An antibody pair against influenza A virus NP protein and its application

Abstract

This application relates to the technical field of biomedicine, and specifically relates to an antibody pair against influenza A virus NP protein and its application. The antibody pair includes two monoclonal antibodies; the monoclonal antibody includes a heavy chain variable region and a light chain variable region; one monoclonal antibody in the monoclonal antibody pair is named 10G10 antibody, and the other monoclonal antibody is named 11F8 antibody. This application also discloses nucleic acid molecules encoding the above monoclonal antibodies and hybridoma cell lines secreting the above monoclonal antibodies. In addition, this application also discloses a kit for detecting influenza virus proteins including the above monoclonal antibody pair, wherein the 10G10 antibody is used as a capture antibody and the 11F8 antibody is used as a labeled antibody. The antibody pair against influenza A virus NP protein provided by this application has good specificity for influenza virus proteins.

Description

Technical Field

[0001] This application relates to the technical field of biomedicine, and particularly relates to an antibody pair against the NP protein of influenza A virus and its application. Background Art

[0002] Influenza refers to an infectious disease caused by influenza viruses (Flu). Influenza viruses are mainly transmitted through droplets in the air, contact between susceptible and infected individuals, or contact with contaminated items, and are characterized by rapid mutation, strong infectivity, fast 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 pathogenic to humans mainly focus on influenza A virus, which has the strongest virus toxicity and is prone to mutation; followed by influenza B virus. Therefore, the detection and research of influenza A virus and influenza B virus have great clinical significance.

[0003] Currently, there are mainly three methods for detecting influenza viruses. Method 1: Isolation and culture method, which is considered the gold standard for influenza virus detection; however, due to its complex operation and long detection time (the detection cycle takes about 14 days), it is difficult to be carried out clinically. Method 2: Nucleic acid detection method, which has high sensitivity, but high cost, requires 4 - 6 hours for the experiment, and has strong professionalism for experimental operation, so its application in the field is limited. Method 3: Rapid antigen detection method, and colloidal gold immunochromatography is commonly used to assist in the diagnosis of influenza A virus and influenza B virus infections. This is the most commonly used detection method in the current market. The whole detection process does not require any instruments, is simple and fast (only takes 10 - 15 minutes), and is especially suitable for self-examination and screening of suspected patients, hospital laboratories, special departments (such as blood transfusion, preoperative, emergency departments, etc.), disease control systems (epidemic prevention stations), and general surveys in places such as entry-exit quarantine.

[0004] The NP protein of influenza virus is very conservative and has a high content in influenza virus, and is the target protein in immunochromatographic detection. Although the immunochromatographic reagents in related technologies can achieve the detection of early infection of influenza virus, they are not ideal enough. Summary of the Invention

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

[0006] Through technological iteration, continuously improving the sensitivity and specificity of influenza virus detection is an important R & D direction in the diagnostic reagent industry. Antibodies against the influenza virus NP protein are the main bioactive 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 the NP protein of influenza A virus, adopting the following technical solution:

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

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

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

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

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

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

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

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

[0016] Another 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 an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having 1 or 2 conservative amino acid substitutions as compared with the sequence shown in SEQ ID NO: 7, or an amino acid sequence containing the above sequence;

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

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

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

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

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

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

[0024] The light chain sequence of the 10G10 antibody comprises an amino acid sequence as 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, adopting the following technical solution:

[0028] A nucleic acid molecule encoding the amino acid sequence of the above 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, adopting the following technical solution:

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

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

[0034] A kit for detecting influenza virus protein, which includes the above 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 the use of the above antibody pair, the above nucleic acid molecule or the above kit in the preparation of a reagent for detecting influenza virus protein.

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

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

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

[0040] To make the purpose, technical solutions and advantages of the present application clearer, the technical solutions 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 creative efforts belong to the scope protected by the present application. The embodiments described below are exemplary and are only used to explain the present application and should not be construed as a limitation of the present application.

[0041] For those not specifying specific techniques or conditions in the embodiments, follow the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained commercially.

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

[0043] Embodiment

[0044] Embodiment 1

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

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

[0047] I. Animal immunization

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

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

[0050] (1) Preparation of immunogen: Mix the influenza virus protein recombinantly expressed by Escherichia coli with Freund's complete adjuvant (product number F5881, sigma company) in equal volume to obtain the first immunogen; mix the influenza virus protein recombinantly expressed by Escherichia coli with Freund's incomplete adjuvant (product number F5506, sigma company) in equal volume to obtain the second immunogen; mix the influenza virus protein recombinantly expressed by Escherichia coli with physiological saline in equal volume to obtain the third immunogen.

[0051] (2) Immunization process:

[0052] Primary immunization: Inject 100 μg of the first immunogen subcutaneously at 3 - 4 points on the back of female BALB / C mice.

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

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

[0055] Two weeks later, perform the fourth immunization - boost immunization by injecting 100 μg of the third immunogen intraperitoneally into female BALB / C mice.

[0056] Twenty - four hours later, perform the fifth immunization by injecting 50 μg of the third immunogen into the tail vein of female BALB / C mice.

[0057] II. Cell fusion

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

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

[0060] (1) Preparation of splenocyte suspension: On the third day after the fifth immunization of female BALB / C mice, collect blood by eye - ball removal from female BALB / c mice, and separate the serum of female BALB / c mice as the positive control for antibody detection; at the same time, sacrifice female BALB / c mice by cervical dislocation, take their spleens, and prepare splenocyte suspension. ]

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

[0062] Preparation of feeder layer cells: One day before cell fusion, take peritoneal macrophages and splenocytes of blank female BALB / C mice, add them to a 96 - well plate for culture, and obtain a cell plate containing feeder layer cells (cell concentration is 1×10 4 / well), and prepare feeder layer cells.

[0063] (2) Cell fusion process:

[0064] Use polyethylene glycol (PEG) to mediate cell fusion. Take the splenocyte suspension and the myeloma cell suspension, mix them in a serum - free 1640 medium (catalog number C3010 - 0500, VivaCell) at a cell number ratio of 5:1, centrifuge at 1200 rpm for 5 min, and discard the supernatant.

[0065] Flick the bottom of the centrifuge tube gently with your finger to loosen and mix the two types of cells evenly. Place it in a beaker containing water at 37°C for incubation. Add 1 mL of 50% PEG1500 (pH 8.0, catalog number 10783641001, Roche) for cell fusion within 1 minute, adding while shaking, and let it stand for 30 s after adding; add serum-free 1640 medium (catalog number C3010-0500, VivaCell) to terminate the fusion, centrifuge at 800 rpm for 5 minutes, suspend the precipitate with HAT medium, aliquot it into a cell plate containing feeder cells to obtain a cell plate containing fused cells - feeder cells, and place it in a cell incubator at 37°C and 5% CO2 for culture;

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

[0067] III. Screening of positive hybridoma cells

[0068] Culture the above cell plate containing fused cells - feeder cells until the 4th day for semi-medium change, and continue to culture until the 7th day for full-medium change. When the fused cells cover 10 - 50% of the well bottom, use the conventional indirect ELISA method to screen positive wells.

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

[0070] (1) Plate coating: Use the influenza virus protein recombinantly expressed by Escherichia coli as the coating antigen, dilute it to 2 μg / mL with 0.05 mol / L CB buffer (31.8 g of Na2CO3, 58.8 g of NaHCO3, and make up to 2 L with ultrapure water) with a pH value of 9.6, add it to the enzyme-labeled plate at 100 μL / well, pat dry after coating overnight at 4°C, and block it with 1% gelatin-PBS buffer at 300 μL / well. After blocking at 37°C for 2 h, pat dry and set aside.

[0071] (2) Detection: Add 100 μL of the cell culture supernatant in the cell plate containing fused cells - feeder layer cells into the ELISA plate. Incubate at 37 °C for 60 min, then wash 3 times with 0.01 mol / L PBST buffer containing Tween - 20 and pat dry. Add 100 μL / well of HRP - labeled goat anti - mouse secondary antibody (product number TJ - 211229CN, Xiamen Taijing), incubate at 37 °C for 60 min, wash 3 times and pat dry. Add 100 μL / well of TMB chromogenic solution, incubate at 37 °C in the dark for 10 min, and then add 50 μL / well of 1 mol / L HCl to terminate the reaction.

[0072] Meanwhile, use the serum of BALB / c female mice obtained by eyeball blood collection in "Cell Fusion" as a positive control, and screen out the fused cells with higher antibody titers, which are the positive hybridoma cells.

[0073] IV. 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. Therefore, the antibodies secreted by the screened hybridoma cells are heterogeneous. To obtain a completely homogeneous monoclonal antibody, the positive hybridoma cells need to be cloned.

[0075] One day before cloning, prepare feeder layer cells and plate them according to the method in step (1) of "Cell Fusion" to obtain a cell plate containing feeder layer cells; suspend the screened positive hybridoma cells with HT medium and pipette to mix well, inoculate them into the cell plate containing feeder layer cells, and dilute the cells in the wells of the cell plate to 1 cell per well with HT medium. Incubate at 37 °C and 5% CO₂ in a humidified environment for 7 - 10 d. When visible cloned cells appear, the antibody can be detected.

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

[0077] Observe under an inverted microscope, mark the wells with only single - clone growth and positive ELISA detection, expand and establish cell lines. Finally, 8 cell lines were initially screened and the supernatants were taken for the next functional screening. Name the above 8 hybridoma cell lines as shown in Table 1.

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

[0079]

[0080] Example 2

[0081] This example provides the preparation process and screening process of antibody pairs against influenza A virus NP protein.

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

[0083] I. Preparation of antibody pairs against influenza A virus NP protein

[0084] (1) Preparation of ascites: Inject ascites-specific adjuvant (BioLuminary, product number KX0210048) into the peritoneal cavity of 8 - 12-week-old Balb / c mice. On the 10th day after injection, inject the cell line prepared in Example 1 (1×10 6 cells / mouse) into the peritoneal cavity of Balb / c mice. After another 12 days, collect the ascites of Balb / c mice using a medical syringe.

[0085] (2) Purification of antibodies: Pour the ascites of Balb / c mice collected in step (1) into a centrifuge tube, centrifuge at 12500 rpm for 20 min, collect the supernatant, mix the ascites supernatant with saturated ammonium sulfate solution at a volume ratio of 1:1, centrifuge at 12000 r / min for 5 min, discard the supernatant, resuspend and dissolve the precipitate with the same volume of PBS, and then filter with a 0.22 μm filter membrane and purify by affinity chromatography on a Protein A column (product number AA301307, Bio-Gel).

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

[0087] Column packing: Take 5 mL of Protein A Resin medium and add it to the chromatography column and let it stand. Wash the chromatography column with 10 column volumes of ultrapure water;

[0088] Equilibration: Equilibrate the chromatography column with 10 column volumes of pre-cooled Protein A column equilibration buffer (50 mM Tris-HCl, 100 mM NaCl, solvent is water, pH = 8.0);

[0089] Sample loading: Load the sample filtered through a 0.22 μm filter membrane at a flow rate of 5 mL / min;

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

[0091] Elution: The antibody was eluted 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, a neutralization buffer (2 M Tris-HCl, solvent is water, pH = 9.0) was added to the elution buffer until the solution reached a neutral pH;

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

[0093] II. Preparation of HRP-antibody conjugate complex protein

[0094] (1) The 8 antibodies against influenza A virus NP protein were respectively 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 actual requirements of the molecular weight cut-off of the target protein and the dialysis volume; the dialysis membrane was presoaked with 0.05 mol / L CB buffer at pH = 9.6, and then the dialysis membrane was soaked and washed again with 0.05 mol / L CB buffer at pH = 9.6. 1 mL of the antibody protein solution was transferred into the dialysis membrane. Dialysis was carried out with stirring at 4 °C in 0.05 mol / L CB buffer at pH = 9.6, and the solution was changed every 1 h for a total of 5 times.

[0095] (2) HRP (product number RS20220118, Rees 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 at 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 activated at 4 °C in the dark for 30 min.

[0096] (3) Ethylene glycol was slowly added dropwise to the centrifuge tube with activated HRP while gently shaking (1 μL of ethylene glycol was added for every 1 mg of HRP), and continued to be kept in the dark at 4 °C for 30 min to terminate the activation of HRP.

[0097] (4) The HRP solution after termination of activation was added into the antibody dialysis membrane (1 mg of antibody was added with 1 mg of HRP and 1 mg of NaIO4), and coupling was carried out overnight at 4 °C in the dark in 0.05 mol / L CB buffer at pH = 9.6.

[0098] The next day, after changing the 0.05 mol / L CB buffer at pH = 9.6, dialysis was continued for 2 h. After dialysis, the coupled dialysis solution was transferred into a centrifuge tube to obtain an antibody-HRP conjugate solution.

[0099] (5) Prepare a NaBH4 solution with a concentration of 20 mg / mL using pure water and add it to the antibody-HRP conjugate solution in step (4). The addition amount is 2 μL of NaBH4 solution for every 1 mg of HRP. React at 4 °C for 2 h, and invert the mixture up and down a few times every 0.5 h to obtain a conjugate complex protein solution.

[0100] (6) Precipitate the conjugate complex protein with 50% ammonium sulfate (i.e., mix saturated ammonium sulfate with the same volume as the conjugate complex protein solution obtained in step (5) at a volume ratio of 1:1), precipitate at 4 °C for 15 min, centrifuge at 10000 rpm for 10 min, discard the supernatant, and obtain 8 strains of HRP-antibody conjugate complex proteins, which are specifically named as shown in Table 1.

[0101] III. Screening of anti-influenza protein antibody pairs

[0102] Pair the 8 strains of antibodies against influenza A virus NP protein obtained in step one and the 8 strains of HRP-antibody conjugate complex proteins obtained in step two pairwise, and screen the antibodies using the double antibody sandwich ELISA method.

[0103] The specific steps are as follows: Use the 8 strains of antibodies against influenza A virus NP protein as capture antibodies and coat them on the enzyme-labeled plate respectively. Then add influenza A virus NP protein (the same immunogen as in Example 1) to the wells of the enzyme-labeled plate, incubate and wash away the unbound recombinant influenza virus protein; then add the HRP-antibody conjugate complex protein as the labeled antibody, incubate and wash away the unbound HRP-antibody conjugate complex protein, finally add the chromogenic solution for color development, and measure the absorbance at 450 nm using a spectrophotometer. The test results are shown in Table 2.

[0104] Judgment of test results: If color development can occur, it indicates that the labeled antibody and the capture antibody can recognize different epitopes of influenza A virus NP protein, indicating that this group of capture antibody and labeled antibody is a pair of paired antibodies. If no color development occurs, it indicates that the labeled antibody cannot bind to influenza A virus NP protein and is thus eluted, indicating that this group of capture antibody and labeled antibody is not a paired antibody.

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

[0106]

[0107] As can be seen from Table 2, based on the above test results, the pair of antibodies, 10G10 antibody and 11F8 antibody, have high titers and can be applied to the detection of influenza virus or its proteins. Among them, the 10G10 antibody is the capture antibody, and the 11F8 antibody is the labeled antibody. The hybridoma cells that produce the 10G10 antibody are named the 10G10 cell line, and the hybridoma cells that produce the 11F8 antibody are named the 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 respectively expanded in culture, and 5×10 6 cells / ml were collected in a centrifuge tube. The supernatant was aspirated, frozen, and sent to Nanjing Detai Bioengineering Co., Ltd. by dry ice for sequencing of hybridoma cells.

[0111] The sequencing results are as follows:

[0112] (I) 10G10 antibody

[0113] (1) Heavy chain sequence information

[0114] Full-length heavy chain base sequence: including the nucleotide sequence shown in SEQ ID NO: 14.

[0115] Full-length heavy chain amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 13.

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

[0117] (2) Light chain sequence information

[0118] Full-length light chain base sequence: including the nucleotide sequence shown in SEQ ID NO: 16.

[0119] Full-length light chain amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 15.

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

[0121] (2) 11F8 antibody

[0122] (1) Heavy chain sequence information

[0123] Full-length heavy chain base sequence: including the nucleotide sequence shown in SEQ ID NO: 18.

[0124] Full-length heavy chain amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 17.

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

[0126] (2) Light chain sequence information

[0127] Full-length light chain base sequence: including the nucleotide sequence shown in SEQ ID NO: 20.

[0128] Full-length light chain amino acid sequence: including the amino acid sequence shown in SEQ ID NO: 19.

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

[0130] Example 4

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

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

[0133] I. Preparation of the detection pad

[0134] (1) Preparation of the 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 them in 1000 mL of ultrapure water, adjust the pH to 7.4, and store it at 4°C for later use.

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

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

[0137] (4) The nitrocellulose membrane coated with the test line and the control line is the test pad. Stick the test pad on the PVC liner and place it in an oven at 50 °C for drying for 24 ± 2 h.

[0138] II. Preparation of the gold-labeled 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 (product number V900933, Sigma) and 100 μL of proclin300, dissolve them in 100 mL of ultrapure water, and store them at 4 °C for later use.

[0142] Preparation of the reconstitution 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 them in 100 mL of ultrapure water, adjust the pH to 8.5 with concentrated hydrochloric acid, and store them at 4 °C for later use.

[0143] (2) Labeling process:

[0144] Take 100 mL of 0.04% colloidal gold solution (product number C805628, Macklin) and place it in a clean container. Add 500 - 2000 μL of 0.2M K2CO3 solution and stir evenly; then add 500 - 1500 μg of 11F8 antibody and stir for reaction for 10 - 20 min; then add 500 μL of 20% BSA solution and stir to block for 5 - 10 min. Centrifuge at 8000 - 10000 r / min for 30 min, discard the supernatant, and re-dissolve the obtained precipitate with 10 mL of the reconstitution solution to obtain the gold-labeled conjugate for influenza B, and store it at 4 °C for later use.

[0145] III. Preparation of the conjugate pad

[0146] (1) Preparation of the gold-label 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 are 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 the influenza B gold-label conjugate to the gold-label conjugate solution at a concentration of 7 - 15%, mix well, and evenly coat it on the glass fiber membrane (35 mL per sheet), then place it in an oven at 50 °C and dry for 24 ± 2 h.

[0148] IV. Preparation of the sample pad

[0149] (1) Preparation of the 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 proclin300 are dissolved in 100 mL of ultrapure water, adjusted to pH 8.0, and stored at 4 °C for later use.

[0150] (2) Apply 32 mL of the sample pad treatment solution per plate evenly on the glass fiber membrane, place it in an oven at 50 °C and dry for 24 ± 2 h. After passing the detection and identification, store it sealed at room temperature for later use.

[0151] V. Assembly of the colloidal gold test strip

[0152] According to the structure of the colloidal gold test strip, sequentially paste the sample pad and the conjugate pad at one end of the PVC board near the test line, with the sample pad partially overlapping the conjugate pad, and the conjugate pad partially overlapping the detection pad; paste the absorbent paper at the other end of the PVC board away from the test line, with the absorbent paper partially overlapping the detection pad. After forming the large board, cut it into thin strips with a width of 3 mm.

[0153] Example 5

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

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

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

[0157] Detection method: Using an inactivated culture of influenza A virus (Microbix, Influenza A grade 2 antigen, Catalogue Number: EL-13-02) as the detection object, after sequential dilution at ratios of 1:5000, 1:20000, and 1:60000 using a diluent (composition: 10 mM PBS, 0.1% Triton X-100), the dilutions were respectively dropped onto the sample pads of the colloidal gold test strips in the above experimental group and control group, and the results were observed after 15 - 20 minutes. The detection results are shown in Table 3.

[0158] Table 3 Sensitivity Detection Results

[0159]

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

[0161] Example 6

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

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

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

[0165] Detection method: Using the substances provided in Table 4 as the detection objects, they were respectively dropped onto the sample pads of the colloidal gold test strips in the above experimental group and control group, and the results were observed after 15 - 20 minutes. The detection results are shown in Table 4.

[0166] Table 4 Specificity Detection Results

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

[0168] From the detection results, it can be seen that the colloidal gold test strip 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 them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate 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 of the monoclonal antibodies in the antibody pair is named 10G10 antibody, and the sequence information of the 10G10 antibody is as follows: The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 1; The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2; The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 4; The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 5; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 6; The other monoclonal antibody in the antibody pair is named 11F8 antibody, and the sequence information of the 11F8 antibody is as follows: The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 7; The amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 9; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 10; The amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 11; The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

12.

2. The anti-influenza A virus NP protein antibody pair according to claim 1, characterized in that: The heavy chain sequence of the 10G10 antibody includes the heavy chain variable region shown in SEQ ID NO: 13; the light chain sequence of the 10G10 antibody includes the light chain variable region shown in SEQ ID NO: 15; the heavy chain sequence of the 11F8 antibody includes the heavy chain variable region shown in SEQ ID NO: 17; and the light chain sequence of the 11F8 antibody includes the light chain variable region 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 kit for detecting influenza A virus protein, characterized in that: The kit comprises the antibody pair against influenza A virus NP protein according to claim 1 or 2.

6. The kit according to claim 5, characterized in that The 10G10 antibody was used as a capture antibody, and the 11F8 antibody was used as a labeling antibody.

7. 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 5 or 6 in the preparation of a reagent for detecting influenza A virus protein.

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