Antibody pair, reagent and method for detecting influenza A virus

Abstract

The invention discloses an antibody pair, a reagent and a method for detecting influenza A virus, and relates to the field of immunodiagnosis. The antibody pair for detecting the influenza A virus comprises the first antibody and the second antibody, and the reagent and the detection method based on the antibody combination can accurately detect the existence of the influenza A virus.

Description

Technical Field

[0001] The present invention relates to the technical field of immunodiagnosis, and in particular to an antibody pair, a reagent and a method for detecting type A influenza virus. Background Art

[0002] Influenza is an acute respiratory infectious disease that humans have not yet completely conquered. Its incidence rate ranks first among all infectious diseases. It is one of the major public health problems facing humans and the first infectious disease to be monitored globally. Influenza virus belongs to the family of Orthomyxoviridae. It is an enveloped and segmented single-stranded negative-sense RNA virus that is spherical or filamentous. According to the different antigenicity of its internal nucleoprotein (NP) and matrix protein (M), it can be divided into three types: A, B, and C. According to the different antigenicity of the hemagglutinin (HA) and neuraminidase (NA) proteins on the outer membrane of the virus particles, influenza A virus can currently be divided into 16 H subtypes (H1-H16) and 9 N subtypes (N1-N9). Among influenza A viruses, there are reports of human infection with subtypes such as H1, H2, H3, H5, H7, and H9. Since the nucleotide sequences encoding HA and / or NA are prone to mutation, resulting in changes in the antigenic epitopes of HA and / or NA, this antigenic shift renders the original specific immunity of the population ineffective. Therefore, influenza A viruses often cause large-scale or even global influenza epidemics.

[0003] In the past few decades, seasonal influenza viruses have mainly been concentrated in subtypes A-H3N2 and H1N1. In recent years, cases of outbreaks of new subtypes of influenza A viruses have occurred from time to time. For example, in 2009, the new influenza A-H1N1 virus caused a global influenza pandemic, and cases of human infection with highly pathogenic avian influenza (H5 subtype) viruses have been reported from time to time, and avian influenza A-H5N1 subtype viruses are considered to have the potential to cause a large-scale influenza pandemic in humans. New influenza A viruses are usually caused by segmental recombination of genes. This large-scale genetic change can easily lead to major changes in the antigenic properties of the virus, which is called antigenic shift. The new influenza A-H1N1 virus (2009) contains gene fragments of avian influenza, swine influenza, and human seasonal influenza, resulting in significant changes in the virus at the antigenic level. Due to the significant change in antigenicity and the possible increase in viral virulence, the infectivity and severity of the virus have increased, so the new influenza A virus may cause higher morbidity and mortality. Since influenza lacks specificity in clinical manifestations, it is difficult to differentiate it from acute respiratory infections caused by other pathogenic microorganisms. In addition, the clinical reporting time of traditional etiological detection methods is relatively delayed, resulting in clinicians often adopting empirical exploratory treatments based on the patient's condition. Therefore, there is an urgent need for a diagnostic reagent that is time-effective and can quickly confirm the diagnosis of influenza A virus.

[0004] Immunological detection method is an ideal method for detecting influenza A virus, which has the advantages of convenient detection, fast result, high detection sensitivity, etc. After long-term research, the inventor found that sandwich pairing of influenza A virus antibody raw materials is the key to realizing influenza A virus immunological detection in addition to the antibody raw materials themselves. Summary of the invention

[0005] The present application provides an antibody pair, which provides an important source of paired antibody raw materials for the detection of influenza A virus and has good detection performance.

[0006] In order to achieve the above object, according to one aspect of the present invention, an antibody pair for detecting influenza A virus is provided. The antibody pair comprises a first antibody, the first antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:11;

[0007] The antibody pair includes a second antibody, which comprises: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:20 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:22.

[0008] The complementarity determining regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0009] To achieve the above object, according to a second aspect of the present invention, an antibody pair for detecting influenza A virus is provided, the antibody pair comprising a first antibody, the first antibody having HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6;

[0010] The antibody pair includes a second antibody having HCDR1 shown in SEQ ID NO: 13, and HCDR2 shown in SEQ ID NO: 14.

[0011] HCDR2, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, LCDR2 shown in SEQ ID NO: 17,

[0012] LCDR3 shown in SEQ ID NO:18.

[0013] In order to achieve the above object, according to a third aspect of the present invention, an antibody pair for detecting influenza A virus is provided;

[0014] The antibody pair comprises a first antibody, and the first antibody comprises at least one of (1)-(2):

[0015] (1) the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11;

[0016] (2) the heavy chain shown in SEQ ID NO: 10 and the light chain shown in SEQ ID NO: 12;

[0017] The antibody pair comprises a second antibody, wherein the second antibody comprises at least one of (a)-(b):

[0018] (a) the heavy chain variable region shown in SEQ ID NO: 20 and the light chain variable region shown in SEQ ID NO: 22;

[0019] (b) The heavy chain shown in SEQ ID NO:21 and the light chain shown in SEQ ID NO:23.

[0020] In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody pair for detecting influenza A virus;

[0021] The antibody pair comprises a first antibody, wherein the heavy chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 11; or;

[0022] The antibody pair comprises a second antibody, wherein the heavy chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 22;

[0023] In an alternative embodiment, the mutation is the addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody.

[0024] In an alternative embodiment, the framework region of the conservative variant formed by mutation of the first antibody has at least 80% identity with the framework region of the first antibody before mutation; or;

[0025] The framework region of the conservative variant formed by mutation of the second antibody has at least 80% identity with the framework region of the second antibody before mutation.

[0026] In order to achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody pair for detecting influenza A virus;

[0027] The antibody pair comprises a first antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO:11;

[0028] The antibody pair comprises a second antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:20 and a light chain variable region set forth in SEQ ID NO:22.

[0029] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided an antibody pair for detecting influenza A virus, wherein the antibody comprises a constant region, wherein the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and the light chain constant region is selected from a κ-type or λ-type light chain constant region;

[0030] In an alternative embodiment, the heavy chain constant region of the first antibody is SEQ ID NO: 7 or a sequence having at least 80% identity thereto, and the light chain constant region of the first antibody is SEQ ID NO: 8 or a sequence having at least 80% identity thereto;

[0031] In an alternative embodiment, the heavy chain constant region of the second antibody is SEQ ID NO: 19 or a sequence at least 80% identical thereto, and the light chain constant region of the second antibody is SEQ ID NO: 8 or a sequence at least 80% identical thereto.

[0032] In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a reagent for detecting influenza A virus, the reagent comprising a first group of antibodies and a second group of antibodies, the first group of antibodies comprising a first antibody in an antibody pair;

[0033] The second set of antibodies includes the second antibody of the antibody pair;

[0034] Optionally, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeling antibody;

[0035] The reagent comprises the first antibody and the second antibody of the above-mentioned antibody pair; the first antibody is a coating antibody and the second antibody is a labeled antibody, or the second antibody is a coating antibody and the first antibody is a labeled antibody;

[0036] In an optional embodiment, the coated antibody or labeled antibody is coupled to biotin or a biotin derivative;

[0037] In an optional embodiment, the labeling substance coupled to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0038] In an alternative embodiment, the coated antibody is coupled to a solid carrier;

[0039] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0040] In order to achieve the above object, according to an eighth aspect of the present invention, a method for detecting influenza A virus is provided, comprising:

[0041] a) contacting the above antibody pair or reagent with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and

[0042] b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of influenza A virus in the test sample.

[0043] In order to achieve the above object, according to the ninth aspect of the present invention, there is provided use of the above antibody pair or reagent in detecting influenza A virus or preparing a product for detecting influenza A virus. DETAILED DESCRIPTION

[0044] In a first aspect, an embodiment of the present invention provides an antibody pair for detecting influenza A virus, the antibody pair comprising a first antibody, the first antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:11; the antibody pair comprises a second antibody, the second antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:20 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:22.

[0045] The complementarity determining regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0046] In the present invention, the term "antibody" is used in the broadest sense, and the antibody in the method of the present invention may be a whole antibody, an antigen-binding fragment or a polymerized antibody that can bind to influenza A virus.

[0047] The whole antibody may be monoclonal. Such a whole antibody is usually an antibody made by any suitable method known in the art. For example, by immunizing a mammal, usually a rabbit or a mouse, with influenza A virus under suitable conditions and isolating antibody molecules from, for example, the serum of the mammal. Monoclonal antibodies can be obtained by hybridoma or recombinant methods. The antigen-binding fragment includes an antigen-binding site, such as a Fab or F(ab)2 fragment. In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody. The antigen-binding fragment of the above-mentioned antibody usually has the same binding specificity as the antibody from which it is derived. It is easy for a person skilled in the art to understand that the antigen-binding fragment of the above-mentioned antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction splitting disulfide bonds. On the basis of the structure of the complete antibody disclosed in the present invention, it is easy for a person skilled in the art to obtain the above-mentioned antigen-binding fragment. The antigen-binding fragment of the above-mentioned antibody can also be synthesized by recombinant genetics techniques also known to a person skilled in the art or by, for example, an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems. Polymeric antibodies refer to polymers formed by the polymerization of whole antibodies and antigen-binding fragments.

[0048] In a second aspect, an embodiment of the present invention provides an antibody pair for detecting influenza A virus, the antibody pair comprising a first antibody and a second antibody; the first antibody has HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; the second antibody has HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18.

[0049] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0050] The definition methods of CDR are well known in the art, and the CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., US Pept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the annotations in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also belong to the scope of protection of the present disclosure.

[0051] Table 1: CDR Definition 1

[0052]

[0053] The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+numbers" and amino acid numbers on the light chain represented by "L+numbers". One of ordinary skill in the art can unambiguously map the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Patent No. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0054] 2"AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0055] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0056] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0057] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0058] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[0059] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0060] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0061] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0062] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0063] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0064] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0065] The antibodies of the present invention also include a framework region. In the present invention, the "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to the region other than CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, comprising LFR1, LFR2, LFR3 and LFR4 framework regions.

[0066] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0067] In some optional embodiments of the present invention, the antibody described in the present invention can also be an antibody that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity in the FR region with the first antibody and the second antibody in the antibody pair.

[0068] In a third aspect, an embodiment of the present invention provides an antibody pair for detecting influenza A virus.

[0069] The antibody pair comprises a first antibody having a heavy chain variable region shown in SEQ ID NO:9 and a light chain variable region shown in SEQ ID NO:11; the antibody pair comprises a second antibody having a heavy chain variable region shown in SEQ ID NO:20 and a light chain variable region shown in SEQ ID NO:22.

[0070] In an optional embodiment, the antibody pair comprises a first antibody having a heavy chain shown in SEQ ID NO:10 and a light chain shown in SEQ ID NO:12; the antibody pair comprises a second antibody having a heavy chain shown in SEQ ID NO:21 and a light chain shown in SEQ ID NO:23.

[0071] In a fourth aspect, an embodiment of the present invention provides an antibody pair for detecting influenza A virus, the antibody pair comprising a first antibody, the heavy chain variable region of the first antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:9, and the light chain variable region of the first antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:11; the antibody pair comprises a second antibody, the heavy chain variable region of the second antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:20, and the light chain variable region of the second antibody being a conservative variant formed by a mutation of the amino acid sequence shown in SEQ ID NO:22.

[0072] The conservative variant refers to a mutant in which the amino acid at the mutation site is replaced by an amino acid with the same chemical properties as the original amino acid.

[0073] The mutation includes one or more amino acid additions, deletions, substitutions or modifications.

[0074] In a fifth aspect, an embodiment of the present invention provides an antibody pair for detecting influenza A virus, the antibody pair comprising a first antibody, the first antibody binds to an epitope, and the epitope is the same as the epitope bound by the antibody comprising the heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 11; the antibody pair comprises a second antibody, the second antibody binds to an epitope: the epitope is the same as the epitope bound by the antibody comprising the heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 11

[0075] The antibodies with the heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:22 bind to the same epitope.

[0076] The epitope is also called an antigen epitope (AE). The epitope determines the ability of the antigen to specifically bind to the antibody. The antibody binds to the same epitope, that is, the amino acid fragments that the antibody binds to the target antigen are consistent. Whether an antibody recognizes the same epitope as other antibodies can be confirmed by competition between the two for the epitope. Competition between antibodies can be evaluated by competitive binding assays, and the methods include: ELISA, fluorescence energy transfer assay (FRET) or fluorescence microassay technology (FMAT). The amount of the antibody bound to the antigen is indirectly related to the binding ability of the candidate competing antibody (tested antibody) that competes for the same epitope. That is, the greater the binding amount or affinity of the tested antibody to the same epitope, the lower the binding amount of the antibody to the antigen, and the greater the binding amount of the tested antibody to the antigen. Specifically, the antibody and the antibody to be evaluated that have been appropriately labeled are added to the antigen at the same time, and the antibody bound is detected by the label. By pre-labeling the antibody, the amount of the antibody bound to the antigen can be easily determined. There is no particular limitation on the label, and the labeling method corresponding to the measurement technology is selected. Specific labeling methods include: fluorescent labeling, radioactive labeling, enzyme labeling, etc.

[0077] In an optional embodiment, the antibody further comprises a constant region, wherein the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and the light chain constant region is selected from a κ-type or λ-type light chain constant region. The λ-type light chain constant region can be selected from λ1, λ2, λ3, and λ4 subtypes.

[0078] It should be noted that, in some embodiments, the constant region sequence 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% identity with the above-mentioned constant region (SEQ ID NO: 7, 8 or 19).

[0079] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG1, hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM;

[0080] In an optional embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0081] In some optional embodiments, the species of origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0082] In a seventh aspect, an embodiment of the present invention provides a reagent for detecting influenza A virus,

[0083] The reagents include a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody in the above antibody pair;

[0084] The second group of antibodies includes the second antibody in the above antibody pair.

[0085] In an optional embodiment, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other group is a labeling antibody.

[0086] It should be noted that the meaning of reagent in this application and the meaning of kit can be regarded as equivalent to each other.

[0087] In an optional embodiment, the coated antibody or labeled antibody is coupled to biotin or a biotin derivative;

[0088] In an optional embodiment, the labeling substance coupled to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0089] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, 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), pre-chlorophyll protein (preCP), etc.).

[0090] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0091] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0092] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0093] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0094] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0095] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0096] In an optional embodiment, the colloidal metal is colloidal gold.

[0097] In an alternative embodiment, the coated antibody is coupled to a solid carrier;

[0098] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0099] In an eighth aspect, an embodiment of the present invention provides a method for detecting influenza A virus, comprising:

[0100] a) contacting the above antibody pair or reagent with a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and

[0101] b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of influenza A virus in the test sample.

[0102] In order to achieve the above object, according to the ninth aspect of the present invention, there is provided use of the above antibody pair or reagent in detecting influenza A virus or preparing a product for detecting influenza A virus.

[0103] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0105] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0106] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0107] Based on long-term creative research on influenza A virus, the applicant discovered two antibodies that can meet the requirements for influenza A virus detection.

[0108] Example 1 Antibody Preparation

[0109] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting monoclonal antibodies to influenza A virus was an existing hybridoma cell line, which was revived for use.

[0110] (1) Antibody gene preparation

[0111] mRNA was extracted from the hybridoma cell line secreting influenza A virus monoclonal antibodies, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0112] (2) Sequence analysis of influenza A virus antibody variable region genes

[0113] The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was approximately 320bp, and there was a 57bp leader peptide sequence in front of it; in the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was approximately 360bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0114] (3) Construction of recombinant antibody expression plasmid

[0115] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning 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 sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody are designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the LightChain gene fragment and the Heavy Chain gene fragment are amplified by the PCR amplification method.

[0116] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0117] 2. Recombinant Antibody Production

[0118] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, re-dissolve with culture medium, and use it as cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of cells after transfection, and place them in a 35℃ constant temperature incubator for culture, speed 120rmp, CO2 content 8%, and centrifuge after 13 days. The supernatant of the centrifugation was affinity purified using proteinA affinity chromatography column. Take 6ug layer of purified antibody for reducing SDS-PAGE. After reducing SDS-PAGE, two bands were shown, one Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).

[0119] The obtained antibodies were named Anti-Flua-012 and Anti-Flua-013. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0120] Table 2 Antibody sequences

[0121] Antibody Name Heavy chain Light chain Anti-Flua-012 SEQ ID NO:10 SEQ ID NO:12 Anti-Flua-013 SEQ ID NO:21 SEQ ID NO:23

[0122] Example 2 Preparation of detection reagents

[0123] The above Anti-Flua-012 antibody was used as the labeling antibody, and the Anti-Flua-013 antibody was used as the coating antibody. The experimental process is as follows:

[0124] (1) Coating:

[0125] Dilute the T-line coated antibody and C-line coated anti-labeled antibody secondary antibody to 1 mg / ml with coating diluent, and use a gold dot membrane sprayer to evenly draw detection lines on the NC membrane (T line and C line, both 1.0 μl / cm), taking care not to scratch the membrane, and then put it into a 50-degree air drying oven for drying for more than 4 hours before use.

[0126] (2) Marking:

[0127] 1ml 4 / 10,000AU, add a certain amount of 0.2mol / LK 2 CO 3 , shake well for 1 minute, then add 10ug of the antibody to be labeled, shake well for 1 minute, and finally add the blocking agent and shake well for 1 minute. After centrifugation at 10,000 rpm / 7min in a Xiangyi desktop centrifuge, collect the precipitate and concentrate it to 1 / 10 of the labeled volume. The number of centrifugal revolutions can be adjusted appropriately according to the centrifugal effect. The supernatant after centrifugation should be light pink or colorless, and there should be no irresolvable precipitate.

[0128] (3) Gold spraying: The complex of the labeled antibody and colloidal gold in (2) is diluted to a specified concentration and then sprayed with gold, and dried for at least 2 hours.

[0129] (4) Sample pad preparation: Spread 2.4 ml of the prepared sample pad treatment solution on a 1.7 cm wide and 30 cm long glass fiber and dry at 50°C for more than 2 h.

[0130] (5) Assembly: Assemble the sample pad, base plate, absorbent paper, conjugate pad, coating film, etc., cut into 3.0 mm pieces, and make the corresponding immunoassay reagent strips.

[0131] Example 3 Reagent Performance Evaluation

[0132] (1) Dilute the sample containing influenza A virus with sample diluent;

[0133] (2) Use a pipette to transfer 50ul of the diluted sample and drop it onto the immunoassay reagent strip prepared above for chromatography;

[0134] (3) Read the sample with the naked eye after 15-20 minutes, and record the color of the T line according to the reading on the test reagent card.

[0135] The test results for different influenza A virus strains (The National Institute for Biological Standards and Control) are shown in the following table:

[0136]

[0137]

[0138]

[0139] It should be noted that:

[0140] 1. The readings of the colloidal gold card have a total of 10 gradients: 1, 2, 3, 4, 5, 6, 7, 8, 9, and B. The colors of the T lines corresponding to these 10 gradients become lighter from left to right. 1 means the T line color is the darkest, 9 means the T line color is the lightest, B means no color, and B+ means almost no color. In the table, "8+" means a little darker than 8 but not reaching 7, and the others are marked with "+" by analogy. In the table, "8-" means a little lighter than 8 but not reaching 9, and the others are marked with "-" by analogy.

[0141] Example 4 Reagent Cross-Reaction Evaluation

[0142] The cross-reactivity of the test kit was evaluated with other respiratory pathogens other than influenza A virus, negative clinical nasopharyngeal formula, and recombinant influenza B virus antigen.

[0143] (1) Use a pipette to transfer 50ul of the diluted sample and drop it onto the immunoassay reagent strip prepared above for chromatography;

[0144] (2) Read the sample with the naked eye after 15-20 minutes, and record the color of the T line according to the reading on the test reagent card.

[0145]

[0146]

[0147] Note: PK2-FBN1 is a recombinant influenza B virus antigen

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0149] The partial amino acid sequences involved in this application are as follows:

[0150]

[0151]

Claims

1. An antibody pair for detecting influenza A virus, characterized in that: The antibody pair comprises a first antibody, the first antibody comprising: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:11; The antibody pair includes a second antibody, which comprises: three heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region shown in SEQ ID NO:20 and three light chain complementary determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:

22.

2. The antibody pair according to claim 1, wherein the complementarity determining regions are defined by any one of the systems of Kabat, Chothia, IMGT, AbM or Contact or a combination of multiple systems.

3. An antibody pair for detecting influenza A virus, characterized in that: The antibody pair comprises a first antibody, wherein the first antibody has HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; The antibody pair includes a second antibody having HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:

18.

4. An antibody pair for detecting influenza A virus, characterized in that: The antibody pair comprises a first antibody, and the first antibody comprises at least one of (1)-(2): (1) the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11; (2) the heavy chain shown in SEQ ID NO: 10 and the light chain shown in SEQ ID NO: 12; The antibody pair comprises a second antibody, wherein the second antibody comprises at least one of (a)-(b): (a) the heavy chain variable region shown in SEQ ID NO: 20 and the light chain variable region shown in SEQ ID NO: 22; (b) The heavy chain shown in SEQ ID NO:21 and the light chain shown in SEQ ID NO:

23.

5. The antibody pair for detecting influenza A virus according to claim 4, characterized in that: The antibody pair comprises a first antibody, wherein the heavy chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 11; or; The antibody pair comprises a second antibody, wherein the heavy chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 20, and the light chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 22; Optionally, the mutation is the addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody; The framework region of the conservative variant formed by mutation of the first antibody has at least 80% identity with the framework region of the first antibody before mutation; or; The framework region of the conservative variant formed by mutation of the second antibody has at least 80% identity with the framework region of the second antibody before mutation.

6. An antibody pair for detecting influenza A virus, characterized in that: The antibody pair comprises a first antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:9 and a light chain variable region set forth in SEQ ID NO:11; The antibody pair comprises a second antibody that binds to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region set forth in SEQ ID NO:20 and a light chain variable region set forth in SEQ ID NO:

22.

7. The antibody pair for detecting influenza A virus according to any one of claims 1 to 6, characterized in that: The antibody further comprises a constant region; wherein the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; and the light chain constant region is selected from a κ-type or λ-type light chain constant region; Optionally, the heavy chain constant region of the first antibody is SEQ ID NO: 7 or a sequence having at least 80% identity thereto, and the light chain constant region of the first antibody is SEQ ID NO: 8 or a sequence having at least 80% identity thereto; Optionally, the heavy chain constant region of the second antibody is SEQ ID NO: 19 or a sequence at least 80% identical thereto, and the light chain constant region of the second antibody is SEQ ID NO: 8 or a sequence at least 80% identical thereto.

8. A reagent for detecting influenza A virus, characterized in that: The reagents include a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody according to any one of claims 1 to 7; The second group of antibodies comprises the second antibody of any one of claims 1-7; Optionally, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeling antibody; Optionally, the coated antibody or labeled antibody is coupled to biotin or a biotin derivative; Optionally, the label to which the labeled antibody is coupled is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels; Optionally, the coated antibody is coupled to a solid carrier; Optionally, the solid support is selected from microspheres, plates and membranes.

9. A method for detecting influenza A virus, characterized in that: include: a) contacting the antibody pair according to any one of claims 1 to 7 or the reagent according to claim 8 with a sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of influenza A virus in the test sample.

10. Use of the antibody pair according to any one of claims 1 to 7 or the reagent according to claim 8 in detecting influenza A virus or preparing a product for detecting influenza A virus.

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