Antibody pairs, reagents and methods for detecting dengue virus

Abstract

The application discloses an antibody pair, a reagent and a method for detecting dengue virus, and relates to the field of immunodiagnosis. The antibody pair for detecting dengue virus disclosed by the application comprises a first antibody, a second antibody and a third antibody, and the reagent and the detection method based on the antibody pair can accurately detect the presence of dengue virus.

Description

Technical Field

[0001] This invention relates to the field of immunodiagnostic technology, and more specifically, to an antibody pair, reagent, and method for detecting dengue virus. Background Technology

[0002] Dengue fever is an acute infectious disease caused by the dengue virus (DENV), one of the most widespread mosquito-borne infectious diseases globally. The dengue virus belongs to the Flaviviridae family and the Flavivirus genus, and is transmitted by mosquitoes such as Aedes aegypti and Aedes albopictus, and is prevalent in more than 100 tropical and subtropical countries and regions worldwide. According to the WHO, more than 2.5 billion people globally are at risk of dengue fever, with 500 to 100 million people infected annually. Approximately 500,000 of these cases are severe (mainly children), and about 12,500 of these severe cases result in death. Furthermore, in recent years, dengue fever outbreaks have been continuously introduced into my country from neighboring countries, with reports of dengue fever in Hainan, Guangxi, Fujian, and Guangdong provinces. In Guangdong Province alone, more than 45,000 dengue fever cases were reported in 2014. The dengue virus can cause everything from mild fever and dengue hemorrhagic fever (DHF) to the more severe dengue shock syndrome (DSS). Currently, there are no vaccines or effective treatments available for human use, and the harm caused by dengue virus has become a serious global public health problem.

[0003] NS1 protein is a conserved glycoprotein in dengue fever (DENV). High concentrations can be detected in serum early in the infection and are closely related to the severity of dengue fever. Over the past decade, various forms of NS1 antigen-capture-based immunoassays have been established and commercialized. These NS1 antigen detection methods are characterized by early detection, rapid detection, specificity, low cost, and ease of operation. Serologically specific NS1 antigen anti-capture ELISA, developed through careful selection of NS1 antibodies, even has serotyping capabilities. Therefore, NS1 antigen-targeted immunoassays play an important role in the diagnosis of dengue fever.

[0004] In conventional laboratory diagnostic methods, dengue virus detection typically involves virus isolation and genome detection. However, this method is time-consuming and costly, making it unsuitable for handling large volumes of clinical samples. Secondly, dengue virus antigen detection primarily utilizes ELISA and immunochromatography. While ELISA offers high sensitivity, it requires specialized equipment and skilled technicians, making it unsuitable for on-site use. Although a few companies both domestically and internationally have developed products for immunochromatography, their low sensitivity and specificity hinder accurate virus screening. There is a strong demand in this field for highly sensitive and specific immunochromatographic products. Through extensive research, the inventors have discovered that the sandwich pairing of dengue virus antibody raw materials, besides the antibody raw materials themselves, is crucial for achieving dengue virus immunodetection. Summary of the Invention

[0005] This application provides an antibody pair that provides an important source of paired antibody raw materials for the detection of dengue virus and has good detection performance.

[0006] To achieve the above objectives, according to one aspect of the present invention, an antibody pair for detecting dengue virus is provided. The antibody pair includes a first antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:9, and three light chain complementarity-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 complementarity-determining regions, HCDR1, HCDR2 and HCDR3, in the heavy chain variable region shown in SEQ ID NO:21, and three light chain complementarity-determining regions, LCDR1, LCDR2 and LCDR3, in the light chain variable region shown in SEQ ID NO:23.

[0008] The antibody pair includes a third antibody, which comprises: three heavy chain complementarity-determining regions, HCDR1, HCDR2 and HCDR3, in the heavy chain variable region shown in SEQ ID NO:33, and three light chain complementarity-determining regions, LCDR1, LCDR2 and LCDR3, in the light chain variable region shown in SEQ ID NO:35.

[0009] The complementary decision region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0010] To achieve the above objective, according to a second aspect of the present invention, an antibody pair for detecting dengue virus is provided, the antibody pair comprising a 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.

[0011] 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.

[0012] The antibody pair includes a third antibody having HCDR1 as shown in SEQ ID NO:25, HCDR2 as shown in SEQ ID NO:26, HCDR3 as shown in SEQ ID NO:27, LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30.

[0013] To achieve the above objectives, according to a third aspect of the present invention, an antibody pair for detecting dengue virus is provided;

[0014] The antibody pair comprises a first antibody, which 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, the second antibody comprising at least one of (a)-(b):

[0018] (a) The heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23;

[0019] (b) The heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:24;

[0020] The antibody pair comprises a third antibody, which comprises at least one of (A)-(B):

[0021] (A) The heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:35;

[0022] (B) The heavy chain shown in SEQ ID NO:34 and the light chain shown in SEQ ID NO:36.

[0023] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody pair for detecting dengue virus is provided;

[0024] The antibody pair comprises a first antibody, wherein the variable region of the heavy chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:11.

[0025] The antibody pair comprises a second antibody, wherein the variable region of the heavy chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:21, and the variable region of the light chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:23.

[0026] The antibody pair comprises a third antibody, wherein the variable region of the heavy chain of the third antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:33, and the variable region of the light chain of the third antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:35.

[0027] The mutation is the addition, deletion, substitution, or modification of one or more amino acids in the framework region of the first antibody, second antibody, or third antibody.

[0028] In one alternative implementation, the framework region of the conserved variant formed by the mutation of the first antibody has at least 80% identity with the framework region of the first antibody before the mutation; or;

[0029] The framework region of the conserved variant formed by the mutation of the second antibody has at least 80% identity with the framework region of the pre-mutant second antibody; or;

[0030] The framework region of the conserved variant formed by the mutation of the third antibody has at least 80% identity with the framework region of the original third antibody.

[0031] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody pair for detecting dengue virus is provided;

[0032] The antibody pair comprises a first antibody that binds to an epitope, the epitope being 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.

[0033] The antibody pair comprises a second antibody that 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:21 and the light chain variable region shown in SEQ ID NO:23;

[0034] The antibody pair comprises a third antibody that binds to an epitope, the epitope being the same as the epitope bound to the antibody comprising the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:35.

[0035] To achieve the above objectives, according to a sixth aspect of the present invention, an antibody pair for detecting dengue virus is provided, the antibody comprising a constant region, wherein the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, or a combination of multiple constant regions; and the light chain constant region is selected from κ-type or λ-type light chain constant regions.

[0036] In an optional embodiment, the heavy chain constant region of the first antibody is SEQ ID NO:7 or a sequence having at least 80% identity with it, and the light chain constant region of the first antibody is SEQ ID NO:8 or a sequence having at least 80% identity with it.

[0037] In an optional embodiment, the heavy chain constant region of the second antibody is SEQ ID NO:19 or a sequence having at least 80% identity with it, and the light chain constant region of the second antibody is SEQ ID NO:20 or a sequence having at least 80% identity with it.

[0038] In an optional embodiment, the heavy chain constant region of the third antibody is SEQ ID NO:31 or a sequence having at least 80% identity with it, and the light chain constant region of the third antibody is SEQ ID NO:32 or a sequence having at least 80% identity with it.

[0039] In an optional implementation, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0040] In one alternative embodiment, the antibody is a polymer formed by the polymerization of antibody monomers.

[0041] To achieve the above objectives, according to a seventh aspect of the present invention, a reagent for detecting dengue virus is provided.

[0042] The reagents include a first group of antibodies, a second group of antibodies, and a third group of antibodies. The first group of antibodies includes the first antibody in the antibody combination described above; the second group of antibodies includes the second antibody in the antibody combination described above; and the third group of antibodies includes the third antibody in the antibody combination described above.

[0043] In an optional implementation, one of the first group of antibodies, the second group of antibodies, and the third group of antibodies is a coating antibody, and the other two groups are labeled antibodies. For example, the first group of antibodies is a coating antibody, and the second and third groups of antibodies are labeled antibodies.

[0044] In one optional implementation, two of the first group of antibodies, the second group of antibodies, and the third group of antibodies are coating antibodies, and the other group is a labeling antibody. For example, the first group of antibodies and the second group of antibodies are coating antibodies, and the third group of antibodies is a labeling antibody.

[0045] In an optional embodiment, the coated antibody or labeled antibody is conjugated with biotin or a biotin derivative;

[0046] In an optional embodiment, the label conjugated with the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0047] In an optional embodiment, the coated antibody is coupled to a solid-phase support;

[0048] In one alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0049] To achieve the above objectives, according to an eighth aspect of the present invention, a method for detecting dengue virus is provided, comprising:

[0050] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody pair or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0051] b) Detect the presence of the immune complex, the presence of which indicates the presence of dengue virus in the test sample.

[0052] To achieve the above objectives, according to a ninth aspect of the present invention, the use of the above-described antibody pairs or reagents in detecting dengue virus or in preparing dengue virus detection products is provided. Detailed Implementation

[0053] In a first aspect, embodiments of the present invention provide an antibody pair for detecting dengue virus, the antibody pair comprising a first antibody, the first antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:11; the antibody pair comprising a second antibody, the second antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:21 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:23; the antibody pair comprising a third antibody, the third antibody comprising: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:33 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:35.

[0054] The complementary decision region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0055] In this invention, the term "antibody" is used in the broadest sense, and the antibody in the method of this invention can be a whole antibody, an antigen-binding fragment, or a polymeric antibody capable of binding to dengue virus.

[0056] Whole antibodies can be monoclonal. Such whole antibodies are typically prepared by any suitable method known in the art. For example, antibody molecules are isolated from the serum of a mammal, typically a rabbit or mouse, immunized with dengue virus under suitable conditions. 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 alternative embodiment, the antigen-binding fragment is selected from any one of the F(ab')2, Fab', Fab, Fv, and scFv of the antibody. The antigen-binding fragment of the above-described antibody typically has the same binding specificity as the antibody from which it originates. As will be readily understood by those skilled in the art according to the description of the present invention, the antigen-binding fragment of the above-described antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the structure of the complete antibody disclosed in this invention, those skilled in the art can readily obtain the above-described antigen-binding fragment. The antigen-binding fragment of the above-described antibody can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, an automated peptide synthesizer, such as those sold by AppliedBioSystems. Polymerized antibodies are polymers formed by the polymerization of whole antibodies and antigen-binding fragments.

[0057] Secondly, embodiments of the present invention provide an antibody pair for detecting dengue virus, the antibody pair comprising a first antibody, a second antibody, and a third antibody; the first antibody having HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; the second antibody having HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:15, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; the third antibody having HCDR1 as shown in SEQ ID NO:25, HCDR2 as shown in SEQ ID NO:26, HCDR3 as shown in SEQ ID NO:27, LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30.

[0058] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0059] Methods for defining CDRs are well-known in the art and 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., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any 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 lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, with slight variations in labeling across different literature. Given the amino acid sequence of the variable region of an antibody, those 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 those in Table 1, are also within the scope of this disclosure.

[0060] Table 1: CDR Definition 1

[0061]

[0062]

[0063] 1The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0064] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0065] 3 If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0066] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0067] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0068] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0069] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0070] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0071] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0072] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0073] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0074] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0075] The antibody of the present invention also includes a frame region. In the present invention, the "frame region" or "FR" region includes a heavy chain frame region and a light chain frame region, which refers to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR. The heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including LFR1, LFR2, LFR3 and LFR4 frame regions.

[0076] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0077] In some optional embodiments of the present invention, the dengue virus detection antibody provided by the present invention may also be an antibody having 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 first, second, or third antibody in the antibody pair in the FR region.

[0078] Thirdly, embodiments of the present invention provide an antibody pair for detecting dengue virus.

[0079] 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:21 and a light chain variable region shown in SEQ ID NO:23; and the antibody pair comprises a third antibody having a heavy chain variable region shown in SEQ ID NO:33 and a light chain variable region shown in SEQ ID NO:35.

[0080] In an optional embodiment, the antibody pair comprises a first antibody having a heavy chain as shown in SEQ ID NO:10 and a light chain as shown in SEQ ID NO:12; the antibody pair comprises a second antibody having a heavy chain as shown in SEQ ID NO:22 and a light chain as shown in SEQ ID NO:24; and the antibody pair comprises a third antibody having a heavy chain as shown in SEQ ID NO:34 and a light chain as shown in SEQ ID NO:36.

[0081] Fourthly, embodiments of the present invention provide an antibody pair for detecting dengue virus, the antibody pair comprising a first antibody, wherein the variable region of the heavy chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:11; the antibody pair comprising a second antibody, wherein the variable region of the heavy chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:21, and the variable region of the light chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:23; the antibody pair comprising a third antibody, wherein the variable region of the heavy chain of the third antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:33, and the variable region of the light chain of the third antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:35.

[0082] The conserved variant refers to a mutant obtained by replacing the amino acid at the mutation site with an amino acid with the same chemical properties as the original amino acid.

[0083] The mutation is the addition, deletion, substitution, or modification of one or more amino acids in the framework region of the first antibody, second antibody, or third antibody.

[0084] Fifthly, embodiments of the present invention provide an antibody pair for detecting dengue virus, the antibody pair comprising a first antibody that binds to an epitope, the epitope being identical to the epitope bound by an antibody containing 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 comprising a second antibody that binds to an epitope, the epitope being identical to the epitope bound by an antibody containing the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23; and the antibody pair comprising a third antibody that binds to an epitope, the epitope being identical to the epitope bound by an antibody containing the heavy chain variable region shown in SEQ ID NO:33 and the light chain variable region shown in SEQ ID NO:35.

[0085] The epitope, also known as an antigen epitope (AE), determines the ability of an antigen to specifically bind to an antibody. Antibodies bind to the same epitope because the amino acid fragments they bind to the target antigen are identical. Whether an antibody recognizes the same epitope as other antibodies can be confirmed by their competition for the epitope. Competition between antibodies can be evaluated using competitive binding assays, such as ELISA, fluorescence energy transfer assay (FRET), or fluorescence microassay (FMAT). The amount of antibody binding to the antigen is indirectly related to the binding ability of candidate competing antibodies (the antibody being tested) that compete for the same epitope. That is, the greater the binding amount or affinity of the antibody being tested for the same epitope, the lower the amount of antibody binding to the antigen, and the higher the amount of antibody binding to the antigen. Specifically, an appropriately labeled antibody and the antibody to be evaluated are added simultaneously to the antigen, and the binding of the antibody is detected using the labeling. By pre-labeling the antibody, the amount of antibody binding to the antigen can be easily determined. There are no particular limitations on the labeling; a labeling method appropriate to the assay technique is selected. Labeling methods include: fluorescent labeling, radioactive labeling, enzyme labeling, etc.

[0086] In an optional embodiment, the antibody further includes a constant region, wherein the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; the light chain constant region is selected from the κ-type or λ-type light chain constant region. The λ-type light chain constant region may be selected from the λ1, λ2, λ3, and λ4 subtypes.

[0087] 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 aforementioned constant regions (SEQ ID NO: 7, 8, 19, 20, 31, or 32).

[0088] In an optional implementation, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

[0089] In one optional implementation, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans;

[0090] In one alternative embodiment, the antibody is a polymer formed by the polymerization of antibody monomers.

[0091] In a seventh aspect, embodiments of the present invention provide a reagent for detecting dengue virus, the reagent comprising a first group of antibodies, a second group of antibodies, and a third group of antibodies, wherein the first group of antibodies comprises a first antibody in the antibody combination; the second group of antibodies comprises a second antibody in the antibody combination; and the third group of antibodies comprises a third antibody in the antibody combination.

[0092] In an optional implementation, one of the first group of antibodies, the second group of antibodies, and the third group of antibodies is a coating antibody, and the other two groups are labeled antibodies. For example, the first group of antibodies is a coating antibody, and the second and third groups of antibodies are labeled antibodies.

[0093] In one optional implementation, two of the first group of antibodies, the second group of antibodies, and the third group of antibodies are coating antibodies, and the other group is a labeling antibody. For example, the first group of antibodies and the second group of antibodies are coating antibodies, and the third group of antibodies is a labeling antibody.

[0094] It should be noted that the meaning of "reagent" in this application can be considered equivalent to the meaning of "kit".

[0095] In an optional embodiment, the coated antibody or labeled antibody is conjugated with biotin or a biotin derivative;

[0096] In an optional embodiment, the label conjugated with the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0097] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0098] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0099] In optional embodiments, 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.

[0100] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0101] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0102] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0103] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0104] In an optional embodiment, the colloidal metal is colloidal gold.

[0105] In an optional embodiment, the coated antibody is coupled to a solid-phase support;

[0106] In one alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0107] Eighthly, embodiments of the present invention provide a method for detecting dengue virus, comprising:

[0108] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody pair or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0109] b) Detect the presence of the immune complex, the presence of which indicates the presence of dengue virus in the test sample.

[0110] To achieve the above objectives, according to a ninth aspect of the present invention, the use of the above-described antibody pairs or reagents in detecting dengue virus or in preparing dengue virus detection products is provided.

[0111] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0112] 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0113] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0114] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0115] Based on long-term and creative research on dengue virus, the inventors discovered two antibodies that can meet the requirements for dengue virus detection.

[0116] Example 1 Antibody Preparation

[0117] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit and pMD-18T vector were purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. Existing hybridoma cell lines secreting dengue virus monoclonal antibodies were used and were revived for later use.

[0118] (1) Antibody gene preparation

[0119] mRNA was extracted from hybridoma cell lines that secrete dengue virus monoclonal antibodies, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After the cells grew, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0120] (2) Sequence analysis of the dengue virus antibody variable region gene

[0121] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was approximately 320 bp, with a 57 bp leader peptide sequence preceding it. Among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was approximately 360 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0122] (3) Construction of recombinant antibody expression plasmid

[0123] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with 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. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The Light Chain gene fragment and Heavy Chain gene fragment were amplified by PCR.

[0124] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0125] (4) Production of recombinant antibodies

[0126] HEK293 cells were prematurely revived and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁶ cells / ml, meeting the antibody selection concentration and cell viability >95%. Cells were washed by centrifugation, rehydrated with culture medium, and the cell density was adjusted to 2.9 × 10⁶ cells / ml. This was then used as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed, and samples were taken for counting. Cell viability after transfection was recorded and observed. Cells were then incubated at 35°C (120 rpm) with 8% CO₂ for 13 days. Samples were collected by centrifugation. The supernatant was purified using a Protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. Two bands were observed after reducing SDS-PAGE, one with Mr 50 KD (heavy chain) and the other with Mr 28 KD (light chain).

[0127] The resulting antibodies were named Anti-DN Rmb7, Anti-DN Rmb9, and Anti-DN Rmb2. The heavy chain (H) and light chain (L) sequences of the above antibodies are shown in the table below. All of the following antibodies can specifically bind to dengue virus proteins.

[0128] Table 2 Antibody Sequence Light Chain and Heavy Chain Table

[0129] Anti-DN Rmb7 SEQ ID NO:10 SEQ ID NO:12 Anti-DN Rmb9 SEQ ID NO:22 SEQ ID NO:24 Anti-DN Rmb2 SEQ ID NO:34 SEQ ID NO:36

[0130] Example 2 Reagent Preparation

[0131] Anti-DN Rmb7 and Anti-DN Rmb9 were used as coating antibodies, and Anti-DN Rmb2 was used as a labeling antibody.

[0132] 1. Antibody labeling: Take 5 ml of colloidal gold at a concentration of 40,000, add 50-60 μL of 0.2 M K₂CO₃ (pH 7.2-7.4), stir for 5 min, add the antibody to be labeled (antibody volume = 50 μg / antibody concentration), stir for 5 min, then add 50 μL of (5% BSA + 5% casein) for blocking and termination labeling; centrifuge at 10,000 rpm for 7 min, discard the supernatant, reconstitute the precipitate with gold reconstitution solution, and finally adjust the volume to 0.5 ml (i.e., 1 / 10 of the colloidal gold solution volume) with gold reconstitution solution.

[0133] 2. Preparation of gold working solution: Dilute the dengue virus labeled antibody concentrated gold to 12 OD using gold reconstitution solution to prepare gold working solution; spread it on glass fiber;

[0134] 3. Prepare dried gold: Place the spread gold in a freeze dryer to freeze dry (1-2 hours) or place it in a 37-degree drying room to dry overnight;

[0135] 4. Sample pad treatment: Dilute the blocking agent with 20mM PB+1 / 10,000 Tween20 to 0.4mg / ml, spread it on the sample pad, and freeze-dry it in a freeze dryer (1-2h) or dry it overnight in a drying room at 37℃.

[0136] 5. Antibody coating: Dilute the antibody to be coated with coating diluent to a final concentration of 1.5 mg / ml and then coat it. After coating, incubate at 37 degrees Celsius for 2-4 hours or overnight.

[0137] 6. Preparation of gold bar: Cut the gold bar into strips of the required width using a strip cutter, assemble them, and then add samples for testing.

[0138] Example 3 Performance Test

[0139] Place the prepared test strips (3cm wide each) into the card. When testing the quality control antigen, dilute the quality control strip to the corresponding dilution factor using sample diluent, then add 45µl of diluted sample to each test strip. Read the results after 15–30 minutes. When testing positive serum, add 45µl of diluted sample to each test strip, and read the results after 15–30 minutes. The concentration of the test samples and the detection performance results of the antibody combinations are shown in Table 3.

[0140] Table 3 Comparison of Actual Sample Detection Results

[0141]

[0142]

[0143] Note: The colloidal gold card has 10 readings: 1, 2, 3, 4, 5, 6, 7, 8, 9, and B. The corresponding T-line colors of these 10 readings decrease from left to right, with 1 indicating the darkest T-line color, 9 indicating the lightest T-line color, B indicating no color development, and B+ indicating almost no color development. In the table, "8+" means a color slightly darker than 8 but not reaching 7, and so on for the others marked with "+". In the table, "8-" means a color slightly lighter than 8 but not reaching 9, and so on for the others marked with "-".

[0144] The data in the table above show that the antibody labeling and coating regimen in this application achieves a significant improvement in detection activity compared to commercially available products. Specifically, in detecting dengue quality control samples, the detection activity was improved under different detection concentration conditions, with an increase of 0–1.5C. In detecting 20 clinical samples, the detection activity was also improved compared to commercially available products, with an increase of 1–5C. The results of testing 211 random serum samples showed that the antibody labeling and coating regimen in this application achieved 100% specificity in negative samples.

[0145] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0146] The partial amino acid sequence involved in this application is shown below:

[0147]

[0148]

[0149]

Claims

1. An antibody pair for detecting dengue virus NS1 protein, characterized in that, The antibody pair includes a first antibody, whose complementarity-determining regions are: three heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:9, and three light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

11. The antibody pair includes a second antibody, the complementarity-determining regions of which are: three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:21, and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:23; and The antibody pair includes a third antibody, whose complementarity-determining regions are: three heavy chain complementarity-determining regions, HCDR1, HCDR2 and HCDR3, in the heavy chain variable region as shown in SEQ ID NO:33, and three light chain complementarity-determining regions, LCDR1, LCDR2 and LCDR3, in the light chain variable region as shown in SEQ ID NO:

35. The complementary determination region is defined by any one of the following systems: Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody pair for detecting dengue virus NS1 protein, characterized in that, The antibody pair includes a first antibody, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody being shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The antibody pair includes a second antibody, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antibody being shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; and The antibody pair includes a third antibody, the amino acid sequences of which are HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively.

3. An antibody pair for detecting dengue virus NS1 protein, characterized in that, The antibody pair comprises a first antibody, which comprises at least one of (1)-(2): (1) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:9 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:11; (2) The heavy chain with an amino acid sequence as shown in SEQ ID NO:10 and the light chain with an amino acid sequence as shown in SEQ ID NO:12; The antibody pair comprises a second antibody, the second antibody comprising at least one of (a)-(b): (a) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:21 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:23; (b) The heavy chain with the amino acid sequence shown in SEQ ID NO:22 and the light chain with the amino acid sequence shown in SEQ ID NO:24; and The antibody pair comprises a third antibody, which comprises at least one of (A)-(B): (A) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:33 and the light chain variable region with amino acid sequence as shown in SEQ ID NO:35; (B) The heavy chain with the amino acid sequence shown in SEQ ID NO:34 and the light chain with the amino acid sequence shown in SEQ ID NO:

36.

4. The antibody pair for detecting dengue virus NS1 protein according to claim 2, characterized in that, The variable region of the heavy chain of the first antibody is shown in SEQ ID NO:9, and the variable region of the light chain is shown in SEQ ID NO:

11. The framework region of the conserved variant formed by the mutation of the first antibody has at least 80% identity with the framework region of the first antibody before the mutation. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody are shown in SEQ ID NO:1 to 6 in sequence. The variable region of the heavy chain of the second antibody is shown in SEQ ID NO:21, and the variable region of the light chain is shown in SEQ ID NO:

23. The framework region of the conserved variant formed by the mutation of the second antibody has at least 80% identity with the framework region of the second antibody before the mutation. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antibody are shown in SEQ ID NO:13~18 in sequence. The variable region of the heavy chain of the third antibody is shown in SEQ ID NO:33, and the variable region of the light chain is shown in SEQ ID NO:

35. The framework region of the conserved variant formed by the mutation of the third antibody has at least 80% identity with the framework region of the third antibody before the mutation. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the third antibody are shown in SEQ ID NO:25-30, respectively.

5. The antibody pair for detecting dengue virus NS1 protein according to any one of claims 1-4, characterized in that, The antibody further includes a constant region, wherein the heavy chain constant region is selected from any one of the heavy chain constant regions 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 the κ-type or λ-type light chain constant region.

6. The antibody pair for detecting dengue virus NS1 protein according to claim 5, characterized in that, The heavy chain constant region of the first antibody is a sequence with at least 80% identity to SEQ ID NO:7, and the light chain constant region of the first antibody is a sequence with at least 80% identity to SEQ ID NO:8; The heavy chain constant region of the second antibody is a sequence with at least 80% identity to SEQ ID NO:19, and the light chain constant region of the second antibody is a sequence with at least 80% identity to SEQ ID NO:20; The heavy chain constant region of the third antibody is a sequence with at least 80% identity to SEQ ID NO:31, and the light chain constant region of the third antibody is a sequence with at least 80% identity to SEQ ID NO:

32.

7. The antibody pair for detecting dengue virus NS1 protein according to claim 5, characterized in that, The amino acid sequence of the heavy chain constant region of the first antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain constant region of the first antibody is shown in SEQ ID NO:

8. The amino acid sequence of the heavy chain constant region of the second antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain constant region of the second antibody is shown in SEQ ID NO:

20. The amino acid sequence of the heavy chain constant region of the third antibody is shown in SEQ ID NO:31, and the amino acid sequence of the light chain constant region of the third antibody is shown in SEQ ID NO:

32.

8. The antibody pair for detecting dengue virus NS1 protein according to claim 5, characterized in that, The heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM, and / or CH4 of IgM.

9. A reagent for detecting dengue virus, characterized in that, The reagent comprises a first group of antibodies, a second group of antibodies, and a third group of antibodies. The first group of antibodies comprises the first antibody in any one of the antibody pairs of claims 1-8. The second group of antibodies comprises the second antibody in any one of the antibody pairs of claims 1-8. The third group of antibodies comprises the third antibody in any one of the antibody pairs of claims 1-8.

10. The reagent for detecting dengue virus NS1 protein according to claim 9, characterized in that, One group of the first group of antibodies, the second group of antibodies, and the third group of antibodies is a coating antibody, and the other two groups are labeled antibodies; or; two groups of the first group of antibodies, the second group of antibodies, and the third group of antibodies are coating antibodies, and the other group is a labeled antibody.

11. The reagent for detecting dengue virus NS1 protein according to claim 10, characterized in that, The coated antibody or labeled antibody is conjugated with biotin.

12. The reagent for detecting dengue virus NS1 protein according to claim 10, characterized in that, The labeled antibody-conjugated markers are selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent markers, electrochemiluminescent markers, latex and nanoparticle markers.

13. The reagent for detecting dengue virus NS1 protein according to claim 10, characterized in that, The coated antibody is coupled to a solid-phase carrier.

14. The reagent for detecting dengue virus NS1 protein according to claim 13, characterized in that, The solid support is selected from microspheres, plates, and membranes.

15. Use of the antibody pair according to any one of claims 1-8 or the reagent according to any one of claims 9-14 in the preparation of a product for detecting dengue virus NS1 protein.

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