Antibody against dengue virus or NS1 protein thereof, and use thereof

Abstract

Disclosed are an antibody against dengue virus or an NS1 protein thereof, and the use thereof. The disclosed antibody against dengue virus or the NS1 protein thereof comprises heavy chain complementarity determining regions and light chain complementarity determining regions. The antibody provides an important raw material source for the detection of dengue virus or the NS1 protein thereof, and has a good affinity or activity.

Description

Antibody against dengue virus or its NS1 protein and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number 202311110160.7 filed with the Patent Office of China on August 30, 2023, entitled “An antibody against dengue virus or its NS1 protein and its application,” the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present disclosure relates to the field of antibody technology, and in particular to an antibody against dengue virus or its NS1 protein and applications thereof. Background Art

[0004] Dengue fever (DF) is an acute mosquito-borne infectious disease caused by four serotypes of the virus (DENV-1, DENV-2, DENV-3, and DENV-4), primarily transmitted by Aedes aegypti and Aedes albopictus. DF is the most widespread, most common, and most harmful insect-borne viral disease, prevalent in more than 100 countries and regions across tropical and subtropical Africa, the Americas, Southeast Asia, and the Western Pacific.

[0005] Clinically, dengue fever (DF) is a severe, flu-like illness. Its main symptoms are sudden onset, high fever, severe headache, retroorbital pain, muscle and joint pain, and may be accompanied by rash, swollen lymphadenopathy, and leukopenia. It can affect all individuals, but symptoms vary depending on the patient's age. This form of the disease, generally referred to as classical dengue fever, spreads rapidly and can cause large-scale epidemics. During dengue epidemics, the attack rate among susceptible individuals is typically 40%-50%, but can be as high as 80%-90%, but the mortality rate is very low. Dengue hemorrhagic fever is characterized by high fever, bleeding, hepatomegaly, and, in severe cases, circulatory failure. It has a high mortality rate and is a more severe clinical form. The accompanying shock syndrome is called dengue shock syndrome.

[0006] There is no specific treatment for dengue fever. Without appropriate treatment, the mortality rate for dengue hemorrhagic fever can exceed 20%. With effective supportive care, the mortality rate can be reduced to less than 1%. Key points for dengue fever diagnosis include: 1) Epidemiological data, including activity status in the 15 days prior to onset, travel to endemic areas, and history of mosquito bites; 2) Clinical features, including sudden onset, fever, "three pains and three reds," and skin examination; 3) Laboratory tests, including decreased white blood cells and platelets; positive serum specific IgM; a four-fold increase in IgG in the recovery phase compared to the acute phase; and isolation of the virus or specific antigen.

[0007] Current methods for detecting dengue virus include viral culture, serological testing, and immunoassays. Virus isolation requires a long time, making rapid diagnosis impossible. Conventional serological diagnosis is also subject to interference due to extensive cross-reactivity. Immunoassays are rapid, simple, require no critical equipment, and can be performed on-site, making them a hot topic of research in the rapid diagnosis of infectious diseases. Immunoassays are based on antigen-antibody reactions. The NS1 protein is a glycoprotein within the nonstructural proteins of the dengue virus. It is highly antigenic and does not induce ADE, making it a common target for immunoassays.

[0008] Immunoassays require antibodies against dengue virus or its NS1 protein. Therefore, there is a strong demand in the art for antibodies against dengue virus or its NS1 protein with good performance.

[0009] Summary of the Invention

[0010] The present application provides an antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, which provides an important source of raw materials for the detection of dengue virus or its NS1 protein and has good activity or affinity.

[0011] To achieve the above objectives, according to one aspect of the present disclosure, an antibody or an antigen-binding fragment thereof against dengue virus or its NS1 protein is provided, wherein the antibody or the antigen-binding fragment thereof comprises three complementarity determining regions of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and three complementarity determining regions of a light chain variable region having the amino acid sequence of any one of SEQ ID NOs: 22, 23, 24, and 25.

[0012] To achieve the above objectives, according to a second aspect of the present disclosure, an antibody or antigen-binding fragment thereof is provided against dengue virus or its NS1 protein, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions:

[0013] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0014] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0015] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0016] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 17;

[0017] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0018] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0019] To achieve the above objectives, according to the third aspect of the present disclosure, an antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 20; and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 22, 23, 24, and 25.

[0020] To achieve the above objectives, according to the fourth aspect of the present disclosure, an antibody or an antigen-binding fragment thereof against dengue virus or its NS1 protein is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 21; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29.

[0021] In order to achieve the above objectives, according to a fifth aspect of the present disclosure, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or antigen-binding fragment thereof.

[0022] In order to achieve the above-mentioned object, according to the sixth aspect of the present disclosure, a reagent or kit is provided, wherein the reagent or kit comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0023] To achieve the above objectives, according to the seventh aspect of the present disclosure, a method for detecting dengue virus or its NS1 protein is provided, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, or reagent or kit with dengue virus or its NS1 protein in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0024] To achieve the above objectives, according to the eighth aspect of the present disclosure, there is provided a use of the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit in the preparation of a product for detecting dengue virus or its NS1 protein.

[0025] To achieve the above objectives, the present disclosure also provides a nucleic acid, a vector, a cell, and a method for preparing the above antibody or antigen-binding fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0027] FIG1 shows the results of reducing SDS-PAGE of Anti-DN 8G12 Rmb1 to Anti-DN 8G12 Rmb4. DETAILED DESCRIPTION

[0028] In a first aspect, the embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof against dengue virus or its NS1 protein, wherein the antibody or the antigen-binding fragment thereof comprises three complementarity determining regions of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and three complementarity determining regions of a light chain variable region having any one of the amino acid sequences of SEQ ID NO: 22, 23, 24, and 25.

[0029] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect.

[0030] For example, the HCDR1, HCDR2, and HCDR3 have amino acid sequences consistent with those of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO: 20; and the LCDR1, LCDR2, and LCDR3 have amino acid sequences consistent with those of LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO: 22.

[0031] In this disclosure, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0032] In the present disclosure, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more, or even all, of the primary amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibodies.

[0033] In the present disclosure, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0034] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200,255,854,554, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but may 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, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0035] Table 1: CDR Definition 1

[0036] 1 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+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this 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. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0037] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] According to an embodiment of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0042] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0043] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0044] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0045] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0046] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0047] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0048] According to an embodiment of the present disclosure, the Kabat numbering positions corresponding to the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 defined by the Kabat, Chothia, AbM or IMGT systems are as follows:

[0049] In a second aspect, the present disclosure provides an antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions:

[0050] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0051] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0052] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0053] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 17;

[0054] LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 5; and

[0055] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0056] According to an embodiment of the present disclosure, the HCDRs and LCDRs are defined by the Kabat system.

[0057] In the present disclosure, "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0058] In the present disclosure, the heavy chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0059] In an optional embodiment, the antibody or antigen-binding fragment thereof of the first aspect or the second aspect further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0060] The HFR1 comprises / is as SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0061] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0062] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0063] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0064] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0065] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0066] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0067] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0068] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or antigen-binding fragments thereof provided herein against dengue virus or its NS1 protein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0069] In an optional embodiment, the LFR1 includes / is represented by the amino acid sequence of SEQ ID NO: 18 or 19.

[0070] In a third aspect, the embodiments of the present disclosure provide an antibody or an antigen-binding fragment thereof against dengue virus or its NS1 protein, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 22, 23, 24, and 25.

[0071] In an optional embodiment, the heavy chain variable region and the light chain variable region of the first aspect or the third aspect are selected from any one of the following combinations:

[0072] In an optional embodiment, the antibody or antigen-binding fragment thereof described in the first, second, or third aspect further comprises a constant region.

[0073] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0074] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0075] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0076] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0077] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0078] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0079] In an alternative embodiment, the species origin of the constant region is mouse.

[0080] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0081] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0082] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0083] The antigen-binding fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that the antigen-binding fragments of the above-mentioned antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction cleavage of disulfide bonds. Based on the structure of the intact antibody disclosed herein, those skilled in the art can readily obtain the above-mentioned antigen-binding fragments.

[0084] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0085] In a fourth aspect, the present disclosure provides an antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being shown in SEQ ID NO: 21, and the amino acid sequence of the light chain being shown in any one of SEQ ID NOs: 26, 27, 28, and 29.

[0086] In an optional embodiment, the antibody or antigen-binding fragment thereof of the first, second, third or fourth aspect comprises any combination of the following heavy chains and light chains:

[0087] In a fifth aspect, the present disclosure provides an antibody conjugate, which comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0088] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

[0089] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

[0090] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0091] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0092] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present disclosure.

[0093] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and derivatives thereof (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and derivatives thereof (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and derivatives thereof (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and derivatives thereof (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 derivatives thereof (for example, including but not limited to phycoerythrin (PE), Phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0094] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0095] 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.

[0096] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0097] 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.

[0098] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0099] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0100] In an optional embodiment, the colloidal metal is colloidal gold.

[0101] In an optional embodiment, the above-mentioned antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0102] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0103] In a sixth aspect, the present disclosure provides a reagent or kit, which comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0104] As mentioned above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present disclosure can effectively bind to dengue virus or its NS1 protein. Therefore, the reagents or kits containing the anti-dengue virus or its NS1 protein antibodies or antigen-binding fragments can effectively detect dengue virus or its NS1 protein qualitatively or quantitatively. The reagents or kits provided by the present disclosure can be used, for example, for detection involving the use of dengue virus or its NS1 protein and its antibody-specific binding properties in immunoblotting, immunoprecipitation, etc. As mentioned above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present disclosure have higher binding activity or affinity with dengue virus or its NS1 protein. Therefore, the reagents or kits containing the antibodies or antigen-binding fragments thereof have higher detection sensitivity or specificity.

[0105] In a seventh aspect, the present disclosure provides a method for detecting dengue virus or its NS1 protein, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with dengue virus or its NS1 protein in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

[0106] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof.

[0107] In an alternative embodiment, the immune complex further comprises a second antibody that binds to dengue virus or its NS1 protein.

[0108] In an eighth aspect, the present disclosure provides use of the above-mentioned anti-dengue virus or its NS1 protein antibody or antigen-binding fragment thereof, antibody conjugate, or the above-mentioned reagent or kit in the preparation of a product for detecting dengue virus or its NS1 protein.

[0109] In a ninth aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0110] In a tenth aspect, the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

[0111] In an eleventh aspect, the present disclosure provides cells containing the above-mentioned vector.

[0112] In a twelfth aspect, the present disclosure provides a method for preparing an anti-dengue virus or NS1 protein antibody or an antigen-binding fragment thereof, comprising: culturing the cells as described above.

[0113] Based on the amino acid sequence of the anti-dengue virus or its NS1 protein antibody or its antigen-binding fragment disclosed in the present disclosure, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-dengue virus or its NS1 protein antibody or its antigen-binding fragment, for example, separating and purifying the antibody or its antigen-binding fragment from the culture product of a recombinant cell capable of recombinantly expressing the antibody or its antigen-binding fragment as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-dengue virus or its NS1 protein antibody or its antigen-binding fragment of the present disclosure, it falls within the scope of protection of the present disclosure.

[0114] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0116] Practice of the present disclosure 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. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (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); and PCR: The Polymerase Chain Reaction. 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.

[0117] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0118] Example 1 Preparation of Anti-DN 8G12 Monoclonal Antibody

[0119] 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 also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the Anti-DN 8G12 monoclonal antibody was generated in our laboratory and revived for future use.

[0120] (1) Antibody gene preparation

[0121] mRNA was extracted from a hybridoma cell line secreting the Anti-DN 8G12 monoclonal antibody. DNA products were obtained by RT-PCR. This product was PCR-amplified with rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0122] (2) Sequence analysis of the variable region gene of Anti-DN 8G12 antibody

[0123] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and 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 them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 321 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 342 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

[0124] (3) Construction of recombinant antibody expression plasmid

[0125] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.70 kb light chain gene fragment and a 1.37 kb heavy chain gene fragment.

[0126] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and 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.

[0127] 2. Recombinant Antibody Production

[0128] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 μg of culture medium and re-dissolved. This was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and allowed to stand at room temperature for 15 minutes. The mixture was slowly added to the cell diluent over 1 minute, mixed, and sampled. The viability of the cells after transfection was recorded and observed. The cells were cultured in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electrophoresis pattern is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with an Mr of 50 KD (heavy chain) and the other with an Mr of 28 KD (light chain).

[0129] The obtained antibody was named Anti-DN 8G12Rmb1. Anti-DN 8G12Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0130] Table 2 Antibody sequences

[0131] Example 2 Performance testing of antibodies

[0132] 1. Activity identification

[0133] Coating solution (main component NaHCO3) diluted goat anti-mouse IgG 1μg / ml for microtiter plate coating, 100μL per well, 4℃ overnight; the next day, wash twice with washing solution (main component Na2HPO4+NaCl), pat dry; add blocking solution (20% BSA+80% PBS), 120μL per well, 37℃, 1h, pat dry; add diluted purified antibody, 100μL / well, 37℃, 60min; shake off the liquid in the plate, pat dry, add 20% mouse negative blood for blocking, 120μL per well, 37℃, 1h; shake off the liquid in the plate, pat dry, add Dilute dengue NS1 antigen (from Feipeng Biotechnology), 100 μL per well, 37°C, 40 min; wash 5 times with detergent and pat dry; add HRP-labeled anti-dengue NS1 antibody (from Feipeng Biotechnology, paired with purified antibody), 100 μL per well, 37°C, 30 min; add colorimetric solution A (50 μL / well) and colorimetric solution B (50 μL / well), 10 min; add stop solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on a microplate reader.

[0134] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4).

[0135] Table 3 Activity data

[0136] 2. Application of antibodies in colloidal gold detection

[0137] 2.1 Preparation of colloidal gold test paper

[0138] 2.1.1 Preparation of nitrocellulose membrane

[0139] Preparation of nitrocellulose membrane: Dilute anti-dengue NS1 protein antibody Anti-DN (Purchased from Feipeng Biotechnology) to 1-5 mg / mL in coating buffer. Adjust the machine to draw a line (T), the test line, near the end of the gold pad. Dilute goat anti-mouse IgG antibody to 1-5 mg / mL in coating buffer. Adjust the machine to draw a line (C), the control line, near the absorbent pad. Keep the two lines evenly spaced 5-8 mm apart. Dry at 37°C and seal for later use.

[0140] 2.1.2 Preparation of colloidal gold and gold-labeled monoclonal antibodies

[0141] Dilute 1% chloroauric acid to 0.01% with double-distilled deionized water. Bring to a boil on an electric stove. Add 2 mL of 1% trisodium citrate per 100 mL of 0.01% chloroauric acid. Continue boiling until the liquid turns bright red. Remove from heat and allow to cool to room temperature before adding water. Prepared colloidal gold should be pure, translucent, and free of precipitates and floating matter.

[0142] Adjust the pH of the colloidal gold to 8.2 with 0.1M potassium carbonate, then add the aforementioned antibodies Anti-DN 8G12Rmb1 to Anti-DN 8G12Rmb4 and mainstream commercial raw materials. Mix using a magnetic stirrer for 30 minutes. Add BSA to a final concentration of 1% while stirring and let stand for 1 hour. Centrifuge at 13,000 rpm and 4°C for 30 minutes, discard the supernatant, and wash the pellet twice with labeling wash buffer. Resuspend the pellet in labeling wash buffer at one-tenth the initial colloidal gold volume. Store the prepared colloidal gold-labeled antibodies Anti-DN 8G12Rmb1 to Anti-DN 8G12Rmb4 at 4°C until ready for use.

[0143] 2.1.3 Preparation of gold label pad

[0144] Soak the gold-labeled pad in blocking solution for 30 minutes and then dry it at 37°C. Then evenly spread the prepared gold-labeled antibody on the gold-labeled pad, with 20 square centimeters per milliliter of solution, freeze-dry, package, and store at 4°C for later use.

[0145] 2.1.4 Assembly of test strips

[0146] An absorption pad (purchased from Millipore), a nitrocellulose membrane, a gold-labeled pad, and a sample pad were placed on a non-absorbent support sheet and cut into 3 mm wide strips. Ten strips were packed into a pack, a desiccant was added, and the packing was vacuum-packed to obtain the test paper.

[0147] 2.2 Application of antibodies in colloidal gold detection

[0148] The assembled test strips were used to detect whether the test material contained dengue NS1 protein, thereby determining the effectiveness of antibodies Anti-DN 8G12Rmb1 to Anti-DN 8G12Rmb4 in detecting dengue NS1 protein.

[0149] A double-antibody sandwich method is used to detect the presence of dengue NS1 protein in the test material. During testing, the dengue NS1 protein first binds to the colloidal gold-labeled antibody to form a dengue NS1 protein-colloidal gold-labeled antibody complex. Due to capillary action, the dengue NS1 protein-colloidal gold-labeled antibody complex migrates along the nitrocellulose membrane. Upon reaching the test line, the dengue NS1 protein-colloidal gold-labeled antibody complex binds to the anti-dengue NS1 protein antibody Anti-DN prepared in step 3.1.1 of the above example, forming an Anti-DN-dengue NS1 protein-colloidal gold-labeled antibody complex. This complex is then enriched on the test line, forming a red precipitate line. The dengue NS1 protein-colloidal gold-labeled antibody complex that is not bound to the dengue NS1 protein antibody on the test line passes through the test line and is captured by goat anti-mouse IgG antibodies, enriching on the quality control line, forming a red precipitate line. A positive result is determined when both the test line and the quality control line have red precipitate lines. If the sample does not contain dengue NS1 protein, the colloidal gold-labeled antibody that has not bound to dengue NS1 protein will not form an Anti-DN-dengue NS1 protein-colloidal gold-labeled antibody complex when it reaches the test line. The colloidal gold-labeled antibody complex that has not bound to dengue NS1 protein will pass through the test line and will only be enriched on the quality control line to form a red precipitate line. In this case, it is judged as a negative result.

[0150] Note: The gold label colorimetric assay consists of C plus a number. The smaller the number after C, the stronger the color and the higher the activity; the higher the number after C, the weaker the color and the lower the activity. A “+” after the number indicates a slightly stronger color of 0.5C, a “-” after the number indicates a slightly weaker color of 0.5C, and “B” represents negative.

[0151] Table 4 Performance evaluation data

[0152] 3. Stability assessment

[0153] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under all three test conditions for 21 days, and the activity did not decrease with increasing test temperature, indicating that the above-mentioned antibody is stable. Table 5 below shows the OD results of the enzyme immunoassay activity assay of the antibody Anti-DN 8G12Rmb4 after 21 days of testing.

[0154] Table 5 Stability data

[0155] The foregoing description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure.

[0156] Some of the amino acid sequences involved in this application are shown in Table 6: Industrial Applicability

[0157] The antibodies against dengue virus or its NS1 protein provided herein can specifically recognize and bind to dengue virus, have high sensitivity and specificity, and can be used for dengue virus detection. Therefore, the antibodies against dengue virus or its NS1 protein provided herein have excellent practical performance and broad market application prospects.

Claims

1. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, wherein the antibody or antigen-binding fragment thereof comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 20 and three complementary determining regions of a light chain variable region having an amino acid sequence of any one of SEQ ID NO: 22, 23, 24, and 25.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, characterized in that: The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or 17; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; Optionally, the antibody or antigen-binding fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 22, 23, 24, and 25; Optionally, the combination of the heavy chain variable region and the light chain variable region is selected from any one of the following combinations: Optionally, the antibody or antigen-binding fragment thereof further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

5. An antibody or antigen-binding fragment thereof against dengue virus or its NS1 protein, comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 21; the amino acid sequence of the light chain is shown in any one of SEQ ID NOs: 26, 27, 28, and 29.

6. A nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment according to any one of claims 1 to 5.

7. A vector, wherein The vector comprises the nucleic acid of claim 6.

8. A cell, characterized in that The cell comprises the nucleic acid of claim 6 or the vector of claim 7.

9. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 5, characterized in that: The method comprises culturing the cell of claim 8.

10. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof; Optionally, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

11. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7 or the antibody conjugate according to claim 10.

12. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 10, or the reagent or kit according to claim 11 in the preparation of a product for detecting dengue virus or its NS1 protein.

13. The use according to claim 12, characterized in that The product is used to detect dengue virus or dengue NS1 protein, diagnose dengue virus infection or diseases related to dengue virus infection.

14. A method for detecting dengue virus or its NS1 protein, characterized in that: include: a) contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 10, or the reagent or kit according to claim 11 with dengue virus or its NS1 protein in 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, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment thereof; Optionally, the immune complex further comprises a second antibody, which binds to the dengue virus or its NS1 protein.

15. A method for diagnosing a subject infected with dengue virus or a disease associated with dengue virus infection, comprising: a) contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the antibody conjugate according to claim 10, or the reagent or kit according to claim 11 with a sample from the subject under conditions sufficient for a binding reaction to occur to perform a binding reaction; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment thereof; Optionally, the immune complex further comprises a second antibody, which binds to the dengue virus or its NS1 protein.

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