cell fusion. Splenocyte suspension and myeloma cell suspension were taken and mixed in serum-free 1640 medium (Cat. No. C3010-0500, VivaCell) at a cell number ratio of 5:1. The mixture was centrifuged at 1200 rpm for 5 min and the supernatant was removed.

[0065] The bottom of the centrifuge tube was flicked with a finger to loosely mix the two cells, and the tube was placed in a beaker filled with 37°C water to keep warm. Within 1 min, 1 mL of 50% PEG1500 (pH 8.0, product number 10783641001, Roche) was added to fuse the cells, and the mixture was shaken while adding. After the addition, the mixture was allowed to stand for 30 s. Serum-free 1640 culture medium (product number C3010-0500, VivaCell) was added to terminate the fusion, and the mixture was centrifuged at 800 rpm for 5 min. The precipitate was suspended with HAT culture medium and distributed into a cell plate containing feeder layer cells to obtain a cell plate containing fused cells and feeder layer cells, and the cell plate was placed in a cell culture incubator at 37°C and 5% CO2 for culture.

[0066] Among them, the following reagents are needed to prepare 500mL of HAT culture medium: 100mL of fetal bovine serum (catalog number 11011-8611, Sijiqing), 5mL of penicillin-streptomycin double antibody for cell culture (100×, catalog number E607011-0100, Shanghai Shenggong), 10mL of HAT culture medium additive (50×, catalog number H0262, Sigma) and 385mL of 1640 culture medium (catalog number C3010-0500, VivaCell).

[0067] 3. Screening of positive hybridoma cells

[0068] The cell plate containing the fused cells and feeder layer cells was cultured until the 4th day for half medium replacement, and continued to be cultured until the 7th day for full medium replacement. When the fused cells covered 10-50% of the bottom of the well, the positive wells were screened by conventional indirect ELISA method.

[0069] The indirect ELISA method specifically includes the following steps:

[0070] (1) Coating: Influenza virus protein recombinantly expressed in Escherichia coli was used as the coating antigen, diluted to 2 μg / mL with 0.05 mol / L CB buffer (31.8 g of Na2CO3, 58.8 g of NaHCO3, and made up to 2 L with ultrapure water) at a pH of 9.6, and added to the ELISA plate at 100 μL / well. After coating at 4°C overnight, pat dry, and block with 1% gelatin-PBS buffer at a mass volume ratio of 300 μL / well. Block at 37°C for 2 h, pat dry and set aside.

[0071] (2) Detection: The cell culture supernatant in the cell plate containing fused cell-feeder layer cells was added to the ELISA plate at 100 μL / well, incubated at 37°C for 60 min, washed three times with 0.01 mol / L PBST buffer containing Tween-20 and patted dry, added with 100 μL / well HRP-labeled goat anti-mouse secondary antibody (Cat. No. TJ-211229CN, Xiamen Taijing), incubated at 37°C for 60 min, washed three times and patted dry, added with 100 μL / well TMB colorimetric solution, incubated at 37°C in the dark for 10 min, and terminated by adding 50 μL / well 1 mol / L HCl.

[0072] At the same time, the BALB / c female mouse serum obtained by eye blood sampling and separation in the "cell fusion" was used as a positive control to screen out fusion cells with higher antibody titers, namely positive hybridoma cells.

[0073] 4. Cloning of Positive Hybridoma Cells

[0074] The positive hybridoma cells screened from the cell plate containing fused cells-feeder layer cells are derived from more than two hybridoma cells, so the antibodies secreted by the screened hybridoma cells are of different nature. In order to obtain completely homogeneous monoclonal antibodies, the positive hybridoma cells need to be cloned.

[0075] The day before cloning, feeder cells are prepared and plated according to the method of step (1) in "Cell Fusion" to obtain a cell plate containing feeder cells; the positive hybridoma cells obtained by screening are suspended in HT culture medium and mixed by pipetting, and inoculated into the cell plate containing feeder cells, and the cells in the wells of the cell plate are diluted to 1 cell in each well using HT culture medium, and the plate is placed at 37°C and 5% CO2 for 7 to 10 days. When cloned cells visible to the naked eye appear, antibodies can be detected.

[0076] Among them, the following reagents are needed to prepare 500mL of HT culture medium: 100mL of fetal bovine serum (catalog number 11011-8611, Sijiqing), 5mL of penicillin-streptomycin double antibody for cell culture (100×, catalog number E607011-0100, Shanghai Biotech), 10mL of HT culture medium additive (catalog number H0137, Sigma) and 385mL of 1640 culture medium (catalog number C3010-0500, VivaCell).

[0077] Observe under an inverted microscope, mark the wells where only a single clone grows and is positive in ELISA test, expand the strain, and finally obtain 8 cell strains through preliminary screening, and take the supernatant for the next functional screening. The above 8 hybridoma cell strains are named as shown in Table 1.

[0078] Table 1 Hybridoma cell lines obtained by screening and antibodies produced

[0079]

[0080] Example 2

[0081] This example provides a preparation process and a screening process for an antibody pair against influenza A virus NP protein.

[0082] The above process specifically includes the following steps:

[0083] 1. Preparation of Antibody Pairs Against Influenza A Virus NP Protein

[0084] (1) Preparation of ascites: 8-12 week old Balb / c mice were intraperitoneally injected with an ascites-specific adjuvant (Beijing Biolon, Catalog No. KX0210048). Ten days after injection, the cell line prepared in Example 1 (1×10 6 The cells / mouse were injected into the peritoneal cavity of Balb / c mice, and the ascites of the Balb / c mice was collected with a medical syringe 12 days later.

[0085] (2) Antibody purification: The Balb / c mouse ascites collected in step (1) was poured into a centrifuge tube and centrifuged at 12500 rpm for 20 min. The supernatant was collected and mixed with saturated ammonium sulfate solution at a volume ratio of 1:1. The mixture was centrifuged at 12000 rpm for 5 min. The supernatant was discarded and the precipitate was resuspended and dissolved with the same volume of PBS, filtered through a 0.22 μm filter membrane, and purified by affinity chromatography using a Protein A column (Cat. No. AA301307, Boglon Biotech).

[0086] The specific steps of purification using Protein A column affinity chromatography are as follows:

[0087] Column loading: Add 5 mL of Protein AResin medium to the chromatography column and let it stand, then rinse the column with 10 column volumes of ultrapure water;

[0088] Balance: Use 10 column volumes of pre-cooled Protein A column balance solution (50 mM Tris-HCl, 100 mM NaCl, solvent is water, pH = 8.0) to balance the chromatography column;

[0089] Loading: Load the sample after filtering with a 0.22 μm filter membrane at a flow rate of 5 mL / min;

[0090] Washing: Wash the column with 10 column volumes of pre-cooled Protein A column equilibration solution;

[0091] Elution: Elute the antibody with an elution buffer (100 mM Glycine, 150 mM NaCl, solvent is water, pH = 3.0) to obtain an elution buffer containing the antibody; immediately after elution, add a neutralization buffer (2 M Tris-HCl, solvent is water, pH = 9.0) to the elution buffer until the solution has a neutral pH;

[0092] Dialysis: The eluted antibodies were dialyzed three times in 1000 times the elution volume of PBS (pH=7.4) solution to obtain 8 purified antibodies against influenza A virus NP protein, whose specific names are shown in Table 1.

[0093] 2. Preparation of HRP-antibody conjugated complex protein

[0094] (1) Eight anti-influenza A virus NP protein antibodies were diluted to 2 mg / mL with 0.05 mol / L CB buffer at pH = 9.6; the dialysis membrane was selected and cut to an appropriate length according to the molecular weight of the target protein and the actual retention requirements and the dialysis volume; the dialysis membrane was soaked in 0.05 mol / L CB buffer at pH = 9.6 in advance, and the dialysis membrane was soaked and washed once with 0.05 mol / L CB buffer at pH = 9.6. 1 mL of antibody protein solution was transferred to the dialysis membrane. The dialysis was performed in 0.05 mol / L CB buffer at pH = 9.6 at 4 ° C with stirring, and the solution was changed every 1 h for a total of 5 times.

[0095] (2) HRP (Cat. No. RS20220118, Rise Reagent) was dissolved in ultrapure water to prepare an HRP solution with a concentration of 20 mg / mL, and NaIO4 was dissolved in ultrapure water to prepare a NaIO4 solution with a concentration of 20 mg / mL; after vortexing to fully dissolve, the HRP solution and the NaIO4 solution were mixed in a volume ratio of 1:1, that is, the NaIO4 solution was slowly added to the HRP solution, and the centrifuge tube was immediately wrapped with tin foil, and the HRP was activated at 4°C for 30 min in a dark environment.

[0096] (3) Slowly add ethylene glycol to the HRP-activated centrifuge tube while gently shaking (add 1 μL of ethylene glycol for every 1 mg of HRP). Continue to incubate at 4°C in the dark for 30 min to terminate HRP activation.

[0097] (4) Add the terminated activated HRP solution to the antibody dialysis membrane (add 1 mg of HRP and 1 mg of NaIO4 to 1 mg of antibody) and couple in 0.05 mol / L CB buffer at pH 9.6 at 4°C in the dark overnight.

[0098] On the second day, the 0.05 mol / L CB buffer solution with a pH of 9.6 was replaced and dialysis was continued for 2 hours. After dialysis, the coupled dialysate was transferred to a centrifuge tube to obtain an antibody-HRP coupled solution.

[0099] (5) Prepare a 20 mg / mL NaBH4 solution with pure water and add it to the antibody-HRP coupling solution in step (4). Add 2 μL of NaBH4 solution for every 1 mg of HRP. React at 4°C for 2 h. Mix by inverting the solution several times every 0.5 h to obtain a coupled protein solution.

[0100] (6) The coupled complex protein was precipitated with 50% ammonium sulfate (i.e., an equal volume of saturated concentration of ammonium sulfate and the coupled complex protein solution obtained in step (5) were mixed in a volume ratio of 1:1), precipitated at 4°C for 15 min, centrifuged at 10000 rpm for 10 min, and the supernatant was removed to obtain 8 strains of HRP-antibody coupled complex proteins, whose specific names are shown in Table 1.

[0101] 3. Screening of Anti-Influenza Protein Antibody Pairs

[0102] The 8 anti-influenza A virus NP protein antibodies obtained in step 1 and the 8 HRP-antibody coupled complex proteins obtained in step 2 were paired in pairs, and the antibodies were screened using the double antibody sandwich ELIS method.

[0103] The specific steps are as follows: 8 antibodies against influenza A virus NP protein are used as capture antibodies and coated on ELISA plates respectively, and then influenza A virus NP protein (same as the immunogen in Example 1) is added to the wells of the ELISA plate, and the unbound recombinant influenza virus protein is washed away after incubation; HRP-antibody coupled complex protein is added as a labeled antibody, and the unbound HRP-antibody coupled complex protein is washed away after incubation, and finally a color developing solution is added for color development, and the absorbance is measured at 450nm using a spectrophotometer. The test results are shown in Table 2.

[0104] Test result judgment: If color can be developed, it means that the labeled antibody and the capture antibody can recognize different epitopes of the influenza A virus NP protein, indicating that the capture antibody and the labeled antibody are a pair of paired antibodies. If color cannot be developed, it means that the labeled antibody cannot bind to the influenza A virus NP protein and is eluted, indicating that the capture antibody and the labeled antibody are not paired antibodies.

[0105] Table 2 Test results of capture antibody and labeling antibody pairing

[0106]

[0107] As shown in Table 2, the above test results show that the 10G10 antibody and the 11F8 antibody have high titers and can be used in the detection of influenza virus or its protein. Among them, the 10G10 antibody is a capture antibody and the 11F8 antibody is a labeling antibody. The hybridoma cell producing the 10G10 antibody is named 10G10 cell line, and the hybridoma cell producing the 11F8 antibody is named 11F8 cell line.

[0108] Example 3

[0109] In this example, the antibodies obtained by the above screening were sequenced.

[0110] The 10G10 cell line and the 11F8 cell line were expanded and cultured separately, and 5×10 6 The cells / ml were placed in a centrifuge tube, the supernatant was aspirated, frozen, and sent on dry ice to Nanjing Detai Bioengineering Co., Ltd. for hybridoma cell sequencing.

[0111] The sequencing results are as follows:

[0112] (I) 10G10 antibody

[0113] (1) Heavy chain sequence information

[0114] The full-length base sequence of the heavy chain includes the nucleotide sequence shown in SEQ ID NO:14.

[0115] Heavy chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO:13.

[0116] The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 1. The heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 2. The heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 3.

[0117] (2) Light chain sequence information

[0118] The full-length base sequence of the light chain includes the nucleotide sequence shown in SEQ ID NO:16.

[0119] Light chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO:15.

[0120] The light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3. The light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4. The light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5. The light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0121] (II) 11F8 antibody

[0122] (1) Heavy chain sequence information

[0123] The full-length base sequence of the heavy chain includes the nucleotide sequence shown in SEQ ID NO:18.

[0124] Heavy chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO:17.

[0125] The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3. The heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 7. The heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 8. The heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 9.

[0126] (2) Light chain sequence information

[0127] The full-length base sequence of the light chain includes the nucleotide sequence shown in SEQ ID NO: 20.

[0128] Light chain full-length amino acid sequence: includes the amino acid sequence shown in SEQ ID NO:19.

[0129] The light chain variable region includes a light chain CDR1, a light chain CDR2 and a light chain CDR3. The light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 10. The light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 11. The light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 12.

[0130] Example 4

[0131] This embodiment provides a colloidal gold test strip for detecting influenza A virus. The colloidal gold test strip is prepared using 10G10 antibody and 11F8 antibody.

[0132] The preparation process of the test strip specifically includes the following steps:

[0133] 1. Preparation of the test pad

[0134] (1) Preparation of coating buffer: Weigh 2.901 g of Na2HPO4·12H2O, 0.2914 g of NaH2PO4·2H2O, 8.5 g of NaCl, and 25 g of trehalose, dissolve in 1000 mL of ultrapure water, adjust the pH to 7.4, and store at 4°C until use.

[0135] (2) Preparation of test line: 10G10 antibody was added to the coating buffer to a final concentration of 1 mg / mL and sprayed onto the nitrocellulose membrane to prepare the test line. The spraying volume was 1.2 μL / cm and the spraying length was 30 cm.

[0136] (3) Preparation of quality control line: Sheep anti-mouse IgG polyclonal antibody (Hangzhou Longji Biotechnology Co., Ltd.) was added to the coating buffer with a final concentration of 1 mg / mL and sprayed onto the nitrocellulose membrane to prepare the quality control line. The spraying volume was 1.2 μL / cm, the spraying length was 30 cm, and the distance between the quality control line and the test line was 5 mm.

[0137] (4) The nitrocellulose membrane coated with the test line and the quality control line is the test pad. The test pad is attached to the PVC backing board and placed in an oven at 50°C to dry for 24±2h.

[0138] 2. Preparation of Gold Label Conjugate

[0139] (1) Preparation of relevant solutions:

[0140] Preparation of 0.2M K2CO3 solution: Weigh 2.7642 g of anhydrous potassium carbonate and dissolve it in 100 mL of pure water.

[0141] Preparation of 20% BSA solution: Weigh 20 g of BSA (Cat. No. V900933, Sigma) and 100 μL of proclin 300, dissolve in 100 mL of ultrapure water, and store at 4° C. for future use.

[0142] Preparation of the complex solution: weigh 0.36 g of Tris, 0.1 g of sodium caseinate, 0.1 g of PEG20000, 100 μL of TW-20, 2 g of sucrose and 100 μL of proclin300, dissolve in 100 mL of ultrapure water, adjust the pH to 8.5 with concentrated hydrochloric acid, and store at 4°C for later use.

[0143] (2) Marking process:

[0144] Take 100mL of 0.04% colloidal gold solution (Cat. No. C805628, McLean) and place it in a clean container, add 500-2000μL of 0.2M K2CO3 solution and stir evenly; then add 500-1500μg of 11F8 antibody and stir for 10-20min; then add 500μL of 20% BSA solution and stir, block for 5-10min, centrifuge at 8000-10000r / min for 30min, discard the supernatant, re-dissolve the precipitate with 10mL of re-solution to obtain the acetyl-gold-labeled conjugate, and store it at 4°C for later use.

[0145] 3. Preparation of conjugate pad

[0146] (1) Preparation of gold-labeled conjugate solution: 0.362 g of Tris, 0.05 g of sodium caseinate, 0.1 g of PEG20000, 100 μL of TW-20, 5 g of sucrose, and 100 μL of proclin300 were dissolved in 100 mL of ultrapure water, adjusted to pH 8.5 with concentrated hydrochloric acid, and stored at 4°C for later use.

[0147] (2) Add 7-15% acetylene gold-labeled conjugate to the gold-labeled conjugate solution, mix well, and evenly coat the solution on the glass fiber membrane (35 mL / sheet), and dry the membrane in an oven at 50° C. for 24±2 h.

[0148] 4. Preparation of sample pad

[0149] (1) Preparation of sample pad treatment solution: 0.242 g of Tris, 0.85 g of NaCl, 0.5 g of sodium caseinate, 2 g of sucrose and 100 μL of proclin 300 were dissolved in 100 mL of ultrapure water, the pH was adjusted to 8.0, and stored at 4°C for later use.

[0150] (2) 32 mL of sample pad treatment solution is evenly coated on the glass fiber membrane, and the membrane is placed in an oven at 50°C for drying for 24±2 hours. After passing the test, the membrane is sealed and stored at room temperature for future use.

[0151] 5. Assembly of colloidal gold test paper

[0152] According to the structure of colloidal gold test paper, the sample pad and the conjugate pad are sequentially pasted on the end of the PVC board close to the test line, and the sample pad is partially overlapped on the conjugate pad, and the conjugate pad is partially overlapped on the test pad; the absorbent paper is pasted on the end of the PVC board away from the test line, and the absorbent paper is partially overlapped on the test pad. After forming a large board, cut it into thin strips with a width of 3mm.

[0153] Example 5

[0154] This example is used to illustrate the detection sensitivity of the antibody pair for detecting influenza A virus provided in the present application.

[0155] Experimental group: colloidal gold test paper provided in Example 4;

[0156] Control group: mainstream influenza B virus antigen detection reagent on the market (colloidal gold method).

[0157] Detection method: Inactivated influenza A virus culture (Microbix, Influenza A grade 2antigen, Catalogue Number: EL-13-02) was used as the detection object, and diluted with diluent (ingredients: 10mM PBS, 0.1% Triaton X-100) in accordance with 1:5000, 1:20000, and 1:60000, respectively, and then dripped onto the sample pad of the colloidal gold test paper of the above experimental group and control group, and the results were observed for 15 to 20 minutes. The test results are shown in Table 3.

[0158] Table 3 Sensitivity test results

[0159]

[0160] From the test results, it can be seen that the colloidal gold test paper provided in Example 4 of the present application can still observe obvious bands when the original influenza virus culture solution is diluted to 60,000 times, while the colloidal gold test paper in the control group can no longer detect influenza virus when the original influenza virus culture solution is diluted to 60,000 times. Compared with the control group reagent, the antibody pair for detecting influenza A virus provided in the present application has better detection sensitivity.

[0161] Example 6

[0162] This example is used to illustrate the detection specificity of the antibody pair provided in the present application for detecting influenza A virus.

[0163] Experimental group: colloidal gold test paper provided in Example 4;

[0164] Control group: mainstream influenza A / B virus antigen detection reagent (colloidal gold method) on the market.

[0165] Detection method: The substances provided in Table 4 were used as the detection objects, and were added dropwise to the sample pads of the colloidal gold test papers of the above experimental group and the control group, and the results were observed for 15-20 minutes. The detection results are shown in Table 4.

[0166] Table 4 Specificity test results

[0167] name Example 4 Control group Respiratory syncytial virus Negative Negative Parainfluenza virus Negative Negative Adenovirus Negative Negative Staphylococcus aureus Negative Negative Neisseria encephalitis Negative Negative Streptococcus pneumoniae Negative Negative SARS-CoV-2N Protein Full Length Negative Negative SARS-CoV-2S Protein Full Length Negative Negative

[0168] It can be seen from the test results that the colloidal gold test paper provided in Example 4 does not react with other respiratory pathogens in the table and has good specificity.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An antibody pair against influenza A virus NP protein, characterized in that: The antibody pair comprises two monoclonal antibodies; the monoclonal antibodies comprise a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3; One monoclonal antibody in the antibody pair is named 10G10 antibody, and the sequence information of the 10G10 antibody is as follows: The heavy chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 1, or an amino acid sequence comprising the above sequence; The heavy chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 2, or an amino acid sequence comprising the above sequence; The heavy chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 3, or an amino acid sequence comprising the above sequence; The light chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 4, or an amino acid sequence comprising the above sequence; The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 5, or an amino acid sequence comprising the above sequence; The light chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 6, or an amino acid sequence comprising the above sequence; The other monoclonal antibody in the antibody pair is named 11F8 antibody, and the sequence information of the 11F8 antibody is as follows: The heavy chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 7, or an amino acid sequence comprising the above sequence; The heavy chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 8, or an amino acid sequence comprising the above sequence; The heavy chain CDR3 comprises the amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 9, or an amino acid sequence comprising the above sequence; The light chain CDR1 comprises the amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence as shown in SEQ ID NO: 10, or an amino acid sequence comprising the above sequence; The light chain CDR2 comprises the amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 11, or an amino acid sequence comprising the above sequence; The light chain CDR3 comprises the amino acid sequence shown in SEQ ID NO: 12, or an amino acid sequence having 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 12, or an amino acid sequence comprising the above sequence.

2. The antibody pair against influenza A virus NP protein according to claim 1, characterized in that: The heavy chain sequence of the 10G10 antibody includes the amino acid sequence shown in SEQ ID NO: 13; the light chain sequence of the 10G10 antibody includes the amino acid sequence shown in SEQ ID NO: 15; the heavy chain sequence of the 11F8 antibody includes the amino acid sequence shown in SEQ ID NO: 17; the light chain sequence of the 11F8 antibody includes the amino acid sequence shown in SEQ ID NO:

19.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the amino acid sequence of the monoclonal antibody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule encoding the 10G10 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 14; the nucleic acid molecule encoding the 10G10 antibody light chain sequence includes the nucleotide sequence shown in SEQ ID NO: 16; the nucleic acid molecule encoding the 11F8 antibody heavy chain sequence includes the nucleotide sequence shown in SEQ ID NO: 18; the nucleic acid molecule encoding the 11F8 antibody light chain sequence includes the nucleotide sequence shown in SEQ ID NO:

20.

5. A hybridoma cell line, characterized in that: The hybridoma cell line secretes the monoclonal antibody according to claim 1 or 2.

6. A kit for detecting influenza virus protein, characterized in that: The kit comprises the antibody pair against influenza A virus NP protein according to claim 1 or 2.

7. The kit according to claim 6, characterized in that The 10G10 antibody was used as a capture antibody, and the 11F8 antibody was used as a labeling antibody.

8. Use of the antibody pair according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the kit according to claim 6 or 7 in preparing a reagent for detecting influenza virus protein.

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