CN115947835B

CN115947835B - Antibody targeting influenza B virus nucleoprotein and application thereof

Info

Publication number: CN115947835B
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: 2023-07-28
Anticipated expiration: 2042-11-15
Also published as: CN115947835A

Abstract

The invention provides an antibody targeting influenza 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 immunodiagnosis field.

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 virus nucleoprotein antibody and application thereof.

Background

A total of 7 virus members of the Orthomyxoviridae family (Orthomyxoviridae), 4 of which are Influenza A, B, C, D viruses, respectively. Influenza virus samples are generally oval or spherical (about 100nm in diameter). Influenza viruses are in some cases in the form of filaments (about 20 μm long axis), and virus samples isolated clinically are often in the form of filaments.

Among them, influenza viruses are named according to the host, the place of isolation, the number of the isolate, and the year of isolation. Unlike influenza a (a), influenza B (B) viruses are classified mainly based on the similarity of various traits found and between strains, for example, one of which is represented by B/Yamagata/16/88, which is called a "B/Yamagata-like" strain (B/Yamagata linear-like virus), influenza B (B) viruses generally cause only localized influenza outbreaks, and influenza B (B) viruses are mainly human-infected but cannot infect a variety of hosts.

Influenza virus is an enveloped virus whose genome comprises segmented (segment 1-segment 8) single-stranded negative-sense viral ribonucleic acid (viral ribonucleic acid, vRNA). The major proteins encoded by the viral genome are: polymerase protein subunits (RNA polymerase subunit 1,2, A-X and 1-F2; PB1, PB2, PA, PA-X and PB 1-F2), nucleoprotein (NP), 2 surface glycoproteins (hemagglutinin HA and neuraminidase NA), 2 matrix proteins (M1 and M2; wherein M2 is also a transmembrane ion channel protein), nonstructural proteins (non-structural protein 1, NS1) and nuclear export proteins (nuclear export protein, NEP).

The life cycle of influenza virus is divided into the following steps: virus adsorption and entry into host cells, virus uncoating, viral ribonucleoprotein (viral ribonucleoprotein, vRNP) entry into the nucleus, viral genome transcription and replication in the nucleus, viral ribonucleoprotein (vRNP) exit, viral assembly and viral budding release.

The envelope of influenza virus is a bilayer lipid membrane obtained from the host cell membrane when the progeny virus buds, and the envelope is provided with a layer of most abundant viral matrix protein (M1) surrounding the viral genome: each viral ribonucleic acid (vRNA) exists in the form of a viral ribonucleoprotein (vRNP) and can express one or more viral proteins; each viral ribonucleoprotein (vRNP) consists of three parts, an oligomeric Nucleoprotein (NP) and a viral ribonucleic acid (vRNA) and a heterotrimeric viral RNA polymerase (RNA polymerase), respectively.

In addition, three viral proteins are embedded on the viral envelope: hemagglutinin (HA), neuraminidase (NA) and transmembrane channel protein (M2). Among them, 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 releasing budding progeny virions out of the host cell; the ion channel protein (M2) is responsible for acidification of the virus as it enters the host cell and plays an important role in budding release of the progeny virus.

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

Nucleoprotein (NP) is crescent-shaped, contains 560 amino acids in total, consists of a head, an intermediate domain and a long tail loop of a specific number of amino acids, is rich in arginine, glycine, serine and a small number of cysteine residues, and has a net positive charge in a neutral pH environment. Nucleoprotein (NP) is a monomeric polypeptide, in both phosphorylated and non-phosphorylated forms, whose conformational changes are regulated by host cell phosphokinase and phosphatase to phosphorylate and dephosphorylate the Nucleoprotein (NP) and in turn affect Nucleoprotein (NP) oligomerization, as well as interactions with other macromolecules.

The Nucleoprotein (NP) can interact with a variety of other macromolecules, such as viral RNA and heterotrimeric viral RNA polymerase (comprising subunits PB1, PB2 and PA) to form viral ribonucleoprotein (vRNP). The smallest functional units of the common viral ribonucleoprotein (vRNP) include a viral RNA polymerase complex that binds viral RNA and 9 Nucleoprotein (NP) monomers arranged in a rod-like structure.

Viral ribonucleoprotein (vRNP) is too largeCannot pass the nucleus of the host cell +.>And diffusing the nuclear pores. The nuclear import protein α (importin- α) of a host cell binds to the Nucleoprotein (NP) of influenza virus through its nuclear localization sequence. During the viral replication cycle, viral ribonucleoprotein (vRNP) is actively transported into and out of the nucleus of the host cell.

Thus, during the proliferation of the virus in the host cell, whether or not the Nucleoprotein (NP) is phosphorylated within the host cell, and the corresponding conformational changes, are critical factors affecting the host response profile of the influenza virus.

Phylogenetic analysis is carried out on influenza virus strains separated from different hosts, and the amino acid sequence translated by the Nucleoprotein (NP) gene is found to have better conservation. The amino acid sequences of the Nucleoprotein (NP) of influenza A (A), B (B) and C (C) viruses are compared, and obvious similarity exists between the amino acid sequences of the Nucleoprotein (NP) of the three viruses, wherein the conservation degree of each Nucleoprotein (NP) of the influenza A (A) and B (B) viruses is the highest. Nucleoprotein (NP) has at least 3 independent epitopes, with the amino acid sequence of one epitope exhibiting high conservation among multiple influenza strains.

The Nucleoprotein (NP) of influenza virus is capable of inducing the host to produce non-neutralizing antibodies under natural conditions, and monoclonal antibodies directed against highly conserved epitopes of Nucleoprotein (NP) can inhibit transcription of viral RNA in the host cell nucleus. At the same time, the body produces a cytotoxic lymphocyte response, which acts to kill the host cell-infected influenza virus by recognizing viral antigens (e.g., nucleoprotein, NP) presented by major histocompatibility complex (major histocompatibility complex, MHC) molecules on the surface of the infected cell.

Compared with other proteins on the surface of the viral envelope, such as Hemagglutinin (HA) and Neuraminidase (NA), the amino acid sequence of Nucleoprotein (NP) is relatively more conserved, HAs stable immunogenicity and is not affected by antigen drift (antigenic drift), is a main specific target for identifying influenza A (A), B (B) and C (C) viruses respectively, and HAs strong differentiation when being used as a diagnostic target antigen.

The Nucleoprotein (NP) of influenza virus is typically the most abundant viral protein expressed in the infected host cell 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, development of monoclonal antibodies targeting influenza virus Nucleoprotein (NP) antigen is of great importance for diagnosis of different influenza virus subtypes and discovery of antibody drugs with strong therapeutic effects against specific subtypes of influenza virus.

Colloidal gold is one of the first labeling materials used in immunochromatography assay kits. The absorption wavelength is in the visible light region, so that the visible light is easy to directly observe by naked eyes, and the color is red, blue or purple along with the change of the size and the shape of the colloidal gold particles. The colloidal gold raw material is usually prepared by simply and reliably reducing chloroauric acid by trisodium citrate. The colloidal gold prepared by the method carries a considerable amount of negative charges on the surface, so that the property of the colloidal gold is very stable in a dry state even in a solution. In addition, the large amount of negative charge surface aggregation enables colloidal gold to perform mild coupling combination with biological macromolecules such as nucleotide, polypeptide or protein which are positively charged under specific pH and other conditions in a simple manner of electrostatic adsorption, and the coupling combination can keep the activity of the biological macromolecules to the maximum extent. The advantages make the colloidal gold become a marking material in most commercial immunochromatography test strips in the market at present.

Although colloidal gold is widely used in immunochromatography, there are many defects such as poor accuracy of detection results 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, such as preparing colloidal gold particles with spike-shaped geometric structures, or modifying carboxyl-terminal polyethylene glycol (PEG) molecules on the surface of the colloidal gold, and combining labeled antibodies in a covalent manner, so that the detection sensitivity or the anti-interference capability of the colloidal gold test strip is improved. The transformation of the traditional colloidal gold further expands the application of the colloidal gold in the lateral flow immunochromatography test strip.

The lateral flow immunochromatography rapid diagnosis kit is a test paper-based result reporting method suitable for instant detection. The core element is a lateral flow immunochromatographic test strip (figures 1, a and b), which is generally in a narrow strip shape, and the width is generally 4-6 mm, and the length is not more than 6-7 cm. A standard lateral flow immunochromatographic test strip should comprise four main parts, 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 detected when in use;

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

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

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

When the lateral chromatographic strip is inserted into the solution to be tested, the solution flows 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 bind to the colloidal gold (conjugated) antigen or the colloidal gold (conjugated) antibody of the binding pad region. The colloidal gold (conjugate) antibody or the colloidal gold (conjugate) antigen rehydrated by the solution or the complex formed by the colloidal gold (conjugate) antigen and the sample to be tested sequentially passes through the detection area (NC membrane) and the water absorption pad area under the action of capillary force. At this time, the lateral chromatography test paper will have two results display models, respectively a standard model (fig. 1 a) and a competition model (fig. 1 b).

FIG. 1a when a standard model (for detecting nucleoprotein antigens of influenza virus) is used for lateral flow immunochromatographic strips, the detection line (comprising specific antibodies to nucleoprotein antigens of influenza virus) can only capture complexes of the antigen to be detected and colloidal gold (conjugate) primary antibodies: if the antigen to be detected exists in the solution, the detection line can capture a complex formed by the antigen to be detected and the colloidal gold (coupled) primary antibody, and the colloidal gold (coupled) primary antibody is always captured by the quality control line (comprising the corresponding secondary antibody), so that the detection line and the quality control line have color changes at the moment, and the result is judged to be positive reaction. If the antigen to be detected does not exist in the solution, only the quality control line (comprising the corresponding secondary antibody) can capture the colloidal gold (coupled) primary antibody and generate color change, and at the moment, the quality control line develops color while the detection line does not develop color, and the result is judged to be negative reaction.

FIG. 1b shows that when a competition model (IgM/IgG antibodies for detecting nucleoprotein [ NP ] antigen of the targeted influenza virus) is adopted by the lateral chromatographic test paper, 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 can be captured by a detection line (antibody containing anti-IgG/IgM): when the antibody to be detected exists, the antibody can be captured by the detection line, the colloidal gold (coupling) antigen can not be captured by the detection line (because of competitive closure of the antibody to be detected), and further the color change can not occur, but the quality control line (comprising the nucleoprotein antigen pairing antibody) can still capture the colloidal gold (coupling) antigen, at the moment, the detection line does not develop color, the quality control line develops color, and the result is judged to be positive reaction; if the antibody to be detected does not exist in the solution, the detection line and the quality control line can both capture colloidal gold (coupled) antigen, at the moment, the detection line and the quality control line can both generate color change, and the result is judged to be negative reaction.

However, there are also some disadvantages to the lateral flow immunochromatographic rapid diagnostic kit: firstly, the detection means can only provide qualitative results, and has weak applicability to pathogens (specific bacteria, mycoplasma, viruses and the like) needing to diagnose the infection according to the fine quantitative results; second, the detection means requires that the test sample must be in a liquid state and that there be a certain viscosity to flow through the porous nitrocellulose membrane.

Nevertheless, the lateral flow immunochromatography rapid diagnosis kit is still a detection technology with important application value, and can realize the general 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 significance, especially for units or areas lacking specialized equipment and not well-trained technicians.

Disclosure of Invention

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

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 the light chain hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody (mab G4E 1) are RSSEPISKYLA, SASTLQS and QQHNRYPWT, respectively, and the amino acid sequences of the heavy chain hypervariable regions CDR-H1, CDR-H2 and CDR-H3 of the antibody are SVWMH, YISSASITIYYAETVDG 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 for coding the light chain and the heavy chain of the antibody are respectively shown as SEQ ID NO. 8 and SEQ ID NO. 9.

In a second aspect, the invention provides the use of said antibody in the preparation of an influenza B virus (nucleoprotein) detection reagent or kit.

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 application of the antibody in preparing a colloidal gold immunochromatography detection test strip or a kit for influenza B virus.

In a fifth aspect, the invention provides the use of said antibody in the preparation of a lateral flow immunochromatographic diagnostic kit for influenza B virus.

In a sixth aspect, the invention provides a colloidal gold immunochromatography detection test strip for influenza virus type B (B), which comprises a sample pad, a colloidal gold binding pad, an NC membrane, a water absorption pad and a bottom plate, wherein a detection line and a quality control line are arranged on the reaction membrane, and the sample pad, the colloidal gold binding pad, the NC membrane and the water absorption pad are sequentially adhered to the bottom plate.

Wherein, the colloidal gold binding pad is coated with a colloidal gold-labeled antibody I targeting influenza 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 binding pad is 10-15 mug/mL.

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

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

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

the invention provides an antibody for targeting and recognizing influenza B virus nucleoprotein (FluB N), which has high specificity and high affinity property on the influenza B virus nucleoprotein, and can be used for designing a therapeutic target of the influenza B virus, wherein the therapeutic target design comprises but is not limited to target design of therapeutic antibodies.

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

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

The colloidal gold-based lateral flow immunochromatography rapid diagnosis kit has the advantages of low detection limit and high sensitivity; and the selected antibody is specifically targeted to an epitope with a highly conserved amino acid sequence, is not influenced by common antigen protein mutation of a virus mutant strain, reduces detection cost, shortens detection time and improves detection efficiency. The lateral flow immunochromatography rapid diagnosis kit is a detection technology with important application value, and can realize the general standards of sensitivity, stability, economy, user friendliness, no need of 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 areas lacking professional equipment and lacking well-trained technicians.

Drawings

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

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

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

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

FIG. 5 shows the results of testing a nasal swab sample from a normal person using the lateral flow immunochromatographic rapid test reagent developed with the antibody of the present invention in a preferred embodiment of the present invention.

FIG. 6 is a graph showing the binding of antibodies to recombinant influenza B virus nucleoprotein antigen at gradient concentrations, respectively, in accordance with a preferred embodiment of the invention. Wherein 0.5. Mu.g (/ mL) is the binding curve of 50ng (100. Mu.L per well) of the coated antigen per well, and 1. Mu.g (/ mL) is the binding curve of 100ng (100. Mu.L per well) of the coated antigen per well.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all 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 translating the influenza B virus nucleoprotein gene (SEQ ID NO:5; the optimized gene sequence is shown as SEQ ID NO: 6) on GenBank, the nucleoprotein amino acid sequence of the influenza B virus (B/Lee/1940) is selected for sequence comparison, and the sequence has very high homology with other influenza A virus nucleoproteins. Through immunogenicity, hydrophilicity and hydrophobicity and surface accessibility analysis, the full length 560 amino acids of the nucleoprotein CDS region of the influenza B virus (B/Lee/1940) are finally screened as the subsequent recombinant protein, namely the sequence of the antigen (SEQ ID NO: 7).

Information about the epitope and/or functional localization of influenza B virus nucleoprotein is shown in table 1:

TABLE 1

2. Preparation of recombinant influenza B virus nucleoprotein

Through gene synthesis and molecular cloning design, the recombinant influenza B virus nucleoprotein, i.e. antigen, is finally obtained by expression in an E.coli system. Recombinant proteins corresponding to the expressed proteins were identified by SDS-PAGE (FIG. 2).

3. Immunized mice

After mixing 1mg/mL of recombinant influenza B virus nucleoprotein with Freund's complete adjuvant uniformly at a ratio of 1:1 and emulsifying at 500. Mu.L, 6-8 week old female Balb/c mice were subcutaneously multi-injected, each with 100. Mu.g of recombinant influenza A virus nucleoprotein (antigen). After three weeks, 1mg/mL antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified and injected subcutaneously at multiple points at an antigen dose of 50. Mu.g per mouse, which was used as booster. Three weeks, six weeks, and nine weeks apart were followed by a second immunization following the procedure of boosting previously described, for a total of 4 boosting.

4. Immune serum potency assay

The mice were bled from the tail vein 10 days after the fourth booster immunization and the immune serum titers were determined by indirect ELISA. 50 μg of synthetic antigen recombinant influenza A virus nucleoprotein (A) was dissolved in 10mL of 0.05M phosphate buffer, pH9.6, coated in polystyrene 96-well plates, 100 μl/well, overnight at 4deg.C. Plates were washed three times with PBST (0.02M PBS containing 0.05% v/v Tween-20) with 10mM PBS contained 100. Mu.l/well of 1% BSA blocking solution, blocked for 2h at 37℃and plates were washed three times with PBST (0.02M PBS containing 0.05% v/v Tween-20) and prepared for use. Murine immune serum was subjected to 10 with 10mM PBS containing 1% BSA ² ～10 ⁶ Multiple dilutions, 96-well plates, 100 μl/well, followed by incubation at 37 ℃ for 1h, after three plate washes with PBST (0.02 MPBS containing 0.05% v/v Tween-20), 1:10000-fold dilutions of horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, inc.) were added, 100 μl/well, followed by incubation at 37 ℃ for 30min, and plate washes were performed. TMB was then added to each well to develop 100. Mu.L/well, and the wells were protected from light at 37℃for 10min at room temperature, after which 2M H was added in an amount of 50. Mu.L/well ₂ SO ₄ The reaction was terminated. The absorbance at 450nm was measured, and the immune serum titer was determined by using the serum of mice before immunization as a negative control and the ratio of the measured value to the control value of 2.1 or more as a positive judgment value (Table 2).

TABLE 2

Dilution factor	Immunized mouse 1	Immunized mouse 2	Immunized mouse 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 titers of greater than 1:10 were taken ⁵ 3 days before fusion, and then, uniformly mixing the synthesized antigen B (B) type influenza virus nucleoprotein with an equal volume of PBS, and performing intraperitoneal injection of BALB/c to-be-fused mice at the amount of 50 mug/500 mug for enhancing immunity. The preparation flow of the feeder layer cells comprises the following steps: balb/c (about 4 weeks) mice were euthanized by orbital venous exsanguination, 75% ethanol soaked for 3 min with ventral side facing up; the chest was opened to isolate thymus, ground using a cell sieve, and cells were resuspended using a pre-warmed basal medium. Preparation of celiac macrophages: on the day of fusion, 1 healthy mouse (one from 1 spleen) was selected, and the eyeballs were collected for exsanguination until no blood drop was produced. Killing cervical vertebra dislocation, soaking in 75% alcohol for 5min, sterilizing, transferring to ultra-clean workbench, and fixing abdomen upwardsOn an anatomic plate. The skin of the mouse abdomen was lifted with forceps, a small opening was cut with scissors (note that the peritoneum was not damaged, so as not to flow out of the peritoneal fluid), the peritoneum was fully exposed by blunt dissection, and the skin was sterilized by wiping with alcohol. 5-10 mL of basic culture solution is sucked by a disposable sterile syringe and injected into the abdominal cavity of the mouse, the fixed syringe is kept still by the right hand, and the abdomen of the mouse is gently rubbed by the left hand by clamping the alcohol cotton ball with forceps for 1-2 minutes, so that macrophages are promoted to swim out. The culture medium in the abdominal cavity was aspirated by syringe and transferred to a 15mL centrifuge tube. After the peritoneal macrophages and the thymus cells were mixed uniformly, the mixture was centrifuged at 1200r/min for 10 minutes, and the supernatant was discarded. The suspension was resuspended in a medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, sigma)) containing 20% FBS 1 XHAT, which was pre-warmed, and kept at 37 ℃.

Cell fusion the preparation procedure of splenocytes at morning: mice 3-4 days after having been boosted were taken, the eyeballs were removed to collect blood, and the isolated serum was collected as a positive control serum at the time of anti-detection. Meanwhile, the mice are killed by cervical dislocation, soaked in 75% alcohol for 5 minutes for sterilization, and immediately placed in an ultra clean bench. Fixing a mouse on a dissecting platen, aseptically opening the abdomen, then lifting the skin on the right side, allowing the spleen to be seen, changing the eye scissors, cutting the peritoneum by using an aseptic operation scissors, taking out the spleen by using forceps, flushing the spleen with normal saline, cutting the spleen into pieces by using scissors, placing the pieces in a disposable cell sieve, lightly squeezing the spleen by using a syringe inner core, repeatedly flushing the cell sieve with normal saline until only connective tissue remains in the cell sieve, and then filtering the cell suspension by using the disposable cell sieve again. The spleen cell suspension was harvested, centrifuged at 1200r/min for 10min and washed 1 time (as much as possible to remove the red blood cell mass) with 30-40mL of heavy suspension centrifugation. The spleen cells are resuspended in basic culture medium containing 10% FBS, and placed in T75 cell bottle at 37deg.C and 5% CO ₂ Culturing in an incubator for 2-3 hours to adhere macrophages in the cell suspension.

Preparation procedure of confluent cells at the afternoon, myeloma cells: 3 bottles of T75 myeloma cells were discarded, and the supernatant was blown down with 50mL of pre-warmed physiological saline, centrifuged together with spleen cells, and centrifuged at 1200r/min for 10 minutes. Spleen cells were thoroughly mixed with 3 bottles of T75 myeloma cells, the supernatant was discarded, resuspended in 40mL of pre-warmed saline, and centrifuged at 1200r/min for 10 min.

Cell fusion process: the supernatant was discarded, the supernatant was discarded as much as possible, and the residual liquid was pipetted to remove the erythrocytes as much as possible without affecting the concentration of the cell fusion agent. The bottom of the centrifugal tube is lightly flicked by a finger to mix evenly, so that the precipitated cells are loose and uniform into paste. Fusion at room temperature: the cell fusion agent and the culture medium containing the feeder layer are placed into an incubator for heat preservation when preparing spleen cells. The packed 1mL of the cell fusion agent solution (added as close to the wall of the centrifuge tube at the cell site as possible in one revolution) was aspirated with a Pasteur pipette. Gently mix for 60s-90 s. After timing, 30mL of basal medium preheated to 37 ℃ is added at a time to dilute the cell fusion agent to lose the fusion promoting effect, and the cell fusion agent is kept stand at 37 ℃ for 5min. Centrifuge at 800r/min for 6 min, discard supernatant. The precipitated cells were gently aspirated by adding pre-warmed 20% FBS 1 xhlet medium, suspended and mixed well (thoroughly mixed to reduce cell mass) with gentle action. This experiment was followed by preparation of 10 96-well cell culture plates, 200. Mu.L per well, requiring 200mL of 20% FBS 1 XHAT medium.

37℃、5％CO ₂ Incubator culture, observation under a microscope 7 days after cell fusion, counting culture wells in 96-well cell culture plates in which obvious cell clones appear, and calculating the fusion rate, fusion rate= (number of fused cells/total number of cells) ×100%.

6. Screening hybridoma cells secreting anti-influenza B virus nucleoprotein monoclonal antibody

The indirect ELISA method screens cell culture supernatant, selects positive clone hybridoma cells with higher titer for subcloning, and uses a limiting dilution method to clone continuously for 2-3 times until the cell positive rate reaches 100%. The culture supernatants of the 20 hybridoma cell lines with higher cost of efficiency obtained finally are detected by an indirect ELISA method, and meanwhile, are subjected to dilution detection by using 0.02M PBS, and the detection results are shown in Table 3:

TABLE 3 Table 3

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

7. Preparation and purification of ascites

Hybridoma cell line G4E1 was grown at 1X 10 ⁶ The abdominal cavity of 8-10 week old BALB/c female mice pretreated with liquid paraffin is injected into the amount of the liquid paraffin, and the ascites is extracted when the abdomen of the mice expands after 10-14 days of feeding observation. Purifying the monoclonal antibody by adopting an affinity chromatography Protein G Sepharose Fast Flow, and determining the purity of the monoclonal antibody by SDS-PAGE, wherein the purity reaches more than 90%.

EXAMPLE 2 characterization of monoclonal antibodies

1. Determination of antibody concentration: the ascites prepared by hybridoma cell G4E1 is purified to obtain a monoclonal antibody against influenza B virus nucleoprotein, and the monoclonal antibody is measured by using a Nanodrop nucleic acid protein measuring instrument manufactured by Thermofisher company, and the concentration of the monoclonal antibody is more than 1mg/ml.

2. Identification of antibody subtypes: subtype of hybridoma cell strain is identified by using a mouse monoclonal antibody subtype identification kit of Thermofisher, subtype of G4E1 secretion antibody is IgG1 type, and light chain is kappa chain.

3. Identification of titers of purified antibodies: 50 μg of synthetic influenza B virus nucleoprotein was dissolved in 10mL of 0.05M carbonate coating buffer pH9.6, and 96-well plates were added at 100 μl per well overnight at 4deg.C. PBS (containing 0.05% v/v Tween-20) plates were washed three times, 1% BSA blocking solution in 10mM PBS was used for 150. Mu.L/well, blocking was performed at 37℃for 2h, PBS (containing 0.05% v/v Tween-20) was used for washing plates three times, 100. Mu.L of purified antibody was added to each well, incubated at 37℃for 1h, PBS (containing 0.05% v/v Tween-20) was used for washing plates three times, horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was added as secondary antibody, incubated at 37℃for 30min, PBS (containing 0.05% v/v Tween-20) was used for washing plates three times, and each well was washed with PBSAdding 100 μl, developing TMB, incubating at 37deg.C for 15min, adding 2M H ₂ SO ₄ The reaction was stopped and the microplate reader was tested at an absorbance of 450 nm.

4. Antibody binding force test:

the influenza B virus nucleoprotein was diluted to 0.5. Mu.g/mL and 1. Mu.g/mL with 1 XCB, added to the wells of the ELISA plate at 100. Mu.L/well, and multiplexed, and adsorbed at 4℃overnight or 37℃for 2 hours. The coated microplates were spin-dried, washed once according to the procedure set by the plate washer (AFP procedure), and blocking solution was added in an amount of 200 μl/well, placed in a 37 ℃ incubator for 2 hours, and then placed at 4 ℃ overnight. Before use, taking out the closed microplate from the temperature of 4 ℃, spin-drying, and adding a cleaning solution (1 XPBS-T) to moisten the enzyme-labeled plate; the monoclonal antibodies of the invention were pre-diluted to 30. Mu.g/mL with 1 XPBS and the fold m of pre-dilution was recorded, at 10-fold dilution, i.e.3. Mu.g/mL as (S1) maximum concentration and then diluted with a 1:3 gradient (dilution in 96 deep well plates) for a total of 8 dilution gradients (S1-S8).

Adding 100 mu L of diluted antibody into a 96-well microplate which is cleanly beaten on absorbent paper, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, beating on absorbent paper, cleaning the ELISA plate 3 times with a plate washer, and adding 100 μl of 1×PBS into each hole of 1-4 columns; adding 200 mu L of urea treatment solution into each of 5 columns and 6 columns of holes, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, beating the ELISA plate on absorbent paper, cleaning the ELISA plate 3 times by using a plate washer, adding 100 mu L of GAM-HRP ELISA secondary antibody which is diluted 10000 times by secondary antibody diluent in advance into each hole, and incubating for 30min at 37 ℃; spin-drying the ELISA plate after incubation, beating the ELISA plate on absorbent paper, cleaning the ELISA plate 3 times by using a plate washer, taking TMB color development liquid, adding 100 mu L color development liquid into each hole, and incubating for 5-10min at 37 ℃; after development, 50. Mu.L of stop solution was added to each well. The read at 450nm/630nm was set up on a microplate reader. The read at 450nm/630nm was set up on a microplate reader.

ELISA antigen-antibody binding experiments were tested under the conditions of 0.5. Mu.g/mL and 1. Mu.g/mL antigen coating, data were obtained by testing absorbance OD (FIG. 6), and corresponding polynomial curves were obtained by fitting, and the corresponding binding was seen to have a significant gradient with variation of antibody dilution ratios, as inAt the time, the affinity binding reflected by the two curves under the conditions of 0.5 mug/mL and 1 mug/mL antigen coating shows the binding specificity, and the concentration corresponding to the binding force of the antibody respectively reaches 8.59E-09mol/L (under the condition of 0.5 mug/mL antigen coating, the highest OD reading is 50 percent, namely, when the concentration of the antigen-antibody complex is half of the total concentration, the concentration value 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 the antigen-antibody complex is half of the total concentration, the corresponding concentration value of the antibody, K) ₁ )。

Binding force calculation, namely calculating the average OD reading value of multiple wells of the S1-S8 antibody concentration before and after urea treatment under the antigen coating conditions of 0.5 mug/mL and 1 mug/mL respectively. Substituting the read value into a binding force calculation formula:

5. and (3) specificity detection: similar to ELISA antibody titer detection, detection using multiple influenza virus recombinant nucleoproteins of orthogonal design showed good antibody specificity of the invention (Table 4A-Table 4I).

TABLE 4A

TABLE 4B

TABLE 4C

TABLE 4D

TABLE 4E

TABLE 4F

TABLE 4G

TABLE 4H

TABLE 4I

As can be seen from tables 4A to 4I, the experimental data corresponding to the antibody pairing experiments show that, except for the fact that the detection hole corresponding to the enzyme-labeled antibody identical to the coated antibody is 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 binding specificity of the antibody G4E1 is reflected, and the sensitivity of the antibody G4E1 to the influenza B virus antigen is optimal when the antibody G4E 7 is matched with the E4H7 antibody to form an immune sandwich reaction.

6. Monoclonal antibody sequencing

Sequencing the light chain variable region and the heavy chain variable region of the monoclonal antibody against influenza B virus nucleoprotein prepared by the purified hybridoma cell G4E1 can show that the sequences of the light chain variable region of the monoclonal antibody against influenza B 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 against influenza B virus NP protein are CDR-H1 (SVWMH), CDR-H2 (YISSASITIYYAETVDG) and CDR-H3 (SGWLNAMKY). The antibody with the corresponding variable region sequence has high affinity and good specificity; the corresponding antibody variable regions can thus be used for the development of recombinant antibodies, single chain antibodies, bispecific antibodies, for diagnostic or therapeutic use in the development of related products.

Example 3 development of lateral flow immunochromatographic detection reagents Using influenza Virus N protein monoclonal antibodies

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

1. Preparation of colloidal gold:

the gold preparation is carried out by adopting a reduction method under the conditions of 2% chloroauric acid 5mL+10mL 1% trisodium citrate.

2. Colloidal gold marks the recombinant nucleoprotein monoclonal antibody of the mouse anti-influenza B virus. By physical adsorption, colloidal gold is combined with antibodies (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) by regulating pH value. The specific marking conditions are as follows: at pH7.5, 10. Mu.L of 0.1M potassium carbonate solution is added to each mL 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) detecting lines: a detection line is prepared by coating a nitrocellulose membrane with a recombinant nucleoprotein monoclonal antibody G4E1 of the mouse anti-influenza B virus (B) with a proper concentration (1.5 mg/mL). Drying at 37 ℃. The amount of the spray point was 0.1. Mu.L/mm.

Quality control line: a quality control line was prepared on a fibrous membrane from 0.5mg/mL goat anti-mouse IgG polyclonal antibody and dried at 37 ℃. The amount of the spray point was 0.1. Mu.L/mm.

4. Inspection method

The test card (test strip), sample diluent and sample are brought back 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, making a sample mark, and placing the sample mark on a horizontal workbench surface;

b. taking 20 mu L of nasopharynx or oropharynx swab sample extracting solution, and directly adding the extracting solution into a sample adding hole (a detection card) or a sample adding part (a 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, judging the result within 15-20 minutes, and invalidating the test result after 20 minutes.

5. Interpretation of test results

a. Detection line positive: and (5) developing the color of the detection line and the quality control line. The sample is prompted to detect the recombinant nucleoprotein antigen of the influenza B virus, which may be in early infection or present infection, and the final confirmation is needed by combining clinical symptoms.

b. Negative: only one red quality control line appears in the detection window. Indicating that the sample did not detect recombinant nucleoprotein antigen of influenza B virus.

c. Invalidation: the detection window has no red quality control line.

Example 4 influenza Virus antigen lateral flow immunochromatographic reagent Performance test

1. Sensitivity test

a. Inactivated influenza virus culture test

The lateral flow immunochromatography rapid detection reagent developed by using the antibody can detect inactivated influenza virus cultures, and can be tested as low as 500TCID at the lowest ₅₀ Viral culture/mL.

TCID ₅₀ by/mL is meant the amount of virus required to cause half of the cytopathic or dead (cytopathic effect, CPE) in a culture plate well or in a test tube to characterize the titer of the virus. 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, resulted in exactly 50% of the cells being infected, TCID ₅₀ 1000/mL. Indicating that 50% of the cells infected with the virus contained in each mL of sample were requiredMultiple of dilution is to be performed.

Thus, TCID ₅₀ The greater the number, the greater the number of copies of virus in the stock of the inactivated supernatant, the greater the dilution required (to achieve an effect of infecting 50% of the cells), and then the respective test strips of the kit were tested with the corresponding stock of the inactivated supernatant, with the corresponding gradient results shown in figure 3. 10TCID ₅₀ the/mL represents the sample with the least number of copies of the stock virus. I.e. as low as 10TCID as possible ₅₀ Viral culture/mL.

b. Purified influenza B virus nucleoprotein assay

The antibody-developed lateral flow immunochromatography rapid detection reagent is detected by adopting a B (B) type influenza virus nucleoprotein solution standard substance produced by Hangzhou horseradish gold ligand biotechnology limited company. As a result, as shown in FIG. 4, a minimum of 5000pg/mL of the standard recombinant nucleoprotein of influenza B virus could be detected.

2. Specificity test (clinical sample test)

The nasal swab samples of 20 normal persons were tested using the antibody developed lateral flow immunochromatographic rapid test reagent, and the test results are shown in FIG. 5. The graph shows that the detection result is clear and visible, and the background is clean, so that the product has good specificity.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. An antibody targeting influenza b virus nucleoprotein, characterized in that the amino acid sequences of the light chain hypervariable regions CDR-L1, CDR-L2 and CDR-L3 of the antibody are RSSEPISKYLA, SASTLQS and QQHNRYPWT, respectively, and the amino acid sequences of the heavy chain hypervariable regions CDR-H1, CDR-H2 and CDR-H3 of the antibody are SVWMH, YISSASITIYYAETVDG and SGWLNAMKY, 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 comprising 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 immunochromatography detection 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 immunochromatography detection test strip is characterized by comprising a sample pad, a colloidal gold binding pad, an NC film, a water absorption pad and a bottom plate, wherein the NC film is provided with a detection line and a quality control line, and the sample pad, the colloidal gold binding pad, the NC film and the water absorption pad are sequentially adhered to the bottom plate;

wherein, the colloidal gold binding 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;

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

8. The test strip of claim 7, wherein the concentration of the antibody I coated on the colloidal gold conjugate pad is 10-15 μg/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 colloidal gold binding pad is made of glass fiber.

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