CN115947835A

CN115947835A - Antibody targeting influenza B virus nucleoprotein and application thereof

Info

Publication number: CN115947835A
Application number: CN202211430198.8A
Authority: CN
Inventors: 罗海峰; 奚建红; 滕宏; 王文峰; 陈超; 周昊; 鲍勇刚; 万欢
Original assignee: Cnpair Biotech Co ltd
Current assignee: Cnpair Biotech Co ltd
Priority date: 2022-11-15
Filing date: 2022-11-15
Publication date: 2023-04-11
Anticipated expiration: 2042-11-15
Also published as: CN115947835B

Abstract

The invention provides an antibody targeting influenza B (B) virus nucleoprotein and application thereof. The amino acid sequences of the light chain and the heavy chain of the antibody are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The antibody is used for developing a lateral flow immunochromatography detection reagent. The antibody has high sensitivity and good specificity, and has wide application prospect in the field of immunodiagnosis.

Description

Antibody targeting influenza B virus nucleoprotein and application thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to a targeted influenza B (B) virus nucleoprotein antibody and application thereof.

Background

A total of 7 members of the genus Orthomyxoviridae (Orthomyxoviridae) are members of the Orthomyxoviridae (4 of them are Influenza virus (Influenza A/B/C/D virus; respectively Influenza A, B, C and D). Influenza virus samples are generally elliptical or spherical (about 100nm in diameter). Influenza viruses are in some cases long filamentous (long axis about 20 μm), and virus samples isolated from clinical sites are often long filamentous.

Among them, influenza viruses are named according to host, location of isolation, number of isolated strain, and year of isolation. Unlike influenza a viruses, influenza B viruses are classified mainly based on the similarities in various traits found and between strains, for example, one of the lines is represented by B/Yamagata/16/88, and is called a "B/Yamagata-like" line ((B/Yamagata linkage-like viruses). Influenza B viruses usually cause only local outbreaks of influenza, and influenza B viruses mainly infect humans and cannot infect various hosts.

Influenza viruses are enveloped viruses whose genomes comprise single-stranded negative-sense viral ribonucleic acid (vRNA) segmented (segment 1 to segment 8). The major proteins encoded by the viral genome are: polymerase protein subunits (

RNA polymerase subunits

1,2, A-X and 1-F2; PB1, PB2, PA, PA-X and PB 1-F2), nucleoproteins (NP), 2 surface glycoproteins (hemagglutinin HA and neuraminidase NA), 2 matrix proteins (matrix proteins, M1 and M2; wherein M2 is also a transmembrane ion channel protein), nonstructural proteins (non-structural protein 1, NS1) and Nuclear Export Protein (NEP).

The life cycle of influenza virus is divided into the following steps: the method comprises the following steps of adsorbing and entering a host cell by a virus, removing a capsid by the virus, entering a nucleus by a virus ribonucleoprotein (vRNP), transcribing and replicating a virus genome in the nucleus, taking out the virus ribonucleoprotein (vRNP) from the nucleus, assembling the virus and budding and releasing the virus.

The envelope of influenza virus is a double-layered lipid membrane obtained from the host cell membrane when progeny viruses bud, and has a layer of the most abundant viral matrix protein (M1) surrounding the viral genome below its envelope: each viral ribonucleic acid (vRNA) is present in the form of a viral ribonucleoprotein (vRNP) and can express one or more viral proteins; each viral ribonucleoprotein (vRNP) consists of three parts, oligomeric Nucleoprotein (NP) and viral ribonucleic acid (vRNA) and heterotrimeric viral RNA polymerase (RNA polymerase).

In addition, the viral envelope is embedded with three viral proteins: hemagglutinin (HA), neuraminidase (NA) and transmembrane channel protein (M2). Among these, hemagglutinin (HA) and Neuraminidase (NA) are two major viral antigens: hemagglutinin (HA) is responsible for binding to sialic acid receptors on host cells; neuraminidase (NA) is responsible for the release of budding progeny virions out of the host cell; the ion channel protein (M2) is responsible for acidification of the virus upon entry into the host cell and plays an important role in budding release of progeny virus.

The Nucleoprotein (NP) of influenza B virus is the major internal structural protein encoded by segment 5 of the viral genome. The length of Nucleoprotein (NP) gene is about 1762bp, and the molecular weight of expressed protein product is about 62KD.

Nucleoproteins (NPs) are crescent-shaped, comprise 560 amino acids in total, consist of a head, a middle domain, and a long tail loop of a specified number of amino acids, are rich in arginine, glycine, serine, and a small number of cysteine residues, and have a net positive charge at neutral pH. Nucleoprotein (NP) is a monomeric polypeptide, both phosphorylated and unphosphorylated, whose conformational changes are regulated by phosphorylation and dephosphorylation of the Nucleoprotein (NP) by host cell phosphokinases and phosphatases, and in turn, affect Nucleoprotein (NP) oligomerization, and interaction with other macromolecules.

The Nucleoprotein (NP) can interact with a variety of other macromolecules, such as viral ribonucleoproteins (vRNP) that can be composed with viral RNA and heterotrimeric viral RNA polymerase (containing subunits PB1, PB2 and PA). The common minimal functional unit of viral ribonucleoprotein (vRNP) consists of a viral RNA polymerase complex that binds viral RNA and 9 Nucleoprotein (NP) monomers arranged in a rod-like structure.

Viral ribonucleoprotein (vRNP) too large

Is unable to pass through the nucleus of the host cell>

Nuclear pore diffusion. The nuclear import protein alpha (import-alpha) of the host cell binds to the Nucleoprotein (NP) of influenza virus through its nuclear localization sequence. During the viral replication cycle, viral ribonucleoproteins (vrnps) enter and exit the nucleus of the host cell by active transport.

Whether the Nucleoprotein (NP) is phosphorylated in the host cell during viral propagation in the host cell, and the corresponding conformational changes, are therefore critical factors affecting the host response profile of influenza viruses.

Phylogenetic analysis is carried out on influenza virus strains separated from different hosts, and the amino acid sequence translated from the Nucleoprotein (NP) gene is found to have better conservative property. Alignment of the amino acid sequences of the Nucleoproteins (NPs) of influenza a (a), B (B) and C (C) viruses revealed significant similarity between the amino acid sequences of the Nucleoproteins (NPs) of the three viruses, with the highest degree of conservation of each of the Nucleoproteins (NPs) of influenza a (a) and B (B) viruses. The Nucleoprotein (NP) has at least 3 independent antigen epitopes, wherein the amino acid sequence of one epitope is highly conserved in a plurality of influenza virus strains.

The Nucleoprotein (NP) of influenza virus induces the host to produce non-neutralizing antibodies under natural conditions, and monoclonal antibodies against highly conserved epitopes of the Nucleoprotein (NP) inhibit the transcription of viral RNA in the host cell nucleus. Meanwhile, the body generates a cytotoxic lymphocyte reaction, and virus antigens (such as nucleoprotein, NP) presented by Major Histocompatibility Complex (MHC) molecules on the surface of infected cells are recognized, so that a cell-mediated killing effect is exerted, and influenza virus infected by host cells is eliminated.

Compared with other proteins on the surface of the viral envelope, such as Hemagglutinin (HA) and Neuraminidase (NA), the amino acid sequence of the Nucleoprotein (NP) is relatively more conserved, the immunogenicity is stable, and the Nucleoprotein (NP) is not influenced by antigen drift (antigenic drift), so that the main specific targets of influenza a (a), influenza B (B) and influenza C (C) viruses can be respectively identified, and the Nucleoprotein (NP) HAs strong discrimination as a diagnostic target antigen.

The Nucleoprotein (NP) of influenza viruses is generally the most abundantly expressed viral protein in infected host cells, and functions include, but are not limited to, transcription, replication, packaging, and nuclear transport of viral RNA. The multifunctional role of Nucleoprotein (NP), coupled with its high degree of sequence conservation, makes it an ideal molecular target.

In conclusion, the development of monoclonal antibodies targeting influenza virus Nucleoprotein (NP) antigen is of great significance for diagnosing different influenza virus subtypes and discovering antibody drugs with strong curative effect on specific influenza virus subtypes.

Colloidal gold is one of the earliest labeling materials used in immunochromatographic assay kits. The absorption wavelength is in a visible light region, so that the direct observation by naked eyes is easy, and the color is red, blue or purple and the like along with the change of the size and the shape of the colloidal gold particles. The colloidal gold raw material is generally prepared by reducing chloroauric acid by simple and reliable trisodium citrate. The surface of the colloidal gold prepared by the method carries a considerable amount of negative charges, so that the colloidal gold has very stable properties in a dry state and even in a solution. In addition, the aggregation of a large number of negative charge surfaces enables the colloidal gold to be subjected to mild coupling combination with biological macromolecules such as nucleotide, polypeptide or protein with positive charges under the conditions of specific pH and the like in a simple manner of electrostatic adsorption, and the coupling combination can retain the activity of the biological macromolecules to the maximum extent. The colloidal gold becomes a marking material in most commercial immunochromatographic test strips on the market at present due to the advantages.

Although the colloidal gold is widely applied to the immunochromatography method, the method also has more defects, such as poor detection result accuracy and unsatisfactory detection sensitivity due to the fact that the colloidal gold is easily interfered by a matrix. Aiming at the defects, the traditional colloidal gold is modified, for example, the colloidal gold particles with the geometrical structures in spike shapes are prepared, or polyethylene glycol (PEG) molecules at the tail ends of carboxyl groups are modified on the surface of the colloidal gold and combined with a labeled antibody in a covalent mode, so that the detection sensitivity or the anti-interference capability of the colloidal gold test strip is improved. The modification of the traditional colloidal gold further expands the application of the colloidal gold in the lateral flow immunochromatographic test strip.

The lateral flow immunochromatography rapid diagnostic kit is a test paper-based result reporting method suitable for instant detection. The core element is the lateral flow immunochromatographic test strip (fig. 1, a and b), which is generally in the shape of a narrow strip, and the width is usually 4-6 mm, while the length is not more than 6-7 cm. A standard lateral flow immunochromatographic strip should include four main components, respectively:

the main material of the sample pad area is made of cellulose, and the sample pad area is fully immersed into an object to be measured when the sample pad area is used;

the main material of the conjugate pad region is made of glass fiber, and the conjugate pad region generally contains colloidal gold (conjugate) antigen or colloidal gold (conjugate) antibody;

the main material of the detection area is made of a nitrocellulose membrane (NC membrane), the detection area is mainly provided with a detection line and a quality control line, and the detection line and the quality control line respectively contain corresponding antibodies;

the main material of the absorbent pad area is made of cellulose.

When the lateral flow test strip is inserted into the solution to be tested, the solution will flow from the sample pad area into the conjugate pad area. When the solution contains the substance to be detected, the substance to be detected will be bound to the colloidal gold (conjugate) antigen or colloidal gold (conjugate) antibody in the binding pad region. The colloidal gold (conjugate) antibody or colloidal gold (conjugate) antigen rehydrated by the solution or a compound formed by the colloidal gold (conjugate) antigen and a sample to be detected can sequentially pass through a detection area (NC membrane) and a water absorption pad area under the action of capillary force. In this case, the lateral flow strip will have two results, a standard model (FIG. 1 a) and a competitive model (FIG. 1 b).

Fig. 1a when the lateral flow immunochromatographic test strip uses a standard model (detecting nucleoprotein antigen of influenza virus), the detection line (containing specific antibody targeting nucleoprotein antigen of influenza virus) can only capture the complex of the antigen to be detected and the colloidal gold (conjugated) primary antibody: if the antigen to be detected exists in the solution, the detection line can capture a compound formed by the antigen to be detected and the colloidal gold (coupled) primary antibody, and the colloidal gold (coupled) primary antibody is captured by the quality control line (containing corresponding secondary antibodies) all the time, so that the detection line and the quality control line have color changes at the moment, and the result is judged as positive reaction. If the antigen to be detected does not exist in the solution, only the quality control line (containing the corresponding secondary antibody) can capture the colloidal gold (coupling) primary antibody and generate color change, at the moment, the quality control line is colored, the detection line is not colored, and the result is judged as negative reaction.

FIG. 1b when the lateral flow test strip is used in a competitive format (IgM/IgG antibodies for detecting nucleoprotein [ NP ] antigen targeted to influenza virus), the antibody to be detected binds to the colloidal gold (conjugated) nucleoprotein antigen coated on the binding pad region, and the antibody to be detected and the colloidal gold (conjugated) nucleoprotein antigen are each captured by the detection line (antibody against IgG/IgM): when the antibody to be detected exists, the antibody to be detected can be captured by the detection line, the colloidal gold (coupling) antigen cannot be captured by the detection line (because the competitive closure of the antibody to be detected), and further the color change cannot occur, but the quality control line (comprising the nucleoprotein antigen and the antibody to be detected) can still capture the colloidal gold (coupling) antigen, the detection line does not develop color, the quality control line develops color, and the result is judged as positive reaction; if the antibody to be detected does not exist in the solution, the detection line and the quality control line can capture the colloidal gold (coupled) antigen, and the detection line and the quality control line can change color at the moment, so that the result is judged as negative reaction.

However, lateral flow immunochromatographic rapid diagnostic kits also have some disadvantages: firstly, the detection means can only provide qualitative results, and has poor applicability to pathogenic agents (specific bacteria, mycoplasma, viruses and the like) which need to diagnose infection conditions according to fine quantitative results; secondly, this test means requires that the test sample must be in a liquid state and must have a certain viscosity in order to flow through the porous nitrocellulose membrane.

Nevertheless, the lateral flow immunochromatography rapid diagnostic kit is still a detection technology with important application value, and can realize the universal standards of sensitivity, stability, economy, user friendliness, no equipment and the like required by rapid diagnosis. These advantages of the lateral flow immunochromatographic rapid diagnostic kit are of great importance, in particular, for units or regions lacking specialized equipment and not well-trained technicians.

Disclosure of Invention

The invention aims to provide an antibody targeting influenza B (B) virus nucleoprotein and application thereof, in particular application in a lateral flow immunochromatography diagnostic kit.

In order to achieve the object of the present invention, in a first aspect, the present invention provides an antibody targeting influenza B (B) virus nucleoprotein, the amino acid sequences of light chain hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody (mab G4E 1) are rssepeskala, SASTLQS and qqhnryptt, respectively, and the amino acid sequences of heavy chain hypervariable regions CDR-H1, CDR-H2 and CDR-H3 of the antibody are SVWMH, yissiyyavdeg and SGWLNAMKY, respectively.

The amino acid sequences of the light chain and the heavy chain of the antibody are respectively shown as SEQ ID NO 1 and SEQ ID NO 2.

The invention also provides nucleic acids encoding the antibodies. The nucleotide sequences of the light chain and the heavy chain of the coded antibody are respectively shown as SEQ ID NO 8 and 9.

In a second aspect, the invention provides an application of the antibody in preparing a reagent or a kit for detecting influenza B (B) virus (nucleoprotein).

In a third aspect, the present invention provides an influenza B (B) virus (nucleoprotein) detection reagent or kit prepared from the antibody.

In a fourth aspect, the invention provides an application of the antibody in preparation of a colloidal gold immunochromatographic assay test strip or kit for influenza B (B) virus.

In a fifth aspect, the invention provides an application of the antibody in preparing a lateral flow immunochromatography diagnostic kit for influenza B (B) virus.

In a sixth aspect, the invention provides a colloidal gold immunochromatographic assay test strip for influenza B (B) virus, which comprises a sample pad, a colloidal gold conjugate pad, an NC membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the colloidal gold conjugate pad, the NC membrane and the water absorption pad are sequentially adhered to the bottom plate.

Wherein, the colloidal gold conjugate pad is coated with a colloidal gold labeled antibody I targeting influenza B (B) virus nucleoprotein; the detection line is coated with the antibody, and the quality control line is coated with goat anti-mouse polyclonal antibody.

Preferably, the concentration of the antibody I (monoclonal antibody E4H 7) coated on the colloidal gold conjugate pad is 10-15 μ g/mL.

Preferably, the concentration of the antibody coated on the detection line is 1.5-2mg/mL.

Preferably, the sample pad and the absorbent pad are made of cellulose, and the colloidal gold bonding pad is made of glass fiber.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention provides an antibody for targeted recognition of influenza B (B) virus nucleoprotein (FluB N), which has high specificity and high affinity characteristics to the influenza B (B) virus nucleoprotein, and can be used for designing a therapeutic target for influenza B (B) virus, so that a therapeutic preparation for influenza B (B) virus can be designed, and the therapeutic target design comprises but is not limited to target design of therapeutic antibodies.

The antibody can be effectively used for quantitative and/or qualitative detection of the influenza B (B) virus, can also be used for ELISA detection, immunochemiluminescence detection, immunofluorescence detection and other detection methods, and has wide application range and strong applicability.

In addition, the corresponding antigen amino acid sequence is correspondingly adjusted or modified, and the modified material comprises one or more of nano material, fluorescent material, enzyme, biotin and protein. The modified or modified amino acid sequence is favorable for being applied to detection of influenza B (B) virus, immune antigen design of influenza B (B) virus vaccine, evaluation of influenza B (B) virus vaccine and the like.

The lateral flow immunochromatography rapid diagnostic kit based on the colloidal gold has the advantages of lower detection limit and higher sensitivity; the selected antibody is an antigen epitope with a specific target and a highly conserved amino acid sequence, and is not influenced by common antigen protein mutation of virus mutant strains, so that the detection cost is reduced, the detection time is shortened, and the detection efficiency is improved. The lateral flow immunochromatography rapid diagnosis kit is a detection technology with important application value, and can realize the universal standards of sensitivity, stability, economy, user friendliness, no equipment and the like required by rapid diagnosis. These advantages of the lateral flow immunochromatographic rapid diagnostic kit are of great importance, in particular, for units or regions lacking specialized equipment and lacking well-trained technicians.

Drawings

FIG. 1 is a schematic diagram of colloidal gold immunoassay. Wherein, a: a standard model; b: a competition model.

FIG. 2 shows the results of protein electrophoresis of recombinant proteins (antigens) of influenza B virus (B) in examples of the present invention.

FIG. 3 shows the results of the detection of inactivated influenza virus cultures (at different dilutions) using the lateral flow immunochromatographic rapid test reagents developed using the antibodies in the preferred embodiment of the present invention.

FIG. 4 shows the results of the detection (different dilutions) of the lateral flow immunochromatographic rapid detection reagent developed based on the antibody of the present invention using the influenza B virus nucleoprotein solution standard substance in the preferred embodiment of the present invention.

FIG. 5 shows the results of a lateral flow immunochromatographic rapid test reagent developed using the antibody of the present invention in a preferred embodiment of the present invention for testing normal human nasal swab samples.

FIG. 6 is a graph showing the binding of the antibody to the nucleoprotein antigen of the recombinant influenza B (B) virus at a gradient concentration in the preferred embodiment of the invention. Where 0.5. Mu.g (/ mL) is the binding curve for 50ng of antigen coated per well (100. Mu.L per well) and 1. Mu.g (/ mL) is the binding curve for 100ng of antigen coated per well (100. Mu.L per well).

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available.

EXAMPLE 1 preparation of monoclonal antibody-producing hybridomas

1. Design of influenza B virus nucleoprotein antigen

According to the amino acid sequence obtained by translation of influenza B virus nucleoprotein gene (SEQ ID NO:5; optimized gene sequence is shown as SEQ ID NO: 6) on GenBank, the nucleoprotein amino acid sequence of influenza B virus (B/Lee/1940) is selected for sequence comparison, and the homology of the section of sequence with other influenza A virus nucleoproteins is found to be high. Through analysis of immunogenicity, hydrophilicity and hydrophobicity and surface accessibility, 560 amino acids in the full length of the CDS region of nucleoprotein of influenza B (B) virus (B/Lee/1940) are finally screened as the sequence of subsequent recombinant protein, namely antigen (SEQ ID NO: 7).

The related information of epitope and/or functional localization of nucleoprotein of influenza B (B) virus is shown in table 1:

TABLE 1

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2. Preparation of recombinant influenza B (B) virus nucleoprotein

Through gene synthesis and molecular cloning design, expression is finally carried out in an E.coli system to obtain recombinant influenza B (B) virus nucleoprotein, namely the antigen. The corresponding expressed protein recombinant protein was identified by SDS-PAGE (FIG. 2).

3. Immunization of mice

1mg/mL of recombinant influenza A (B) virus nucleoprotein was mixed with Freund's

complete adjuvant

1, 500. Mu.L was emulsified, and then 6-8 week-old female Balb/c mice were injected subcutaneously at multiple points, and 100. Mu.g of recombinant influenza A (A) virus nucleoprotein (antigen) was inoculated per mouse. Three weeks later 1mg/mL antigen was mixed with Freund's

incomplete adjuvant

1 and 500. Mu.L emulsified and injected subcutaneously in multiple spots at an antigen dose of 50. Mu.g per mouse as a booster. Three, six and nine weeks apart, immunizations were performed again according to the procedure of the boosting immunization described above, and 4 boosting immunizations were performed in total.

4. Immune serum potency assay

And (5) collecting blood from the tail vein of the mouse 10 days after the fourth boosting immunization, and measuring the titer of immune serum by adopting an indirect ELISA method. 50 μ g of the synthetic antigen recombinant influenza A (A) virus nucleoprotein was dissolved in 10mL of 0.05M phosphate buffer pH9.6, and coated on a polystyrene 96-well plate at 100 μ L/well overnight at 4 ℃. The plate was washed three times with PBST (0.02M PBS containing 0.05% v/v Tween-20), 1% BSA blocking solution 100. Mu.l/well with 10mM PBS, blocked at 37 ℃ for 2h, and washed three times with PBST (0.02M PBS containing 0.05% v/v Tween-20), ready for use. Mouse immune serum was 10% in 10mM PBS containing 1% BSA ² ～10 ⁶ Double dilution, adding 96 hole plate, 100 u L/hole, then at 37 degrees C conditions were incubated for 1h, after PBST (0.02 MPBS contains 0.05 v/V Tween-20) plate washing three times, adding 1. Then, TMB was added to each well to develop 100. Mu.L/well, and then, 2M H was added thereto in an amount of 50. Mu.L/well in the dark at 37 ℃ for 10min ₂ SO ₄ The reaction was terminated. The 450nm absorbance was measured, and the serum from the preimmune mouse was used as a negative control, and the ratio of the measured value to the control value was not less than 2.1 as a positive judgment value to determine the titer of the immune serum (Table 2).

TABLE 2

Dilution factor	Immunization of mice 1	Immunization of mice 2	Immunization of mice 3	Immunized mouse 4
					10000	3.47	3.32	2.44	2.44
50000	2.83	2.92	1.53	1.62
					100000	2.11	2.10	0.89	0.99
500000	1.24	1.30	0.52	0.56
					1000000	0.12	0.12	0.10	0.12

5. Preparation of hybridomas

Serum titer greater than 10 ⁵ 3 days before fusion, the synthesized influenza B (B) virus nucleoprotein is taken and mixed with PBS with the same volume, and BALB/c is intraperitoneally injected into the mice to be fused according to the amount of 50 mu g/500 mu L for each to strengthen the immunity. Feeder layer cell preparation process: balb/c (about 4 weeks) mice were sacrificed by orbital vein exsanguination, soaked in 75% ethanol for 3 minutes with ventral side facing up; the thorax was opened to isolate the thymus, ground using a cell sieve, and the cells were suspended using a pre-warmed basal medium. Preparing abdominal cavity macrophages: on the day of fusion, 1 healthy mouse (one from 1 spleen) was selected, and its eyeball was removed to bleed blood until no blood was dropped. Dislocation of cervical vertebrae is lethal, soaking in 75% alcohol for 5min for disinfection, transferring into superclean bench, and fixing onto dissecting plate with abdomen upward. The skin of the abdomen of the mouse is lifted by forceps, a small opening (the peritoneum cannot be damaged so as to avoid the outflow of the abdominal cavity fluid) is cut by scissors, the peritoneum is fully exposed by blunt dissection, and the mouse is wiped by alcohol for disinfection. 5-10 mL of basic culture solution is absorbed by a disposable sterile syringe and injected into the abdominal cavity of the mouse, the right hand fixed syringe is kept still, and the left hand pinches an alcohol cotton ball with forceps and gently rubs the abdominal cavity of the mouse for 1-2 minutes to promote the migration of macrophages. The intraperitoneal medium was aspirated by a syringe and transferred to a 15mL centrifuge tube. After the abdominal cavity macrophages and the thymocytes are mixed evenly, the mixture is centrifuged for 10 minutes at 1200r/min, and the supernatant is discarded. The 20-cent FBS 1 XHAT medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, sigma)) was used in a pre-warmed resuspension and was kept at 37 ℃ until use.

Cell fusion in the morning of the day, the procedure for splenocyte preparation: taking a mouse 3-4 days after the boosting immunization, removing an eyeball to collect blood, and collecting separated serum as positive control serum for anti-detection. Meanwhile, mice were killed by cervical dislocation, soaked in 75% alcohol for 5 minutes for sterilization, and immediately placed in a clean bench. Fixing mouse on dissecting table, aseptically opening abdomen, lifting right abdomen skin, seeing spleen, changing ophthalmic scissors, cutting peritoneum with aseptic scissors, taking out spleen with forceps, washing spleen with normal saline, cutting spleen with scissors, placing in disposable cell sieve, gently squeezing spleen with syringe inner core, repeatedly washing cell sieve with normal saline until only connective tissue remains in cell sieveThe cell suspension was filtered again using a disposable cell screen after weaving. Spleen cell suspensions were harvested, centrifuged at 1200r/min for 10 minutes, and washed 1 time with 30-40mL of resuspended centrifugation (to remove red cell clumps as much as possible). Resuspending spleen cells in basal medium containing 10% FBS, placing in T75 cell flask at 37 deg.C, 5% CO ₂ Culturing in an incubator for 2-3 hours to make the macrophage in the cell suspension adhere to the wall.

The preparation process of myeloma cells in the afternoon of the day of fusion cells: 3 bottles of T75 myeloma cells were discarded, blown down with 50mL of pre-warmed saline, and centrifuged together with spleen cells at 1200r/min for 10 minutes. The spleen cells were mixed well with 3 bottles of T75 myeloma cells, the supernatant was discarded, resuspended in 40mL of pre-warmed saline, and centrifuged at 1200 rpm for 10 minutes.

And (3) cell fusion process: discarding the supernatant, discarding the supernatant as much as possible, and sucking up the residual liquid with a dropper to avoid affecting the concentration of the cell fusion agent and remove the erythrocytes as much as possible. Lightly flicking the bottom of the centrifugal tube with fingers to mix uniformly to loosen and mix the settled cells into paste. Fusion at room temperature: the cell fusion agent and the culture medium containing the feeder layer are placed in an incubator for heat preservation when spleen cells are prepared. The dispensed 1mL of cell fusogen solution was aspirated with a Pasteur pipette (added as close as possible to the cell centrifuge wall in a single revolution). Mixing gently within 60s-90 s. After timing is finished, 30mL of basic culture medium preheated to 37 ℃ is added at one time to dilute the cell fusion agent to lose the fusion promoting effect, and the mixture is kept stand for 5min at 37 ℃. Centrifuge at 800r/min for 6 minutes and discard the supernatant. The precipitated cells were gently aspirated by adding preheated 20% FBS 1 XHAT medium, suspended and mixed (well mixed, cell pellet reduction) gently. This experiment was performed by preparing 10 96-well cell culture plates, 200. Mu.L per well, requiring 200mL of 20% FBS 1 XHAT medium.

37℃、5％CO ₂ Culturing in an incubator, observing under a microscope 7 days after cell fusion, counting culture wells with obvious cell clones in a 96-well cell culture plate, and calculating the fusion rate, wherein the fusion rate is = (the number of fused cells/the total number of cells) × 100%.

6. Screening hybridoma cells secreting monoclonal antibody against influenza B (B) virus nucleoprotein

Screening cell culture supernatant by an indirect ELISA method, selecting positive clone hybridoma cells with higher titer for subcloning, and continuously cloning for 2-3 times by a limiting dilution method until the cell positive rate reaches 100%. The culture supernatant of 20 hybridoma cell strains with higher titer which are finally obtained is subjected to indirect ELISA detection, and meanwhile, 0.02M PBS is adopted for dilution detection, and the measured results are shown in Table 3:

TABLE 3

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As can be seen from Table 3, by comparing the data of ELISA method performed on the culture supernatants of hybridoma cell lines, cell lines which stably secrete anti-influenza B (B) virus nucleoprotein monoclonal antibody and have high antibody titer can be further selected and labeled as G4E1. And (5) performing amplification culture on the cells with the positive rate reaching 100% after cloning, and then freezing and storing the cells by liquid nitrogen.

7. Preparation and purification of ascites fluid

Hybridoma cell line G4E1 at 1 × 10 ⁶ Injecting liquid paraffin into abdominal cavity of female BALB/c mouse of 8-10 weeks old, feeding and observing for 10-14 days, and extracting ascites when the mouse abdominal cavity is enlarged. The monoclonal antibody is purified by adopting affinity chromatography Protein G Sepharose Fast Flow, and the purity of the monoclonal antibody is determined by SDS-PAGE and reaches more than 90 percent.

EXAMPLE 2 characterization of monoclonal antibodies

1. Determination of antibody concentration: the ascites fluid prepared from hybridoma G4E1 was purified to obtain a monoclonal antibody against influenza B (B) virus nucleoprotein, and the concentration thereof was >1mg/ml as measured using a Nanodrop nucleic acid protein analyzer manufactured by Thermofisher.

2. And (3) antibody subtype identification: the subtype of the hybridoma cell strain is identified by adopting a mouse monoclonal antibody subtype identification kit of Thermofish, the subtype of the G4E1 secretory antibody is IgG1 type, and the light chain is kappa chain.

3. Titer identification of purified antibody: 50 μ g of synthetic influenza B (B) virus nucleoprotein was dissolved in 10mL of 0.05M carbonate-coated buffer pH9.6, and added to 96-well plates at 4 ℃ overnight at 100 μ L per well. PBS (containing 0.05% v/v Tween-20) three times, with 10mM PBS 1% BSA blocking solution 150. Mu.L/well, 37 degrees C blocking 2H, using PBS (containing 0.05% v/v Tween-20) plate washing three times, each hole adding 100. Mu.L purified antibody, 37 degrees C incubation 1H, PBS (containing 0.05% v/v Tween-20) plate washing three times, adding horseradish peroxidase labeled goat anti-mouse IgG polyclonal antibody as a secondary antibody, 37 degrees C incubation 30min, PBS (containing 0.05% v/v Tween-20) plate washing three times, each hole adding 100. Mu.L, TMB color development, 37 degrees C incubation 15min, adding 2M H ₂ SO ₄ The solution stops the reaction, and the microplate reader detects the absorbance value at 450 nm.

4. Testing the binding force of the antibody:

influenza B virus nucleoprotein (B) was diluted to 0.5. Mu.g/mL and 1. Mu.g/mL with 1 XBB, added to wells of an ELISA plate at 100. Mu.L/well, and subjected to double-well and adsorbed at 4 ℃ overnight or 37 ℃ for 2 hours. Spin-drying the coated microporous plate, washing once according to the operation program (AFP program) set by a plate washer, adding confining liquid according to the amount of 200 muL/hole, placing in an incubator at 37 ℃ for 2h, and then placing at 4 ℃ overnight. Before use, the sealed microporous plate is taken out from 4 ℃, dried, and added with cleaning solution (1 XPBS-T) to moisten the enzyme label plate; the monoclonal antibodies of the invention were pre-diluted with 1 × PBS to 30 μ g/mL and the fold m of the pre-dilution was recorded, with 10-fold dilution, i.e. 3 μ g/mL, as (S1) peak concentration and then diluted with 1.

Adding 100 mu L of diluted antibody into a 96-hole micropore plate which is cleanly beaten on absorbent paper, and incubating for 30min at 37 ℃; after incubation, the ELISA plate is dried by spin-drying, the plate is dried on absorbent paper, the ELISA plate is washed for 3 times by a plate washing machine, and 100 mu L of 1 XPBS is added into each hole of 1-4 rows; adding 200 μ L urea treatment solution into each well of 5 rows and 6 rows, and incubating at 37 deg.C for 30min; spin-drying the ELISA plate after incubation, drying the ELISA plate on absorbent paper, washing the ELISA plate for 3 times by using a plate washing machine, adding 100 mu L of GAM-HRP enzyme-labeled secondary antibody which is diluted by 10000 times by using secondary antibody diluent in advance into each hole, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, drying the ELISA plate on absorbent paper, washing the ELISA plate for 3 times by using a plate washing machine, adding 100 mu L of color developing solution into each hole of TMB color developing solution, and incubating for 5-10min at 37 ℃; after development, 50. Mu.L of stop buffer was added to each well. The 450nm/630nm reading was set on a microplate reader. The reading at 450nm/630nm was set on a microplate reader.

ELISA antigen-antibody binding force experiments are tested under the conditions of 0.5 mu g/mL and 1 mu g/mL antigen coating respectively, data (figure 6) is obtained by testing light absorption value OD, corresponding polynomial curves are obtained by fitting, the corresponding binding can see obvious gradient along with the change of antibody dilution multiple ratio, meanwhile, the two curves reflect the affinity binding of the antibody under the conditions of 0.5 mu g/mL and 1 mu g/mL antigen coating, the binding specificity is embodied, and the concentration corresponding to the antibody binding force respectively reaches 8.59E-09mol/L (under the condition of 0.5 mu g/mL antigen coating, the highest OD reading is 50 percent, namely when the concentration of an antigen-antibody compound accounts for half of the total concentration, the concentration K corresponding to the antibody _0.5 ) And 8.24E-09mol/L (50% of the highest OD reading under 1. Mu.g/mL antigen coating, i.e., the concentration of antigen-antibody complexes is half of the total concentration, the concentration value K corresponding to the antibody ₁ )。

And (4) calculating the binding force, namely calculating the OD reading values of the complex hole average values of the concentrations of the S1-S8 antibodies before and after urea treatment under the antigen coating conditions of 0.5 mu g/mL and 1 mu g/mL respectively. Substituting the reading value into a binding force calculation formula:

5. and (3) specific detection: similar to ELISA antibody titer detection, the detection using orthogonally designed nucleoproteins recombined from various influenza viruses showed that the antibodies of the present invention have good specificity (tables 4A-4I).

TABLE 4A

TABLE 4B

TABLE 4C

TABLE 4D

TABLE 4E

TABLE 4F

TABLE 4G

TABLE 4H

TABLE 4I

It can be seen from tables 4A to 4I that, by comparing the experimental data corresponding to the antibody pairing experiments, it is shown that, except that the detection holes corresponding to the enzyme-labeled antibody as the coated antibody are shielded, other enzyme-labeled antibodies can still bind to the influenza B virus nucleoprotein antigen in the presence of the coated antibody, and the binding effect increases with the increase of the concentration of the added antigen, so that the specificity of the antibody G4E1 binding is embodied, and the optimal sensitivity to the influenza B virus antigen is exhibited when the antibody is matched with the E4H7 antibody to form an immune sandwich reaction.

6. Sequencing of monoclonal antibodies

The monoclonal antibody of anti-influenza B (B) virus nucleoprotein prepared from the purified hybridoma cell G4E1 is taken, and the sequences of a light chain variable region and a heavy chain variable region of the monoclonal antibody of the influenza virus NP protein are known, wherein the sequences of the light chain variable region of the monoclonal antibody of the influenza virus NP protein are CDR-L1 (RSSEPISKYLA), CDR-L2 (SASTLQS) and CDR-L3 (QQHNRYPWT), and the sequences of the heavy chain variable region of the monoclonal antibody of the influenza virus NP protein are CDR-H1 (SVWMH), CDR-H2 (YISSATIYYAVDG) and CDR-H3 (SGNAMKY). The antibody with the corresponding variable region sequence has high affinity and good specificity; the corresponding antibody variable regions can therefore be used for the development of recombinant antibodies, single chain antibodies, bispecific antibodies, for the development of related products for diagnostic or therapeutic use.

Example 3 development of lateral flow immunochromatographic assay reagents Using influenza virus N protein monoclonal antibodies

The antibody can be used for the development of immunodiagnostic reagents such as enzyme-linked immunosorbent assay, chemiluminescence assay, lateral flow immunochromatography assay, immunofluorescence assay and the like.

1. Preparing colloidal gold:

the gold is prepared by a reduction method under the condition that 2% chloroauric acid is 5mL +10mL and 1% trisodium citrate is prepared.

2. The colloidal gold is used for marking the recombinant nucleoprotein monoclonal antibody of the mouse anti-B (B) type influenza virus. The pH value is adjusted by adopting a physical adsorption method to combine the colloidal gold and the antibody (targeted influenza B virus nucleoprotein antibody E4H7, and the amino acid sequences of the light chain and the heavy chain are respectively shown as SEQ ID NO:3 and 4). The specific marking conditions are as follows: under the condition of pH7.5, 10 mu L of 0.1M potassium carbonate solution is added into each milliliter of colloidal gold, and the labeling concentration of the mouse anti-influenza virus N protein monoclonal antibody is 10 mu g/mL.

3. Detection line and quality control line

And (3) detection line: a detection line is prepared by coating a mouse anti-B (B) type influenza virus recombinant nucleoprotein monoclonal antibody G4E1 with a proper concentration (1.5 mg/mL) on a nitrocellulose membrane. Drying at 37 ℃. The spot size was 0.1. Mu.L/mm.

Quality control line: a goat anti-mouse IgG polyclonal antibody of 0.5mg/mL is taken, a quality control line is prepared on a fiber membrane, and the obtained product is dried at 37 ℃. The spot size was 0.1. Mu.L/mm.

4. Inspection method

The test card (test strip), sample diluent and sample are returned to 18-30 ℃. The detection method of the detection card or the detection strip comprises the following steps:

a. taking out the detection card or the detection strip from the aluminum foil bag, marking a sample, and horizontally placing the sample on a horizontal working table;

b. taking 20 mu L of nasopharyngeal or oropharyngeal swab sample extract, directly adding the extract into a sample adding hole (detection card) or a sample adding part (detection strip) at the lower end of an indication arrow;

c. then 100 mu L (2-3 drops) of sample diluent is added;

and d.15-20 minutes, judging the result, and after 20 minutes, the test result is invalid.

5. Interpretation of test results

a. Positive detection line: and the detection line and the quality control line are developed. The sample is suggested to detect the recombinant nucleoprotein antigen of the influenza B (B) virus, which may be in early infection or emergent infection, and needs to be finally confirmed by combining clinical symptoms.

b. Negative: only one red quality control line appears in the detection window. This indicates that no recombinant nucleoprotein antigen of influenza B (B) virus was detected in the sample.

c. And (4) invalidation: the detection window has no red quality control line.

Example 4 testing of influenza Virus antigen lateral flow immunochromatographic reagent Performance

1. Sensitivity testing

a. Inactivated influenza virus culture assay

Detection of inactivated influenza virus cultures using lateral flow immunochromatographic rapid test reagents developed using the antibodies of the invention, with minimum testability down to 500TCID ₅₀ Viral culture/mL.

TCID ₅₀ the/mL is the amount of virus required to cause half of the cytopathic effect or death (CPE) in the wells or tubes of the plate, and is used to characterize the virus titer. It should be noted that the amount of virus here is not a specific concentration, but a multiple of the dilution of the original sample. For example, 1mL of culture medium diluted 1000-fold results in exactly 50% cell infection, TCID ₅₀ The concentration was 1000/mL. Indicating the fold of dilution required for 50% of the cells infected with the virus contained in each mL of sample.

Thus, TCID ₅₀ The larger the value, the more the virus copy number of the original inactivated supernatant, the larger the dilution factor (to achieve the effect of infecting 50% of cells), and then the corresponding original inactivated supernatant is used to detect the reagent kit test paper respectively, and the corresponding gradient result is shown in fig. 3. 10TCID ₅₀ mL represents the sample with the lowest virus copy number in the stock solution. I.e., lowest testable to as low as 10TCID ₅₀ Viral culture in/mL.

b. Purified influenza B virus nucleoprotein assay

The lateral flow immunochromatographic rapid detection reagent developed by the antibody is detected by adopting Hangzhou China sunflower gold and a B (B) type influenza virus nucleoprotein solution standard substance produced by biotechnology limited. As shown in FIG. 4, the standard recombinant nucleoprotein of influenza B (B) virus was detected at a minimum of 5000 pg/mL.

2. Specificity test (clinical sample test)

The nasal swab samples of 20 normal persons were tested using the lateral flow immunochromatographic rapid detection reagent developed using antibodies, and the test results are shown in fig. 5. As can be seen from the figure, the detection result is clear and visible, the background is clean, and the product specificity is good.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims

1. The antibody targeting influenza B virus nucleoprotein is characterized in that the amino acid sequences of the hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the light chain of the antibody are RSSEPISKYLA, SASTLQS and QQHNRYPWT respectively, and the amino acid sequences of the hypervariable regions CDR-H1, CDR-H2 and CDR-H3 of the heavy chain of the antibody are SVWMH, YISSATIYYAETVDG and WLSGNAMKY respectively.

2. The antibody of claim 1, wherein the amino acid sequences of the light chain and the heavy chain of the antibody are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively.

3. Use of the antibody of claim 1 or 2 in the preparation of an influenza b virus detection reagent or kit.

4. An influenza b virus detection reagent or kit prepared from the antibody of claim 1 or 2.

5. Use of the antibody of claim 1 or 2 in the preparation of an influenza b virus colloidal gold immunochromatographic assay test strip or kit.

6. Use of the antibody of claim 1 or 2 for the preparation of a lateral flow immunochromatographic diagnostic kit for influenza b virus.

7. The influenza B virus colloidal gold immunochromatographic assay test strip is characterized by comprising a sample pad, a colloidal gold combination pad, an NC membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the colloidal gold combination pad, the NC membrane and the water absorption pad are sequentially adhered to the bottom plate;

wherein, the colloidal gold conjugate pad is coated with a colloidal gold labeled antibody I targeting influenza B virus nucleoprotein; the detection line is coated with the antibody of claim 1 or 2, and the quality control line is coated with goat anti-mouse polyclonal antibody;

wherein, the amino acid sequences of the light chain and the heavy chain of the targeted influenza B virus nucleoprotein antibody are respectively shown in SEQ ID NO. 3 and SEQ ID NO. 4.

8. The test strip of claim 7, wherein the concentration of the coated antibody I on the colloidal gold conjugate pad is 10-15 μ g/mL;

the concentration of the antibody coated on the detection line is 1.5-2mg/mL.

9. The test strip of claim 7 or 8, wherein the sample pad and the absorbent pad are made of cellulose, and the gold colloidal conjugate pad is made of glass fiber.

10. Nucleic acid encoding the antibody of claim 1 or 2.