WO2022062789A1 - Antibody and detection reagent kit for influenza b virus - Google Patents

Abstract

The present disclosure relates to the technical field of antibodies. Provided are an antibody and a detection reagent kit for an influenza B virus. The antibody against the influenza B virus provided by the present disclosure comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody against the influenza B virus provided by the present disclosure has good affinity for an influenza B virus antigen. Using the antibody to detect the influenza B virus has good sensitivity and specificity, and provides more new antibody choices for the detection of the influenza B virus.

Description

Antibodies and test kits for influenza B virus

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure claims priority to the Chinese patent application entitled "Antibody and Detection Kit Against Influenza B Virus" with application number "202011029407.9" filed with the China Patent Office on September 27, 2020, the entire contents of which are incorporated by reference in this disclosure.

technical field

The present disclosure relates to the technical field of antibodies, and in particular, to an antibody against influenza B virus and a detection kit.

Background technique

Influenza virus (Flu), referred to as influenza virus, is a representative species of Orthomyxoviridae, including human influenza virus, swine influenza virus, equine influenza virus, avian influenza virus, etc. Antigenicity can be divided into three types: A (A), B (B), and C (C), which are the pathogens of influenza. Influenza virus can cause infection and disease in a variety of animals such as humans, poultry, pigs, horses, and bats. Among them, influenza A virus and influenza B virus are the main ones that can infect people, which mainly cause upper respiratory tract infection, but also lower respiratory tract infection in children and adults, mainly pneumonia. Severe influenza in infants and young children is often accompanied by bronchial, high fever .

Influenza B is an influenza caused by influenza B (B) virus, which is characterized by abrupt onset, chills, and fever. . With headache, body aches, fatigue, loss of appetite. Mild respiratory symptoms, dry throat, sore throat, dry cough, and diarrhea. Facial flushing, conjunctival lateral canthus hyperemia, pharyngeal hyperemia, follicles on the soft palate. Amantadine can be used as M2 ion blocker and other drugs for treatment, and traditional Chinese medicine can also be used.

Influenza B virus will produce many subtypes, because the hemagglutinin (HA) and neuraminidase (NA) antigens of influenza virus are prone to change, and their constituent amino acid sites can be mutated. After each pandemic of influenza virus, a new type of influenza virus is generated through the mutation of the above amino acid sites. The human immune system generally lacks resistance to the mutated subtypes, which is easy to cause local epidemics, and can cause large-scale epidemics under special circumstances. Crowd infection. Surveillance data from the U.S. and China Centers for Disease Control and Prevention (CDC) from December 2017 to January 2018 showed an upward trend in the incidence of influenza B virus. Therefore, early and rapid screening of influenza virus is particularly important.

The clinical symptoms of influenza virus infection are not typical, and clinical diagnosis mainly depends on laboratory testing, and there are about 200 kinds of respiratory viruses that can simultaneously infect humans. Therefore, the sensitivity and specificity of detection reagents are very important for clinical diagnosis. The selection of influenza virus screening technology with short detection time and high detection rate is the technical path and prerequisite guarantee to ensure rapid clinical diagnosis and symptomatic treatment.

There are various laboratory detection methods for influenza virus. Referring to the changes in the "Influenza Diagnostic Criteria" of the Ministry of Health, it can be seen that early detection relies on chicken embryo inoculation. Due to the complex operation and high technical requirements, it is rarely used in clinical practice. Modern detection techniques include Antigen detection and nucleic acid PCR detection, the new technology has the characteristics of rapidity, sensitivity and specificity, which provides great help for clinical diagnosis.

Fluorescent PCR amplification technology has the advantages of high sensitivity and specificity, but the PCR method has higher requirements on samples, test environment and operators. The amplification methodology is suitable for the detection of batch samples, and the report time is relatively short. It can not meet the requirements of clinical rapid diagnosis very well. The immunocolloidal gold technology for detection of viral antigens can be used as a preferred method for rapid diagnosis of influenza B. The detection time is short, which can effectively assist clinical diagnosis, and to a large extent help clinical early symptomatic medication.

To strengthen the monitoring of influenza B virus and provide assistance for the rapid and accurate screening of influenza B virus infection, the focus is on optimizing the quality of rapid detection reagents, shortening the sample lysis time, reducing the limit of sample detection concentration, and improving the sensitivity and specificity. However, antibodies against influenza B virus currently on the market have certain defects in specificity and sensitivity.

SUMMARY OF THE INVENTION

The present disclosure provides an antibody against influenza B virus or a functional fragment thereof, the antibody or functional fragment thereof comprising the following complementarity determining regions:

CDR-VH1: G-F-X1-F-S-S-X2-A-M-S; where: X1 is T or S; X2 is F, A or Y;

CDR-VH2: T-X1-S-X2-G-G-X3-Y-T-Y-Y-P-D-S-X4-T-G; where: X1 is I or L; X2 is D or N; X3 is T or S; X4 is I, V or L;

CDR-VH3: X1-R-R-D-X2-G-A-M; wherein: X1 is T or A; X2 is L, V or I;

CDR-VL1: R-X1-S-Q-S-X2-G-L-N-X3-H; wherein: X1 is G or A; X2 is I, V or L; X3 is I, V or L;

CDR-VL2: Y-X1-S-Q-S-X2-S; where: X1 is V or A; X2 is I, V or L;

CDR-VL3: Q-X1-S-Y-S-X2-P-H; where: X1 is N or Q; X2 is F, Y or W.

In some embodiments,

In CDR-VH1, X1 is T;

In CDR-VH2, X1 is I;

In CDR-VH3, X1 is A;

In CDR-VL1, X1 is A;

In CDR-VL2, X1 is V;

In CDR-VL3, X1 is Q.

In some embodiments, in the CDR-VH1, X2 is F.

In some embodiments, X2 is A in the CDR-VH1.

In some embodiments, in the CDR-VH1, X2 is Y.

In some embodiments, X2 is D in the CDR-VH2.

In some embodiments, in the CDR-VH2, X2 is N.

In some embodiments, in the CDR-VH2, X3 is T.

In some embodiments, in the CDR-VH2, X3 is S.

In some embodiments, in the CDR-VH2, X4 is 1.

In some embodiments, in the CDR-VH2, X4 is V.

In some embodiments, in the CDR-VH2, X4 is L.

In some embodiments, in the CDR-VH3, X2 is L.

In some embodiments, in the CDR-VH3, X2 is V.

In some embodiments, in the CDR-VH3, X2 is 1.

In some embodiments, X2 is 1 in CDR-VL1.

In some embodiments, in CDR-VL1, X2 is V.

In some embodiments, in CDR-VL1, X2 is L.

In some embodiments, in CDR-VL1, X3 is 1.

In some embodiments, in CDR-VL1, X3 is V.

In some embodiments, in CDR-VL1, X3 is L.

In some embodiments, in the CDR-VL2, X2 is 1.

In some embodiments, in the CDR-VL2, X2 is V.

In some embodiments, in the CDR-VL2, X2 is L.

In some embodiments, in CDR-VL3, X2 is F.

In some embodiments, in CDR-VL3, X2 is Y.

In some embodiments, in CDR-VL3, X2 is W.

In some embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 1-60:

In some embodiments, the antibody or functional fragment thereof binds to an influenza B virus antigen with an affinity of K _D ≤ 4.21×10 ^-8 mol/L, in some embodiments, K _D ≤ 9.25×10 ^-8 mol/L.

In some embodiments,

In CDR-VH1, X1 is S;

In CDR-VH2, X1 is L;

In CDR-VH3, X1 is T;

In CDR-VL1, X1 is G;

In CDR-VL2, X1 is A;

In CDR-VL3, X1 is N;

In some embodiments, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 61-69:

	CDR-VH1	CDR-VH2	CDR-VH3	CDR-VL1	CDR-VL2	CDR-VL3
Mutation Combo 61	A	D/T/I	L	V/I	L	F
Mutation Combo 62	F	N/T/V	I	L/L	L	Y
Mutation Combo 63	F	D/T/L	V	L/I	I	Y
Mutation Combo 64	A	N/T/V	V	V/V	L	F
Mutation Combo 65	F	N/S/I	V	V/V	V	Y
Mutation Combo 66	F	N/S/L	V	V/V	I	F
Mutation Combination 67	F	N/S/V	L	L/L	L	F
Mutation Combo 68	A	D/T/I	L	L/I	I	F
Mutation Combo 69	Y	N/S/I	V	I/L	V	Y

.

In some embodiments, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L of the sequence shown in SEQ ID NO: 1-4, and/or the sequence of Heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8.

In some embodiments, the antibody further comprises a constant region.

In some embodiments, the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

In some embodiments, the species source of the constant region is bovine, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck , goose, turkey, cockfight or man.

In some embodiments, the constant region is derived from a mouse.

In some embodiments, the light chain constant region sequence of the constant region is set forth in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is set forth in SEQ ID NO:10.

In some embodiments, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

In some embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR1-L, FR2-L, FR3-L, and FR4-L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, FR2-H, FR3-H, and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively H.

The present disclosure provides a reagent or kit for detecting influenza B virus, comprising any one of the antibodies or functional fragments thereof.

In some embodiments, the antibody or functional fragment thereof is labeled with a detectable label.

In some embodiments, the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In some embodiments, the fluorescent dye is selected from fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, and protein dyes and its derivatives.

In some embodiments, the enzyme that catalyzes coloration of the substrate is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6- Phosphoglucose deoxygenase.

In some embodiments, the radioisotope is selected ^from212Bi , ^131I , ^111In , ^90Y , ^186Re , ^211At , ^{125I , 188Re} , ^153Sm , ^213Bi , ^32P , ^94mTc ,99mTc , ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu, and ¹⁸ F.

In some embodiments, the chemiluminescent reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, Oxetane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives.

In some embodiments, the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In some embodiments, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In some embodiments, the colloidal metal is selected from colloidal gold, colloidal silver, and colloidal selenium.

The present disclosure provides a nucleic acid molecule encoding any one of the antibodies or functional fragments thereof.

The present disclosure provides a vector comprising a nucleic acid molecule encoding any one of the antibodies or functional fragments thereof.

The present disclosure provides a recombinant cell containing the vector.

The present disclosure provides the use of the antibody or functional fragment thereof, the reagent or the kit in detecting influenza B virus.

The present disclosure provides the use of the antibody or its functional fragment, the reagent or the kit for detecting influenza B virus.

The present disclosure provides a method for detecting influenza B virus, comprising:

A) contacting the antibody or functional fragment thereof of any of the above with a sample under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The present disclosure provides a method of diagnosing influenza B in a subject, comprising:

A) contacting the antibody or functional fragment thereof of any one of the above with a sample from the subject under conditions sufficient for a binding reaction to occur to effect a binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

The present disclosure provides a method for preparing any one of the antibodies or functional fragments thereof, comprising: culturing the recombinant cells, and isolating and purifying the antibodies or functional fragments thereof from the cultured product.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present disclosure more clearly, the accompanying drawings required in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

FIG. 1 shows the results of reducing SDS-PAGE of the anti-influenza B virus antibody of Example 1. FIG.

detailed description

In order to make the objectives, technical solutions and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out in accordance with the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, techniques employed or considered herein are standard methods. The materials, methods and examples are illustrative only and not restrictive.

Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the purview of those skilled in the art. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( MJGait, ed., 1984); "Animal Cell Culture" (RIFreshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); " Handbook of Experimental Immunology (eds. DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (eds. JMMiller and MPCalos, 1987); Contemporary Molecular Biology Methods (Current Protocols in Molecular Biology)" (FMAusubel et al., ed., 1987); "PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction)" (Mullis et al., ed., 1994); and "Contemporary Current Protocols in Immunology" (JEColigan et al., eds., 1991), each of which is expressly incorporated herein by reference.

Definition of Terms

As used herein, the term "complementarity determining regions" means: an intact or complete antibody comprises two heavy chains and two light chains; each heavy chain comprises a variable region (VH) and a constant region (CH); each The light chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape with the stem of the Y consisting of the second and third of the two heavy chains held together by disulfide bonds The constant region consists of; each arm of Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain; the variable region and heavy chain of the light chain The variable regions of the are responsible for antigen binding; the variable regions in both chains typically contain three hypervariable regions, called complementarity determining regions.

As used herein, when used to refer to an antibody, the term "functional fragment" is intended to refer to a portion of an antibody comprising a heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. These functional fragments may include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single chain Fv (scFv), diabodies, triple chain Antibodies (triabodies), tetrabodies (tetrabodies) and minibodies (minibodies). Other functional fragments may include, for example, heavy or light chain polypeptides, variable region polypeptides or CDR polypeptides or portions thereof, so long as these functional fragments retain binding activity.

As used herein, the term "constant region" refers to a relatively stable region of the light and heavy chains of an antibody molecule near the C-terminal amino acid sequence.

As used herein, the term "variable region" refers to the region of the light and heavy chains of an antibody molecule that varies greatly in amino acid sequence near the N-terminus.

As used herein, the term "colloidal gold detection" refers to a novel immunolabeling technique applied to antigen-antibody with colloidal gold as a tracer marker.

As used herein, the term "naked anti-stability" refers to the change in activity of an antibody in its unmodified and labeled state as a function of storage temperature and time.

Some embodiments of the present disclosure provide an antibody against influenza B virus and a detection kit. The anti-influenza B virus antibody provided by the present disclosure has better affinity for influenza B virus antigen, and the antibody is used to detect influenza B virus It has better sensitivity and specificity, and provides a new antibody choice for the detection of influenza B virus.

One embodiment of the present disclosure provides an antibody against influenza B virus or a functional fragment thereof, wherein the antibody or functional fragment thereof has the following complementarity determining regions:

CDR-VH1: G-F-X1-F-S-S-X2-A-M-S; where: X1 is T or S; X2 is F, A or Y;

CDR-VH2: T-X1-S-X2-G-G-X3-Y-T-Y-Y-P-D-S-X4-T-G; where: X1 is I or L; X2 is D or N; X3 is T or S; X4 is I, V or L;

CDR-VH3: X1-R-R-D-X2-G-A-M; wherein: X1 is T or A; X2 is L, V or I;

CDR-VL1: R-X1-S-Q-S-X2-G-L-N-X3-H; wherein: X1 is G or A; X2 is I, V or L; X3 is I, V or L;

CDR-VL2: Y-X1-S-Q-S-X2-S; where: X1 is V or A; X2 is I, V or L;

CDR-VL3: Q-X1-S-Y-S-X2-P-H; where: X1 is N or Q; X2 is F, Y or W.

The anti-influenza B virus antibody or its functional fragment provided by the present disclosure has the above-mentioned complementarity determining region structure, and the above-mentioned complementarity determining region structure enables the antibody or its functional fragment to specifically bind to the influenza B virus antigen. The influenza virus antigen has good affinity, and the antibody or its functional fragment can be used to detect influenza B virus with good specificity and sensitivity.

In an alternative embodiment,

In CDR-VH1, X1 is T;

In CDR-VH2, X1 is I;

In CDR-VH3, X1 is A;

In CDR-VL1, X1 is A;

In CDR-VL2, X1 is V;

In CDR-VL3, X1 is Q.

In an alternative embodiment, in CDR-VH1, X2 is F.

In an alternative embodiment, X2 is A in CDR-VH1.

In an alternative embodiment, in CDR-VH1, X2 is Y.

In an alternative embodiment, X2 is D in the CDR-VH2.

In an alternative embodiment, X2 is N in CDR-VH2.

In an alternative embodiment, in CDR-VH2, X3 is T.

In an alternative embodiment, in the CDR-VH2, X3 is S.

In an alternative embodiment, in the CDR-VH2, X4 is 1.

In an alternative embodiment, in CDR-VH2, X4 is V.

In an alternative embodiment, in the CDR-VH2, X4 is L.

In an alternative embodiment, in the CDR-VH3, X2 is L.

In an alternative embodiment, in CDR-VH3, X2 is V.

In an alternative embodiment, in the CDR-VH3, X2 is 1.

In an alternative embodiment, in CDR-VL1, X2 is 1.

In an alternative embodiment, in CDR-VL1, X2 is V.

In an alternative embodiment, in CDR-VL1, X2 is L.

In an alternative embodiment, in CDR-VL1, X3 is 1.

In an alternative embodiment, in CDR-VL1, X3 is V.

In an alternative embodiment, in CDR-VL1, X3 is L.

In an alternative embodiment, in CDR-VL2, X2 is 1.

In an alternative embodiment, in CDR-VL2, X2 is V.

In an alternative embodiment, in CDR-VL2, X2 is L.

In an alternative embodiment, in CDR-VL3, X2 is F.

In an alternative embodiment, in CDR-VL3, X2 is Y.

In an alternative embodiment, in CDR-VL3, X2 is W.

In an optional embodiment, each complementarity determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 1-60:

In an optional embodiment, the antibody or its functional fragment binds to influenza B virus with an affinity of K _D ≤4.21×10 ^-8 mol/L, in an optional embodiment, K _D ≤9.25× 10 ^-9 mol/L.

In optional embodiments, K _D ≤9×10 ^-9 mol/L, 8×10 ^-9 mol/L, K _D ≤7×10 ^-9 mol/L, K _D ≤6×10 ^-9 mol /L, K _D ≤5×10 ^-9 mol/L, K _D ≤4×10 ^-9 mol/L, K _D ≤3×10 ^-9 mol/L, K _D ≤2×10 ^-9 mol/L , K _D ≤1×10 ^-9 mol/L, K _D ≤9×10 ^-8 mol/L, K _D ≤8×10 ^-8 mol/L, K _D ≤7×10 ^-8 mol/L, K _D ≤6×10 ^-8 mol/L, K _D ≤5×10 ^-8 mol/L, K _D ≤4×10 ^-8 mol/L, K _D ≤3×10 ^-8 mol/L, K _D ≤ 2×10 ^-8 mol/L, or K _D ≤1×10 ^-8 mol/L.

In an optional embodiment, 2.07×10 ⁻⁹ mol/L≦K _D ≦4.21×10 ⁻⁸ mol/L.

In an optional embodiment, 2.07×10 ⁻⁹ mol/L≦K _D ≦9.25×10 ⁻⁹ mol/L.

The detection of K _D is performed with reference to the methods in the embodiments of the present disclosure.

In an alternative embodiment,

In CDR-VH1, X1 is S;

In CDR-VH2, X1 is L;

In CDR-VH3, X1 is T;

In CDR-VL1, X1 is G;

In CDR-VL2, X1 is A;

In CDR-VL3, X1 is N.

In an alternative embodiment, each complementarity determining region of the antibody or functional fragment thereof is selected from any one of the following mutation combinations 61-69:

	CDR-VH1	CDR-VH2	CDR-VH3	CDR-VL1	CDR-VL2	CDR-VL3
Mutation Combo 61	A	D/T/I	L	V/I	L	F
Mutation Combo 62	F	N/T/V	I	L/L	L	Y
Mutation Combo 63	F	D/T/L	V	L/I	I	Y
Mutation Combo 64	A	N/T/V	V	V/V	L	F
Mutation Combo 65	F	N/S/I	V	V/V	V	Y
Mutation Combo 66	F	N/S/L	V	V/V	I	F
Mutation Combination 67	F	N/S/V	L	L/L	L	F
Mutation Combo 68	A	D/T/I	L	L/I	I	F
Mutation Combo 69	Y	N/S/I	V	I/L	V	Y

.

In an alternative embodiment, the antibody comprises the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, and/or the sequences of the light chain framework regions shown in SEQ ID NOs: 1-4 in sequence. The heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NOs: 5-8 in sequence.

In general, the variable region (VH) of the heavy chain and the variable region (VL) of the light chain can be obtained by linking the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In some embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, FR2-L, FR3-L and FR4- L, and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% homology with sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.

In some embodiments, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L, and FR4-L that are at least 80% identical to the sequences of SEQ ID NOs: 1, 2, 3, 4, respectively, in order , and/or, heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H having at least 80% identity to the sequences of SEQ ID NOs: 5, 6, 7, 8, respectively.

It should be noted that, in other embodiments and examples, the amino acid sequences of the respective framework regions of the antibodies or functional fragments thereof provided by the present disclosure may be the same as those of the corresponding framework regions (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8) may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In alternative embodiments, the antibody further comprises a constant region.

In an alternative embodiment, the constant region is selected from the constant region of any one of IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

In an alternative embodiment, the species source of the constant region is mammalian or poultry. In alternative embodiments, mammals include cows, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, or humans. In alternative embodiments, poultry animals include chickens, ducks, geese, turkeys or fighting cocks.

In an optional embodiment, the species source of the constant region is bovine, equine, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken , duck, goose, turkey, cockfight or man.

In an alternative embodiment, the constant region is derived from a mouse.

In an alternative embodiment, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10.

In an alternative embodiment, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

Functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. Those skilled in the art can easily understand from the contents described in the present disclosure that the functional fragments of the above-mentioned antibodies can be cleaved by methods including but not limited to enzymatic digestion (including but not limited to pepsin or papain) and/or by chemical reduction Sulfur bond method. On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.

Functional fragments of the above-described antibodies can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, automated peptide synthesizers such as those sold by including, but not limited to, Applied BioSystems and the like. One embodiment of the present disclosure provides a reagent or kit for detecting influenza B virus, which comprises the antibody or functional fragment thereof according to any one of the above.

In an optional embodiment, the antibody or functional fragment thereof in the above reagent or kit is labeled with a detectable label.

As used herein, "detectable label" refers to a class of substances having properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, coloration, radioactivity, etc., by which the corresponding Qualitative or quantitative detection of a target.

In alternative embodiments, the detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, those skilled in the art can select a suitable marker according to detection conditions or actual needs, and no matter what marker is used, it falls within the protection scope of the present disclosure.

In an optional embodiment, the fluorescent dyes include, but are not limited to, fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC) hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or its analogs), rhodamine dyes and derivatives thereof (such as, but not limited to, red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or the like compounds), Cy series dyes and their derivatives (such as but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (such as including But not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but Not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoxanthin-chlorophyll protein (preCP), etc.).

In an optional embodiment, the enzymes that catalyze the coloration of the substrate include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase enzyme and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotopes include but are not limited to ^212Bi , ^131I , ^111In , ^90Y , ^186Re , ^211At , ^125I , ^188Re , ^153Sm , ^213Bi , ^32P , ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu and ¹⁸ F.

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives Derivatives, Dioxetane and its Derivatives, Lopine and its Derivatives and Peroxyoxalate and its Derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium. An embodiment of the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof. One embodiment of the present disclosure provides a vector containing the above-mentioned nucleic acid molecule. One embodiment of the present disclosure provides a recombinant cell containing the above-mentioned vector. Some embodiments of the present disclosure provide the use of the above-mentioned antibodies or functional fragments thereof, reagents or kits in detecting influenza B virus.

Some embodiments of the present disclosure provide the use of the above-mentioned antibodies or functional fragments thereof, reagents or kits for detecting influenza B virus.

Some embodiments of the present disclosure provide methods of detecting influenza B virus, comprising:

A) contacting the above-described antibody or functional fragment thereof with a sample under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

A method of diagnosing influenza B in a subject, comprising:

A) contacting the above-described antibody or functional fragment thereof with a sample from the subject under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

B) Detection of immune complexes produced by the binding reaction.

One embodiment of the present disclosure provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells, and separating and purifying the antibody or its functional fragment from the cultured product.

On the basis of the amino acid sequences of antibodies or functional fragments thereof provided in the present disclosure, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (chemical synthesis, hybridoma cells) to prepare the antibodies or functional fragments thereof, such as It is easy for those skilled in the art to separate and purify the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described above. Based on this, No matter what technology is used to prepare the antibody or its functional fragment of the present disclosure, it falls within the protection scope of the present disclosure.

Example

The features and properties of the present disclosure will be further described in detail below with reference to the embodiments.

Example 1

In this example, restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. BD SMART ^™ RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.

1 Construction of recombinant plasmids

(1) Antibody gene preparation

The mRNA was extracted from the hybridoma cell line (5C9) secreting anti-influenza B virus antigen antibody, and the DNA product was obtained by RT-PCR method. The cells were transformed into DH5α competent cells, and after the colonies were grown, the heavy chain (Heavy Chain) and the light chain (Light Chain) gene were cloned respectively, and 4 clones were sent to a gene sequencing company for sequencing.

(2) Sequence analysis of antibody variable region genes

The gene sequences obtained by the above sequencing were placed in the IMGT antibody database for analysis, and the VNTI11.5 software was used for analysis to confirm that the genes amplified by the heavy chain and light chain primers were correct, and the genes amplified by the light chain were correct. In the fragment, the VL gene sequence is 324bp, belonging to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 351bp, belonging to the VH1 gene family, with 57bp in front of it. leader peptide sequence.

(3) Construction of recombinant antibody expression plasmid

^pcDNATM 3.4

vector is the constructed recombinant antibody eukaryotic expression vector. The expression vector has introduced polyclonal restriction sites such as HindIII, BamHI and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the above pMD-18T Based on the results of gene sequencing of the variable region of the antibody, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases on both ends, respectively, and the light chain of 0.73KB was amplified by PCR amplification method. Gene fragment and 1.42kb heavy chain gene fragment.

The heavy chain and light chain gene fragments were digested with HindIII/EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. Recombinant expression plasmids for heavy and light chains were obtained.

2 Screening of stable cell lines

(1) The recombinant antibody expression plasmid was transiently transfected into CHO cells to determine the activity of the expression plasmid

The recombinant expression plasmids of heavy chain and light chain were diluted to 400ng/ml with ultrapure water, adjusted to 1.43×10 ⁷ cells/ml of CHO cells in a centrifuge tube, 100 μL of plasmid was mixed with 700 μL of cells, transferred to an electroporation cup, and electroporated. , on the 3rd, 5th, and 7th days, the samples were counted, and the samples were collected and tested on the 7th day.

The coating solution (the main component NaHCO ₃ ) was diluted with goat anti-mouse IgG 1ug/ml for microplate coating, 100 μL per well, overnight at 4°C; the next day, the washing solution (the main component Na ₂ HPO ₄ +NaCl) was washed twice , pat dry; add blocking solution (20%BSA+80%PBS), 120μL per well, 37℃, 1h, pat dry; add diluted cell supernatant, 100μL/well, 37℃, 60min; shake off the plate liquid, pat dry, add 20% mouse negative blood to block, 120 μL per well, 37 ℃, 1 h; shake off the liquid in the plate, pat dry, add diluted influenza B virus antigen, 100 μL per well, 37 ℃, 40 min; wash Wash 5 times and pat dry; add HRP-labeled anti-influenza B virus antigen monoclonal antibody, 100 μL per well, 37°C, 30 min; add chromogenic solution A (50 μL/well, the main ingredients are citric acid + sodium acetate + Acetanilide + carbamide peroxide), add color developing solution B (50 μL/well, main ingredient citric acid + EDTA 2Na + TMB + concentrated HCl), 10min; add stop solution (composition: EDTA 2Na + concentrated H ₂ SO ₄ ) , 50 μL/well; read the OD value at 450 nm (reference 630 nm) on the microplate reader. The results showed that the reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the unadded cell supernatant was less than 0.1, indicating that the antibody produced by the transient transfection of the plasmid was active against influenza B virus antigen.

(2) Linearization of recombinant antibody expression plasmid

Prepare the following reagents: Buffer 50μL, DNA 100μg/tube, PuvI enzyme 10μL, sterile water to make up to 500μL, digest overnight in a 37°C water bath; first use an equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24 : 1, and then extract with chloroform (water phase) in turn; 0.1 times the volume (water phase) of 3M sodium acetate and 2 times the volume of ethanol precipitate on ice, rinse the precipitate with 70% ethanol, remove the organic solvent, and use an appropriate amount when the ethanol is completely volatilized. The sterilized water was used for reconstitution, and finally the concentration was measured.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Dilute the plasmid obtained by linearization of the recombinant antibody expression plasmid in step 2-(2) with ultrapure water to 400ng/ml, adjust CHO cells to 1.43×10 ⁷ cells/ml in a centrifuge tube, mix 100 μL of plasmid with 700 μL of cells, and transfer Into the electroporation cup, electroporated, counted the next day; 25umol/L MSX 96-well pressurized culture for about 25 days.

Observe the cloned wells marked with cells under a microscope, and record the degree of confluence; take the culture supernatant and send samples for detection; select cell lines with high antibody concentration and relative concentration and transfer them to 24 wells, and transfer them to 6 wells in about 3 days; preserve seeds after 3 days Batch culture, adjust the cell density to 0.5×10 ⁶ cells/ml, 2.2 ml for batch culture, cell density 0.3×10 ⁶ cells/ml, 2 ml for seed preservation; 7-day 6-well batch culture supernatant is sent for sample detection and selection The cell lines with smaller antibody concentration and cell diameter were transferred to TPP for seed preservation and passage.

3 Recombinant Antibody Production

(1) Cell expansion

Stably transfect the recombinant antibody expression plasmid in step 2-(3), pressurize the cells obtained by screening the stable cell line, and then culture them in a 125ml shake flask, the inoculation volume is 30ml, and the medium is 100% Dynamis medium. , placed in a shaker with a rotational speed of 120 r/min, a temperature of 37 °C, and 8% carbon dioxide. After culturing for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/ml, and the expansion volume is calculated according to the production demand, and the medium is 100% Dynamis medium. After that, the culture was expanded every 72h. When the cell volume meets the production requirements, the seeding density is strictly controlled to be about 500,000 cells/ml for production.

(2) Shake flask production and purification

Shaking flask parameters: rotating speed 120r/min, temperature 37°C, carbon dioxide 8%. Feed feeding: start feeding every day when cultured in shake flasks to 72h, HyCloneTM Cell BoostTM Feed 7a medium is fed with 3% of the initial culture volume every day, and Feed 7b is fed daily at 1/1000 of the initial culture volume , until the 12th day (the 12th day feeding). Glucose was supplemented with 3 g/L on the sixth day. Samples were collected on the 13th day. Affinity purification was performed using a protein A affinity chromatography column. Take 4 μg of purified antibody for reducing SDS-PAGE, and 4 μg foreign control antibody as control. The electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO: 14), the other Mr is 28KD (light chain, SEQ ID NO: 13).

Example 2

Antibody performance testing

(1) Activity detection of the antibody of Example 1 and its mutants

The antibody (WT) sequence of Example 1 was analyzed, and its heavy chain variable region was shown in SEQ ID NO: 12, wherein the amino acid sequence of each complementarity determining region on the heavy chain variable region was as follows:

CDR-VH1: G-F-S(X1)-F-S-S-A(X2)-A-M-S;

CDR-VH2: T-L(X1)-S-D(X2)-G-G-T(X3)-Y-T-Y-Y-P-D-S-I(X4)-T-G;

CDR-VH3: T(X1)-R-R-D-L(X2)-G-A-M;

Its light chain variable region is shown in SEQ ID NO:11, wherein, the amino acid sequence of each complementarity determining region on the light chain variable region is as follows:

CDR1-VL: R-G(X1)-S-Q-S-V(X2)-G-L-N-I(X3)-H;

CDR-VL2: Y-A(X1)-S-Q-S-L(X2)-S;

CDR-VL3: Q-N(X1)-S-Y-S-F(X2)-P-H.

On the basis of the anti-influenza B virus antibody (WT) of Example 1, the sites related to antibody activity in the complementarity determining region were mutated, wherein X1, X2, X3, and X4 were all mutation sites. See Table 1 below.

Table 1 Mutation sites related to antibody activity

Detection of antibody binding activity in Table 1:

The coating solution (the main component NaHCO ₃ ) was diluted with goat anti-mouse IgG 1 μg/ml for microplate coating, 100 μl per well, overnight at 4°C; the next day, the washing solution (the main component Na ₂ HPO ₄ +NaCl) was washed twice , pat dry; add blocking solution (20%BSA+80%PBS), 120μl per well, 37℃, 1h, pat dry; add diluted purified antibody in Table 1, 100μl/well, 37℃, 60min; shake off The liquid in the plate, patted dry, add 20% mouse negative blood to block, 120 μl per well, 37°C, 1 h; shake off the liquid in the plate, pat dry, add diluted influenza B virus antigen, 100 μl per well, 37°C, 40min ; Wash 5 times with washing solution and pat dry; add HRP-labeled influenza B virus paired monoclonal antibody (obtained from Philippine Biotechnology), 100 μl per well, 37°C, 30 min; add chromogenic solution A (50 μl/well, Contain 2.1g/L citric acid, 12.25g/L sodium acetate, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), add chromogenic solution B (50μl/well, containing 1.05g/L citric acid, 0.186g/LEDTA·2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl), 10min; add stop solution (50μl/well, containing 0.75g/EDTA·2Na and 10.2ml/L concentrated H ₂ SO ₄ ); Read the OD value at 450nm (reference 630nm) on the microplate reader. The results are shown in Table 2 below.

Table 2 Activity data of WT antibody and its mutants

Antibody concentration (ng/ml)	250	125	31.25	7.813	1.953	0
WT	2.024	1.537	0.493	0.198	0.095	0.031
Mutation 1	2.105	1.617	0.657	0.360	0.187	0.038
Mutation 2	2.031	1.572	0.613	0.337	0.197	0.036
Mutation 3	2.103	1.602	0.665	0.342	0.134	0.076
Mutation 4	2.098	1.593	0.609	0.318	0.145	0.032
Mutation 5	0.635	0.340	0.131	0.045	-	-
Mutation 6	0.612	0.322	0.123	0.033	-	-

From the data in Table 2, it can be seen that the activities of WT and mutation 1 to mutation 4 antibodies are higher than those of mutation 5 and mutation 6, among which, mutation 1 has the best activity.

(2) Affinity detection of antibodies and their mutants

(a) On the basis of mutation 1, other sites are mutated, and the sequence of each mutation is shown in Table 3 below.

Table 3 Mutation sites related to antibody affinity

Affinity analysis

Using AMC sensor, the purified antibody was diluted to 10ug/ml with PBST, and the influenza B virus antigen was serially diluted with PBST (phosphate Tween buffer) (main component Na ₂ HPO ₄ +NaCl+TW-20);

Running process: equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69 GLY solution and buffer 3 to regenerate the sensor and output data. The results are shown in Table 4 below. K _D represents the equilibrium dissociation constant or affinity.

Table 4 Affinity detection data

	K D (M)
Mutation 1	5.13E-09
Mutation 1-1	3.35E-09
Mutation 1-2	2.97E-09
Mutation 1-3	7.88E-09
Mutation 1-4	3.38E-09

Mutation 1-5	2.22E-09
Mutation 1-6	4.44E-09
Mutation 1-7	3.12E-09
Mutation 1-8	4.17E-09
Mutation 1-9	3.02E-09
Mutation 1-10	8.18E-09
Mutation 1-11	6.27E-09
Mutation 1-12	6.42E-09
Mutation 1-13	3.34E-09
Mutation 1-14	6.32E-09
Mutation 1-15	2.92E-09
Mutation 1-16	5.12E-09
Mutation 1-17	2.70E-09
Mutation 1-18	4.90E-09
Mutation 1-19	2.10E-09
Mutation 1-20	2.95E-09
Mutation 1-21	5.66E-09
Mutation 1-22	3.41E-09
Mutation 1-23	5.91E-09
Mutation 1-24	9.25E-09
Mutation 1-25	2.45E-09
Mutation 1-26	5.88E-09
Mutation 1-27	3.24E-09
Mutation 1-28	4.76E-09
Mutation 1-29	2.21E-09
Mutation 1-30	2.77E-09
Mutation 1-31	6.83E-09
Mutation 1-32	3.49E-09
Mutation 1-33	3.00E-09
Mutation 1-34	4.26E-09
Mutation 1-35	4.06E-09
Mutation 1-36	5.07E-09
Mutation 1-37	4.25E-09
Mutation 1-38	3.85E-09
Mutation 1-39	4.22E-09
Mutation 1-40	9.19E-09
Mutation 1-41	2.79E-09
Mutation 1-42	3.89E-09
Mutation 1-43	6.99E-09
Mutation 1-44	4.82E-09
Mutation 1-45	5.70E-09
Mutation 1-46	4.08E-09
Mutation 1-47	4.12E-09
Mutation 1-48	4.20E-09
Mutation 1-49	5.83E-09
Mutation 1-50	3.62E-09
Mutation 1-51	2.72E-09
Mutation 1-52	6.15E-09
Mutation 1-53	2.07E-09

Mutation 1-54	3.37E-09
Mutation 1-55	3.20E-09
Mutation 1-56	2.53E-09
Mutation 1-57	5.09E-09
Mutation 1-58	5.35E-09
Mutation 1-59	2.31E-09

From the data in Table 4, it can be seen that the mutation 1 antibody and its series of mutants have high affinity for influenza B virus antigen, indicating that on the basis of mutation 1, by mutating according to the mutation method in Table 3, the Antibodies against influenza B virus antigens with higher affinity.

(b) On the basis of WT, other sites were mutated, and the affinity of each mutant was detected. The sequence of each mutation is shown in Table 5 below, and the corresponding affinity data is shown in Table 6.

Table 5 Mutations using WT as backbone

Table 6 Affinity test results of WT antibody and its mutants

	K D (M)
WT	2.76E-08
WT1	4.10E-08
WT2	3.39E-08
WT3	3.16E-08
WT4	3.48E-08
WT5	3.86E-08
WT6	3.14E-08
WT7	4.21E-08
WT8	3.41E-08

From the data in Table 6, it can be seen that the WT and WT1 to WT8 antibodies have good affinity to the antigen, indicating that the antibodies obtained by mutating WT according to the mutation method in Table 5 have good affinity.

(3) Bare resistance stability assessment

The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and the 7-day, 14-day, and 21-day samples were taken for state observation, and the 21-day sample was tested for activity. , the results showed that there was no obvious change in the protein state of the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies were stable. The following table 7 mutation 1 antibody is the OD results of the enzyme immunoassay activity detection for 21 days.

Table 7

Antibody concentration (ng/ml)	250	31.25	0
4°C, 21-day samples	1.999	0.704	0.041
-80℃, 21 days sample	1.975	0.718	0.022
37°C, 21-day sample	1.949	0.702	0.031

Example 3

Application of Antibody in Colloidal Gold Detection

1 Preparation of colloidal gold detection test paper

(1) Preparation of nitrocellulose membrane

Preparation of coating buffer: PBS buffer containing 6% methanol and 0.01M pH7.2 is the coating buffer, filtered through a 0.22 μm membrane, and kept at 4° C. for later use, valid for one week. 1000ml 6% methanol in 0.01M pH 7.2 PBS buffer Recipe: NaCL 8g, KCL 0.2g, _{Na2HPO4 12H2O} 2.9g, _{KH2PO4 0.2g} , methanol 60ml, _double distilled deionized water to make up to 1000ml.

Preparation of nitrocellulose membrane: Dilute the purified antibodies in Table 3 and Table 5 to 1-5 mg/ml with coating buffer, adjust the machine, and mark the T line, which is the detection line, and the T line is close to the gold standard. Pad, about 5mm away from the end of the gold label pad; dilute the goat anti-mouse IgG antibody to 1～5mg/ml with coating buffer, adjust the machine, draw the line C, which is the control line, the line C is close to the absorption pad, and the distance The absorbent pad is about 3mm. The distance between the two lines is 5-8mm, evenly. Dry at 37°C and package for later use.

(2) Preparation of colloidal gold and gold-labeled monoclonal antibodies

(a) Preparation of solution

①Preparation of chloroauric acid: Dissolve chloroauric acid with double distilled deionized water to prepare a 1% solution, set it at 4°C for standby, and the validity period is four months. 1000ml 1% chloroauric acid solution formula: 10g chloroauric acid: make up to 1000ml with double distilled deionized water.

②Preparation of trisodium citrate: Dissolve sodium citrate with double distilled deionized water to prepare a 1% solution, filter through a 0.22μm membrane, set it at 4 degrees for use, and the valid capacity is up to 1000ml.

③Preparation of 0.1M potassium carbonate: prepare it with double distilled deionized water, filter it with 0.22μm membrane, set it at 4°C for use, and it is valid for four months. 1000ml 0.1M potassium carbonate solution formula: 13.8g potassium carbonate; make up to 1000ml with double distilled deionized water.

④ Preparation of 2% PEG-20000: Prepared with double distilled deionized water, filtered through a 0.22 μm membrane, set at 4°C for use, valid for four months. 1000ml 2% PEG-20000 solution formula: 20g PEG-20000; make up to 1000ml with double distilled deionized water.

⑤ Preparation of labeling washing preservation solution: 2% bovine serum albumin (BSA), 0.05% sodium azide (NaN ₃ ), 0.01M pH7.2 PBS solution, 0.22μ membrane filtration, set aside at 4°C for use, valid for four moon. 1000ml label washing and preservation solution formula: 20g BSA, 0.5g NaN3, 0.01M pH7.2 PBS solution to 1000ml.

(b) Preparation of colloidal gold.

Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water, set it to boil on an electric stove, add 2ml of 1% trisodium citrate per 100ml of 0.01% chloroauric acid, and continue to boil until the liquid turns bright red, then stop heating, Make up for water loss after cooling to room temperature. The appearance of the prepared colloidal gold should be pure, translucent, free of precipitation and floating matter, and valid for one week.

(c) Preparation of colloidal gold-labeled antibodies.

Adjust the pH of colloidal gold to 8.2 with 0.1 M potassium carbonate, add another paired influenza B virus-labeled antibody at 8-10 μg antibody/ml colloidal gold, mix with a magnetic stirrer for 30 min, and add BSA to The final concentration was 1% for 1 hour. Centrifuge at 13,000 rpm and 4°C for 30 min, discard the supernatant, wash the precipitate twice with labeled washing preservation solution, and resuspend the precipitate with one-tenth the initial volume of labeled washing preservation solution, and set it at 4 °C for later use, valid for one week.

(3) Preparation of gold label pads

(a) Preparation of blocking solution.

PBS solution containing 2% BSA, 0.1% TritonX-100, 0.05% NaN ₃ , 0.01M pH7.2, filtered through a 0.22 μm membrane, and kept at 4°C for use, valid for four months. 1000ml blocking solution formula: 20g BSA, 0.5g NaN3, 1ml TritonX-100, 0.01M pH7.2 PBS solution to 1000ml.

(b) Preparation of gold label pads

The gold label pad was soaked in the blocking solution for 30 min, and then dried at 37°C. Then, the prepared gold-labeled antibody was spread evenly on the gold-labeled pad, 20 square centimeters per ml of the solution, freeze-dried, packaged, and stored at 4°C for later use.

(4) Preparation of test strip sample pads

(a) Preparation of blocking solution.

Contains 2% BSA, 0.1% TrtionX-100, 0.05% NaN ₃ , 0.01M pH7.2 PBS solution, 0.22 μm membrane filtration, set aside at 4 degrees for use, valid for four months. 1000ml blocking solution formula: 20g BSA, 0.5g NaN ₃ , 1ml TrtionX-100, 0.01M pH7.2 PBS solution to 1000ml.

(b) Preparation of sample pads.

Immerse the sample pad in the blocking solution for 30 min, then dry it at 37°C, seal it, and store it at 4°C for later use.

(5) Assembly of test strips

The absorbent pad (purchased from Millipore Company), nitrocellulose membrane, gold standard pad, and sample pad were placed on a non-absorbent support sheet, and cut into 3 mm wide strips. A pack of ten small strips is added with a desiccant and vacuum-sealed to obtain a colloidal gold detection test paper for detecting influenza B virus.

2 Application of antibodies in colloidal gold detection

The above-mentioned assembled test strips are used to detect whether the tested material contains influenza B virus antigen, so as to determine the effect of the antibody obtained in the foregoing embodiment on the detection of influenza B virus antigen. The double antibody sandwich method is used to detect whether the tested material contains influenza B virus antigen. During detection, influenza B virus antigen first combines with colloidal gold-labeled influenza B virus antibody to form a complex of influenza B virus antigen-colloidal gold labeling-influenza B virus antibody. Due to capillary action, influenza B virus antigen-colloid The gold-labeled influenza B virus antibody complex moves forward along the nitrocellulose membrane, and when it reaches the detection line, the influenza B virus antigen-colloidal gold-labeled-influenza B virus antibody complex will interact with the influenza B virus obtained in the example. The antibody binds to form a complex of influenza B virus antibody-influenza B virus antigen-colloidal gold label-influenza B virus antibody, which is enriched on the detection line to form a red precipitation line. The influenza B virus antigen-colloidal gold label-influenza B virus antibody complex that is not bound to the influenza B virus antibody on the detection line passes through the detection line, is captured by the goat anti-mouse IgG antibody, and is enriched on the quality control line to form Red precipitation line. When the test line and the quality control line have a red precipitation line at the same time, it is judged as a positive result. If the sample does not contain influenza B virus antigen, when the colloidal gold-labeled influenza B virus antibody that is not bound to the influenza B virus antigen reaches the detection line, it will not form influenza B virus antibody-influenza B virus antigen-colloidal gold The complex of labeled-influenza B virus antibody, the colloidal gold-labeled influenza B virus antibody complex that is not bound to the influenza B virus antigen passes the detection line, and is only enriched on the quality control line to form a red precipitation line. negative result.

The results for some of the antibodies are shown in Table 8 below.

Table 8 Antibody detection activity

Remarks: The color development of the gold standard is composed of C plus a number. The smaller the number after C, the stronger the color and the higher the activity; the higher the number after C, the weaker the color and the lower the activity; the number is followed by a "+" It is 0.5-1C stronger than without color rendering, and the "-" after the number is slightly lower than that without color rendering by 0.5-1C. B means inactive.

From the results in Table 8 above, it can be seen that the antibodies provided in the embodiments of the present disclosure have good activity in the double-antibody sandwich method detection on the gold-labeled platform.

The above descriptions are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Industrial Applicability

The present disclosure provides an antibody against influenza B virus and a detection kit. The anti-influenza B virus antibody provided by the present disclosure has better affinity for influenza B virus antigen, and the use of the antibody to detect influenza B virus has better Sensitivity and specificity, providing new antibody options for influenza B virus detection. At the same time, the detection kit provided by the present disclosure also has the same technical effect as the antibody, and has broad application prospects and high market value.

Claims (16)

1. A kind of antibody or its functional fragment of anti-influenza B virus, it is characterized in that, described antibody or its functional fragment comprise following complementarity determining region:

   CDR-VH1: G-F-X1-F-S-S-X2-A-M-S; where: X1 is T or S; X2 is F, A or Y;

   CDR-VH2: T-X1-S-X2-G-G-X3-Y-T-Y-Y-P-D-S-X4-T-G; where: X1 is I or L; X2 is D or N; X3 is T or S; X4 is I, V or L;

   CDR-VH3: X1-R-R-D-X2-G-A-M; wherein: X1 is T or A; X2 is L, V or I;

   CDR-VL1: R-X1-S-Q-S-X2-G-L-N-X3-H; wherein: X1 is G or A; X2 is I, V or L; X3 is I, V or L;

   CDR-VL2: Y-X1-S-Q-S-X2-S; where: X1 is V or A; X2 is I, V or L;

   CDR-VL3: Q-X1-S-Y-S-X2-P-H; where: X1 is N or Q; X2 is F, Y or W.
2. The anti-influenza B virus antibody or its functional fragment according to claim 1, wherein,

   In CDR-VH1, X1 is T;

   In CDR-VH2, X1 is I;

   In CDR-VH3, X1 is A;

   In CDR-VL1, X1 is A;

   In CDR-VL2, X1 is V;

   In CDR-VL3, X1 is Q;

   Preferably, in CDR-VH1, X2 is F;

   Preferably, in CDR-VH1, X2 is A;

   Preferably, in CDR-VH1, X2 is Y;

   Preferably, in CDR-VH2, X2 is D;

   Preferably, in CDR-VH2, X2 is N;

   Preferably, in CDR-VH2, X3 is T;

   Preferably, in CDR-VH2, X3 is S;

   Preferably, in CDR-VH2, X4 is 1;

   Preferably, in CDR-VH2, X4 is V;

   Preferably, in CDR-VH2, X4 is L;

   Preferably, in CDR-VH3, X2 is L;

   Preferably, in CDR-VH3, X2 is V;

   Preferably, in CDR-VH3, X2 is 1;

   Preferably, in CDR-VL1, X2 is 1;

   Preferably, in CDR-VL1, X2 is V;

   Preferably, in CDR-VL1, X2 is L;

   Preferably, in CDR-VL1, X3 is 1;

   Preferably, in CDR-VL1, X3 is V;

   Preferably, in CDR-VL1, X3 is L;

   Preferably, in CDR-VL2, X2 is 1;

   Preferably, in CDR-VL2, X2 is V;

   Preferably, in CDR-VL2, X2 is L;

   Preferably, in CDR-VL3, X2 is F;

   Preferably, in CDR-VL3, X2 is Y;

   Preferably, in CDR-VL3, X2 is W;

   Preferably, each complementarity determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 1-60:

   

   

   
3. The anti-influenza B virus antibody or its functional fragment according to claim 2, wherein the antibody or its functional fragment and the influenza B virus antigen have K _D ≤4.21×10 ^-8 mol/L The affinity binding, preferably, K _D ≤ 9.25×10 ^-8 mol/L.
4. The anti-influenza B virus antibody or its functional fragment according to claim 1, wherein,

   In CDR-VH1, X1 is S;

   In CDR-VH2, X1 is L;

   In CDR-VH3, X1 is T;

   In CDR-VL1, X1 is G;

   In CDR-VL2, X1 is A;

   In CDR-VL3, X1 is N;

   Preferably, each complementarity determining region of the antibody or its functional fragment is selected from any one of the following mutation combinations 61-69:

   
5. The anti-influenza B virus antibody or its functional fragment according to any one of claims 1-4, wherein the antibody comprises a light chain framework region whose sequence is sequentially shown in SEQ ID NOs: 1-4 FR1-L, FR2-L, FR3-L and FR4-L, and/or the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4 whose sequences are shown in SEQ ID NOs: 5-8 in turn -H;

   Preferably, the antibody further comprises a constant region;

   Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD;

   Preferably, the species source of the constant region is bovine, horse, dairy cattle, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkeys, cockfights or people;

   Preferably, the constant region is derived from mice;

   Preferably, the light chain constant region sequence of the constant region is shown in SEQ ID NO:9, and the heavy chain constant region sequence of the constant region is shown in SEQ ID NO:10;

   Preferably, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
6. The anti-influenza B virus antibody or its functional fragment according to any one of claims 1-4, wherein the antibody comprises sequences with sequence SEQ ID NOs: 1, 2, 3, and 4 in sequence, respectively. Light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L having at least 80% homology, and/or, respectively, have at least 80 % homology to the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H.
7. A reagent or kit for detecting influenza B virus, characterized in that it comprises the antibody or its functional fragment according to any one of claims 1-6.
8. The reagent or kit according to claim 7, wherein the antibody or its functional fragment is labeled with a detectable marker;

   Preferably, the detectable label is selected from fluorescent dyes, enzymes that catalyze the color development of substrates, radioisotopes, chemiluminescent reagents and nanoparticle labels;

   Preferably, the fluorescent dye is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives ;

   Preferably, the enzyme that catalyzes the coloration of the substrate is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and glucose 6-phosphate deoxygenase enzymes;

   Preferably, the radioisotope is selected from ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi, ³² P, ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb , ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu and ¹⁸ F;

   Preferably, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine esters and its derivatives, dioxetane Alkane and its derivatives, Lofenine and its derivatives, and peroxyoxalate and its derivatives;

   Preferably, the nanoparticle marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles;

   Preferably, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex;

   Preferably, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium.
9. A nucleic acid molecule encoding the antibody or functional fragment thereof according to any one of claims 1-6.
10. A vector, characterized in that it comprises a nucleic acid molecule encoding the antibody or functional fragment thereof according to any one of claims 1-6.
11. A recombinant cell, characterized in that it contains the vector according to claim 10 .
12. Use of the antibody or its functional fragment according to any one of claims 1-6 or the reagent or kit according to claim 7 or 8 in detecting influenza B virus.
13. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6, or the reagent or kit according to claim 7 or 8, for detecting influenza B virus.
14. A method of detecting influenza B virus comprising:

    A) contacting the antibody or functional fragment thereof of any one of claims 1-6 with a sample under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

    B) Detection of immune complexes produced by the binding reaction.
15. A method of diagnosing influenza B in a subject, comprising:

    A) contacting the antibody or functional fragment thereof of any one of claims 1-6 with a sample from the subject under conditions sufficient for the binding reaction to occur to effect the binding reaction; and

    B) Detection of immune complexes produced by the binding reaction.
16. A method for preparing the antibody or its functional fragment according to any one of claims 1-6, characterized in that it comprises: culturing the recombinant cell according to claim 11, and separating and purifying the culture product to obtain the Antibodies or functional fragments thereof.

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