WO2023078452A1 - Antibody against dengue ns1 protein and use thereof - Google Patents

Abstract

Provided are an antibody against a dengue NS1 protein and a preparation method therefor. The antibody provides a raw material source for the diagnosis of the dengue fever disease.

Description

A kind of antibody against dengue NS1 protein and its application

Cross References to Related Applications

This disclosure claims priority and rights to patent application number 202111315863.4 filed with the State Intellectual Property Office of China on November 8, 2021, and is hereby incorporated by reference in its entirety.

technical field

The present disclosure belongs to the technical field of antibodies. More specifically, it relates to an antibody against dengue NS1 protein and its application.

Background technique

Dengue fever (DF) is an acute mosquito-borne infectious disease caused by four serotypes of viruses (DENV-1, DENV-2, DENV-3, DENV-4), mainly transmitted by Aedes aegypti and Aedes albopictus DF is an arbovirus disease with the widest distribution, the most incidence, and great harm. It is widely prevalent in more than 100 countries and regions in tropical and subtropical Africa, America, Southeast Asia and the Western Pacific.

There is no specific treatment for dengue fever. If there is no appropriate treatment, the case fatality rate of dengue hemorrhagic fever can exceed 20%, and after effective supportive treatment, the case fatality rate can be less than 1%. Key points for dengue fever diagnosis: 1) epidemiological data, activities within 15 days after onset, whether you have been to endemic areas, history of mosquito bites; 2) clinical features, sudden onset, fever, "three pains and three reds", skin diagnosis ; 3) Laboratory tests showed a decrease in white blood cells and platelets; the serum characteristic IgM was positive; IgG in the convalescent phase was 4 times higher than that in the acute phase; viruses or specific antigens were isolated. Clinically used dengue virus detection methods include virus culture, serological detection, and viral nucleic acid detection. It takes a long time to isolate the virus, which fails to achieve the purpose of rapid diagnosis, and the conventional serological diagnosis is interfered due to the existence of extensive cross-reaction. The colloidal gold-labeled immunochromatographic method is fast, simple, does not need to rely on important equipment, and can realize on-site detection. It has become a research hotspot in the rapid diagnosis of infectious diseases.

NS1 protein is the only glycoprotein in the non-structural protein of dengue virus. It has strong antigenicity and does not cause antibody-dependent infection enhancement (Antibody-dependent enhancement, ADE), so it is used as the target of colloidal gold detection. The detection of colloidal gold requires specific monoclonal antibodies against the NS1 protein, and traditional clinical use is of mouse-derived monoclonal antibodies. Now the mainstream anti-dengue NS1 monoclonal antibody raw materials in the market are imported, the price is high, and the specificity and sensitivity need to be improved.

Contents of the invention

The present disclosure provides an antibody or an antigen-binding fragment, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1-HCDR3 include or are any of SEQ ID NO: 15, SEQ ID NO: 21-23 The amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region is shown; the LCDR1-LCDR3 includes or is an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.

Optionally, the CDRs are defined by the Kabat, Chothia, IMGT, Lesk, AbM or Contact numbering system.

Optionally, the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.

The present disclosure provides an antibody or antigen-binding fragment comprising the following CDRs:

HCDR1, which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;

HCDR2, which comprises the amino acid sequence shown in SEQ ID NO.2, or consists of it;

HCDR3, which comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19, or consists of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19;

LCDR1, which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;

LCDR2, which comprises the amino acid sequence shown in SEQ ID NO.5, or consists of it;

LCDR3, which comprises the amino acid sequence shown in SEQ ID NO.6, or consists of it.

Optionally, the antibody or antigen-binding fragment further comprises framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region, the HFR1 Comprising any amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:20, having more than 90% homology with SEQ ID NO:7 or having more than 80% homology with SEQ ID NO:20; HFR2 includes SEQ ID NO:8 or an amino acid sequence having more than 80% homology with SEQ ID NO:8; HFR3 comprises SEQ ID NO:9 or an amino acid sequence having more than 80% homology with SEQ ID NO:9; HFR4 comprises SEQ ID NO:10 or an amino acid sequence having more than 80% homology with SEQ ID NO:10; and LFR1 comprising SEQ ID NO:11 or an amino acid sequence having more than 80% homology with SEQ ID NO:11; LFR2 Comprising SEQ ID NO: 12 or an amino acid sequence having more than 80% homology with SEQ ID NO: 12; LFR3 comprising SEQ ID NO: 13 or an amino acid sequence having more than 80% homology with SEQ ID NO: 13; LFR4 Comprising SEQ ID NO: 14 or an amino acid sequence having more than 80% homology with SEQ ID NO: 14.

The present disclosure provides an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the above-mentioned HCDR1-3 and the above-mentioned HFR1-4, the light chain variable region comprises the above-mentioned LCDR1-3 and the above-mentioned LFR1-4.

Optionally, the heavy chain variable region comprises an amino acid sequence selected from any of SEQ ID NO: 15 and SEQ ID NO: 21-23, or consists of it.

The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or consists of it.

Optionally, the antibody or antigen-binding fragment further comprises a constant region.

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region.

Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD. The light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

Optionally, the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , Turkey, Fighting Cock Or Man. Optionally, the species source of the constant region is mouse.

Optionally, the heavy chain constant region is SEQ ID NO: 24 or an amino acid sequence having more than 80% homology with SEQ ID NO: 24; the light chain constant region is SEQ ID NO: 25 or an amino acid sequence with SEQ ID NO: 24 NO:25 amino acid sequence with more than 80% homology.

Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

The present disclosure also provides an antibody or an antigen-binding fragment, comprising a heavy chain and/or a light chain, the heavy chain comprising the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain comprising the above-mentioned light chain chain variable region and the aforementioned light chain constant region.

Optionally, the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 17, 26, 27, 28; the amino acid sequence of the light chain is shown in SEQ ID NO: 18.

The disclosure also provides a nucleic acid encoding the antibody or antigen-binding fragment of any of the above.

The present disclosure also provides a cell comprising the above-mentioned nucleic acid.

The present disclosure also provides a method for preparing the antibody or antigen-binding fragment described in any one of the above, the method comprising culturing the above-mentioned cells.

The present disclosure also provides an antibody conjugate comprising the antibody or antigen-binding fragment of any one of the above and a conjugation moiety conjugated thereto.

In an optional embodiment, the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, radioactive label, luminescent substance, colored substance, enzyme, such as a fluorescent label, a chromophore label, an electron-dense label , such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar One or more of oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin label.

In an optional embodiment, the coupling moiety is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

The present disclosure also provides a kit or a diagnostic reagent, which comprises the antibody or antigen-binding fragment described in any one of the above or the antibody conjugate described above.

In an optional embodiment, the kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to an antigen fragment other than dengue NS1 protein.

The present disclosure also provides the use of any one of the antibodies or antigen-binding fragments or antibody conjugates described above in the preparation of kits or diagnostic reagents.

In an optional embodiment, the reagent or kit is used for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or diseases related to dengue virus infection.

The present disclosure also provides the antibody or antigen-binding fragment or the antibody conjugate described in any one of the above or the kit or diagnostic reagent for detecting dengue virus or dengue NS1 protein and diagnosing dengue Use in virus infection or diseases related to dengue virus infection.

In an optional embodiment, the diseases associated with dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.

The present disclosure also provides a method for diagnosing a subject infected with dengue virus or a disease related to dengue virus infection, comprising:

A) Under conditions sufficient for a binding reaction to occur, combine any of the above-mentioned antibodies or functional fragments thereof, or the antibody conjugates, or the reagents or kits with those from the test subject contact with a sample of the patient for a binding reaction; and

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

In an optional embodiment, the diseases associated with dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.

The present disclosure also provides a method of detecting dengue virus or dengue NS1 protein in a test sample, comprising:

a) Under conditions sufficient for an antibody/antigen binding reaction to occur, the dengue virus or dengue NS1 protein antigen in the test sample is combined with the antibody or its functional fragment described in any one of the above, or the described The antibody conjugate, or said reagent or kit is contacted to form an immune complex; and

b) detecting the presence of said immune complex indicating the presence of said antigen in said test sample;

Optionally, in step a), the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment;

Optionally, in step a), the immune complex further includes a second antibody that binds to the dengue virus or dengue NS1 protein antigen.

Description of drawings

Fig. 1 is the reducing SDS-PAGE result of DF-5M32RMb1 antibody.

Detailed ways

Some embodiments of the present disclosure provide an antibody or an antigen-binding fragment, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include any of SEQ ID NO: 15, SEQ ID NO: 21-23 An amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region shown; said LCDR1-LCDR3 includes an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.

In an optional embodiment, the HCDR1-HCDR3 are amino acid sequences consistent with the HCDR1-HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO: 15 and SEQ ID NO: 21-23; the LCDR1-HCDR3 LCDR3 is an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.

In alternative embodiments, the CDRs are defined by the Kabat, Chothia, IMGT, Lesk, AbM or Contact numbering system.

In alternative embodiments, the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.

Some embodiments of the present disclosure relate to an antibody or antigen-binding fragment comprising the following CDRs:

HCDR1, which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;

HCDR2, which comprises the amino acid sequence shown in SEQ ID NO.2, or consists of it;

HCDR3, which comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19, or consists of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19;

LCDR1, which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;

LCDR2, which comprises the amino acid sequence shown in SEQ ID NO.5, or consists of it;

LCDR3, which comprises the amino acid sequence shown in SEQ ID NO.6, or consists of it.

As used herein, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

The term "antigen-binding fragment" is a substance comprising part or all of the CDRs of an antibody which lacks at least some of the amino acids present in the full-length chain but which is still capable of specifically binding to the antigen. Such fragments are biologically active in that they bind to the antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Such fragments are selected from Fab (composed of complete light chain and Fd), Fv (composed of VH and VL), scFv (single chain antibody, VH and VL are connected by a linker peptide) or single domain antibody ( Consists of VH only). Such fragments can be produced by recombinant nucleic acid techniques, or can be produced by enzymatic or chemical cleavage of antigen-binding molecules, including intact antibodies.

As used herein, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more, or even all, of the antibodies or The region of major amino acid residues responsible for the binding affinity of an antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

As used herein, the heavy chain complementarity determining region is denoted by "HCDR", which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is denoted by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. The accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues. The present disclosure adopts the Kabat annotation standard to mark the CDR region, but the CDR region marked by other methods also belongs to the protection scope of the present disclosure.

As used herein, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region and refers to an antibody heavy chain variable region (which may be denoted as VH) and a light chain variable region (which may be denoted as VL) Regions other than the CDRs in ; where the heavy chain framework region is denoted by "HFR" and can be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework The regions are indicated by "LFR" and can be further subdivided into contiguous regions separated by CDRs, comprising the LFR1, LFR2, LFR3 and LFR4 framework regions.

As used herein, the heavy chain variable region is obtained by joining the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs The following combinations and permutations are obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

As used herein, the terms "subject" and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.

In an alternative embodiment, the antibody further comprises the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the framework regions LFR1, LFR2, LFR3 and LFR4 of the light chain variable region, wherein:

HFR1 comprises any amino acid sequence of SEQ ID NO:7 or SEQ ID NO:20, or consists of it;

HFR2-4 comprises the amino acid sequence of SEQ ID NO:8-10 successively, or consists of the amino acid sequence of SEQ ID NO:8-10 successively;

LFR1-4 comprises the amino acid sequence of SEQ ID NO:11-14 successively, or consists of the amino acid sequence of SEQ ID NO:11-14 successively.

It should be noted that, in other embodiments, the amino acid sequence of each framework region of the antibody or antigen-binding fragment provided by the present disclosure may be identical to the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% homology.

In an optional embodiment, the binding affinity K _D of the antibody or antigen-binding fragment to Dengue NS1 is ≤6.36×10 ^-8 M.

Some embodiments of the present disclosure also provide an antibody or an antigen-binding fragment, the antibody or antigen-binding fragment comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprising the above-mentioned HCDR1 -3 and the aforementioned HFR1-4, the light chain variable region comprising the aforementioned LCDR1-3 and the aforementioned LFR1-4.

In an optional embodiment, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NO: 15, SEQ ID NO: 21-23; the light chain variable region comprises SEQ ID NO : the amino acid sequence shown in 16.

In an optional embodiment, the amino acid sequence of the heavy chain variable region consists of any amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 21-23; the light chain variable region consists of SEQ ID NO: Amino acid sequence composition shown in ID NO:16.

In an optional embodiment, the antibody further comprises a constant region;

In an alternative embodiment, the constant region includes a heavy chain constant region and/or a light chain constant region; as used herein, a "heavy chain constant region" can be represented as CH; a "light chain constant region" can be represented as CL .

In an optional embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

In an optional embodiment, the species sources of the heavy chain constant region and the light chain constant region are cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, Deer, mink, chicken, duck, goose, turkey, gamecock or human.

In an alternative embodiment, the species origin of the heavy chain constant region and the light chain constant region is mouse.

In an optional embodiment, the heavy chain constant region is SEQ ID NO:24 or an amino acid sequence having more than 80% homology with SEQ ID NO:24; the light chain constant region is SEQ ID NO:25 or an amino acid sequence with SEQ ID NO:24 NO:25 amino acid sequence with more than 80% homology.

It should be noted that, in other embodiments, the amino acid sequence of the constant region of the antibody or antigen-binding fragment provided by the present disclosure may have at least 80%, 81%, or 82% of the above-mentioned corresponding constant region (SEQ ID NO: 24, 25). %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In an alternative embodiment, the amino acid sequence of the heavy chain of the antibody consists of SEQ ID NO: 17; the amino acid sequence of the light chain of the antibody consists of SEQ ID NO: 18.

In an optional embodiment, the above antigen-binding fragment is selected from Fab, Fab', F(ab')2, scFv, Fv, Fd, single chain antibody, diabody or domain antibody.

The present disclosure also relates to nucleic acids encoding the antibodies or antigen-binding fragments described above.

Nucleic acids are usually RNA or DNA, and nucleic acid molecules can be single- or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence. DNA nucleic acid is used when it is ligated into a vector.

Some embodiments of the present disclosure also relate to vectors containing the above-mentioned nucleic acids.

Some embodiments of the present disclosure also relate to cells containing the nucleic acids or vectors described above.

Some embodiments of the present disclosure also relate to an antibody conjugate, comprising the above-mentioned antibody or antigen-binding fragment and a coupling moiety coupled thereto;

In an optional embodiment, the coupling moiety includes a label selected from purification tags (such as His tags), detectable labels, such as colloidal gold, radioactive labels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels , electron-dense labels, such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, Lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin-labeled.

In an alternative embodiment, the coupling moiety is selected from magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

Some embodiments of the present disclosure also relate to a kit or diagnostic reagent comprising the above-mentioned antibody or antigen-binding fragment or antibody conjugate, wherein:

In an optional embodiment, the above kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to a protein other than dengue NS1.

Some embodiments of the present disclosure also provide the use of the above antibodies or antigen-binding fragments or antibody conjugates in the preparation of kits or diagnostic reagents.

In an optional embodiment, the above reagent or kit is used for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or diseases related to dengue virus infection.

Some embodiments of the present disclosure also provide the above-mentioned antibodies or antigen-binding fragments, antibody conjugates or kits or diagnostic reagents for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or related to dengue virus infection use in disease.

In an optional embodiment, the above-mentioned diseases related to dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.

Some embodiments of the present disclosure also provide a method for diagnosing a subject infected with dengue virus or a disease associated with dengue virus infection, comprising:

A) contacting the above-mentioned antibody or functional fragment thereof, antibody conjugate, reagent or kit with a sample from the subject to carry out the binding reaction under conditions sufficient for the binding reaction to occur; and

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

In an optional embodiment, the above-mentioned diseases related to dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.

Some embodiments of the present disclosure also provide a method for detecting dengue virus or dengue NS1 protein in a test sample, comprising:

a) Under conditions sufficient for the antibody/antigen binding reaction to occur, the dengue virus or dengue NS1 protein antigen in the test sample is contacted with the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit to form immune complexes; and

b) detecting the presence of immune complexes, the presence of which is indicative of the presence of said antigen in said test sample.

Optionally, in step a), the immune complex further includes a second antibody, which binds to the above-mentioned antibody or antigen-binding fragment;

In an optional embodiment, in step a), the immune complex further includes a second antibody, which binds to the above dengue virus or dengue NS1 protein antigen.

In order to make the objectives, technical solutions and advantages of the embodiments disclosed in the present invention more clear, the following will clearly and completely describe the technical solutions in the embodiments disclosed in the present invention. For those who do not indicate specific conditions in the implementation manner, carry out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

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

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 skill of the art. This technique is fully explained in the literature, e.g., Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( M.J.Gait edited, 1984); "Animal Cell Culture (Animal Cell Culture)" (R.I.Freshney edited, 1987); "Methods in Enzymology" (Academic Press, Inc.); " "Handbook of Experimental Immunology" (D.M.Weir and C.C.Blackwell edited); "Gene Transfer Vectors for Mammalian Cells" (J.M.Miller and M.P.Calos edited, 1987); "Contemporary Methods in Molecular Biology (Current Protocols in Molecular Biology)" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction)" (Mullis et al., eds., 1994); and Contemporary Immunology Methods (Current Protocols in Immunology)" (J.E. Coligan et al. eds., 2011), each of which is expressly incorporated herein by reference.

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

The disclosure provides an anti-dengue NS1 antibody, a reagent and a kit for detecting dengue virus, the antibody can specifically bind to the dengue NS1 protein, and has a high affinity for it, and the antibody can be used to detect dengue virus or dengue NS1 protein has better sensitivity or specificity. The present disclosure provides a richer selection of antibodies for the detection of dengue virus.

Example

The features and performances disclosed in the present invention will be further described in detail below in conjunction with the examples.

Embodiment 1 The preparation of the antibody of anti-dengue NS1 protein

1. Expression plasmid construction

(1) Anti-DF 5M32 antibody gene preparation

The mRNA was extracted from the hybridoma cell line secreting Anti-DF 5M32 monoclonal antibody prepared in our laboratory, and the DNA product was obtained by RT-PCR method, and the product was inserted into the pMD-18T vector after adding A reaction with rTaq DNA polymerase , transformed into DH5α competent cells, and after the colonies grew out, four heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were respectively taken and sent to a gene sequencing company for sequencing.

(2) Sequence analysis of Anti-DF 5M32 antibody variable region gene

Put the gene sequence obtained by the above sequencing into the Kabat antibody database for analysis, and use VNTI11.5 software to analyze and confirm that the genes amplified by the heavy chain and light chain primer pairs are correct, and the genes amplified by the light chain In the fragment, the VL gene sequence is 339bp, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 351bp, which belongs to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.

(3) Construction of recombinant antibody expression plasmid

pcDNA ^™ 3.4

Vector is a recombinant antibody eukaryotic expression vector constructed. The expression vector has been introduced with HindIII, BamHI, EcoRI and other polyclonal restriction sites, and named as pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T According to the sequencing results of the variable region gene of the Chinese antibody, the VL and VH gene-specific primers of the Anti-DF 5M32 antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, and 0.74KB was amplified by PCR amplification The light chain gene fragment and the 1.4kb heavy chain gene fragment.

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

2. Screening of stable cell lines

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells to determine the activity of the expression plasmid

Dilute the plasmid prepared in step 1-(3) to 40 μg/100 μL with ultrapure water, adjust the CHO cells to 1.43×10 ⁷ cells/mL in a centrifuge tube, mix 100 μL of the above plasmid with 700 μL of cells, and transfer to the electroporation cup , electroporation, sampling and counting on the 3rd, 5th, and 7th day, and sampling and testing on the 7th day.

Dilute goat anti-mouse IgG 1ug/ml in the coating solution (main component NaHCO3) to coat the microplate, 100uL per well, overnight at 4°C; the next day, wash twice with the washing solution (main component Na2HPO4+Nacl), and pat dry; Add blocking solution (20% BSA+80% PBS), 120uL per well, 37°C, 1h, pat dry; add diluted cell supernatant, 100uL/well, 37°C, 60min; shake off the liquid in the plate, pat dry , add 20% mouse negative blood to seal, 120ul per well, 37°C, 1h; shake off the liquid in the plate, pat dry, add diluted DENV positive serum, 100uL per well, 37°C, 40min; wash 5 times with washing solution, pat Dry; add HRP-labeled DENV monoclonal antibody, 100uL per well, 37°C, 30min; add chromogenic solution A (50uL/well, main components citric acid + sodium acetate + acetanilide + carbamide peroxide), add chromogenic solution Solution B solution (50uL/well, main component citric acid + EDTA 2Na + TMB + concentrated HCL), 10min; add stop solution (EDTA 2Na + concentrated H2SO4), 50uL/well; read at 450nm (reference 630nm) on a microplate reader OD value.

The results showed that the reaction OD of the cell supernatant was still greater than 1.0 after dilution of 1000 times, and the reaction OD of the wells without cell supernatant was less than 0.1, indicating that the antibody produced after plasmid transient transfer was active against the DENV antigen.

(2) Linearization of recombinant antibody expression plasmid

Prepare the following reagents: 50 μL of Buffer, 100 μg/tube of the plasmid prepared in step 1-(3), 10 μL of PvuI enzyme, make up to 500 μL with sterile water, digest overnight in a water bath at 37°C; first use an equal volume of phenol/chloroform/iso Amyl alcohol (lower layer) 25:24:1, and then extracted with chloroform (water phase) in sequence; 0.1 volume (water phase) of 3M sodium acetate and 2 volumes of ethanol were precipitated on ice, and 70% ethanol was used to rinse the precipitate to remove organic Solvent, after the ethanol volatilizes completely, remelt with an appropriate amount of sterilized water, and finally measure the concentration.

(3) Stable transfection of recombinant antibody expression plasmids, pressurized selection of stable cell lines

Step 2-(2) Dilute the plasmid prepared in step 2 to 40 μg/100 μL with ultrapure water, adjust CHO cells to 1.43× ^{10 7} cells/mL in a centrifuge tube, mix 100 μL of the above plasmid with 700 μL of cells, and transfer to the electroporation cup, Electroporation, counting the next day; 25μmol/LMSX 96-well pressurized culture for about 25 days.

Observe and mark the clone wells with cells under a microscope, and record the confluence; take the culture supernatant and send samples for detection; select cell lines with high antibody concentration and relative concentration and transfer to 24 wells, and transfer to 6 wells in about 3 days; preserve the species after 3 days Batch culture, adjust the cell density to 0.5×106cells/mL, 2.2mL for batch culture, cell density 0.3× ¹⁰⁶ cells/mL, 2mL for seed preservation; 7-day 6-well batch culture supernatant for sample detection, select antibody concentration and Cell lines with smaller cell diameters were transferred to TPP for preservation and passage.

3. Production of recombinant antibodies

(1) Cell expansion

After recovery, the cells were first cultured in a 125mL shake flask with an inoculation volume of 30mL, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate and expand the culture at an inoculation density of 500,000 cells/mL. The expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the amount of cells meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.

(2) Shake flask production and purification

Shake flask parameters: rotational speed 120r/min, temperature 37°C, carbon dioxide 8%. Fed-batch feeding: Feed feeding starts every day when the shake flask is cultured for 72 hours. HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day). Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day. Affinity purification was performed with a proteinA affinity chromatography column. Take 6.6 μg of the purified antibody for reducing SDS-PAGE, and the electropherogram is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with a Mr of 50KD (heavy chain) and the other with a Mr of 28KD (light chain). The inventors named the purified DF-5M32 monoclonal antibody as "DF-5M32RMb1".

The amino acid sequences of HCDR1-3 of DF-5M32RMb1 obtained through the above steps are shown in SEQ ID NO.1-3 respectively; the amino acid sequences of LCDR1-3 are shown in SEQ ID NO.4-6 respectively. The amino acid sequences of the heavy chain variable region, the light chain variable region, the heavy chain and the light chain are respectively shown in SEQ ID NO.15-18.

Analyze the structure of DF-5M32RMb1, design mutation primers, repeat steps 1-(3) to 3-(2), and obtain DF-8F13RMb2-4 through activity identification and screening. After sequencing analysis, the heavy chain variable region of DF-8F13RMb2-4 is shown in SEQ ID NO.21-23, the heavy chain constant region is shown in SEQ ID NO.24, and the light chain is the same as DF-5M32RMb1.

Example 2 Affinity Analysis and Activity Identification

1. Affinity analysis

Using the AMC sensor, the purified antibody was diluted to 10ug/ml with PBST, and the DENV-IV antigen (an NS1 protein, purchased from Fapon) was serially diluted with PBST;

Operation process: Equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69GLY solution And buffer 3 for sensor regeneration, output data.

Table 1

sample name	KD(M)	Kon(1/Ms)	Kdis(1/s)
control antibody	8.11E-07	2.34E+03	1.90E-03
DF-5M32RMb1	5.96E-08	2.14E+04	1.28E-03
DF-5M32RMb2	5.78E-08	3.19E+04	1.84E-03
DF-5M32RMb3	6.36E-08	2.93E+04	1.86E-03
DF-5M32RMb4	5.29E-08	3.32E+04	1.76E-03

Note: KD means the equilibrium dissociation constant, that is, affinity; Kon means the association rate constant; Kdis means the dissociation rate constant.

2. Activity identification

Dilute goat anti-mouse IgG 1ug/ml in the coating solution (main component NaHCO3) to coat the microplate, 100uL per well, overnight at 4°C; the next day, wash twice with the washing solution (main component Na ₂ HPO ₄ +Nacl), Pat dry; add blocking solution (20%BSA+80%PBS), 120uL per well, 37°C, 1h, pat dry; add diluted purified antibody, 100uL/well, 37°C, 60min; shake off the liquid in the plate, Pat dry, add 20% mouse negative blood to seal, 120ul per well, 37℃, 1h; shake off the liquid in the plate, pat dry, add diluted DENV positive serum, 100uL per well, 37℃, 40min; wash 5 times with washing solution , pat dry; add DENV monoclonal antibody labeled with HRP (purchased from Fapon), 100uL per well, 37°C, 30min; add chromogenic solution A (50uL/well), add chromogenic Solution B solution (50uL/well), 10min; add stop solution, 50uL/well; read OD value at 450nm (refer to 630nm) on a microplate reader.

Table 2

Sample concentration (ng/ml)	12.5	6.25	3.125	1.5625	0.78125	0
control antibody	1.537	0.996	0.692	0.480	0.317	0.045
DF-5M32RMb1	2.098	1.520	0.963	0.654	0.489	0.039
DF-5M32RMb2	2.042	1.474	0.960	0.603	0.471	0.031
DF-5M32RMb3	1.965	1.496	0.952	0.622	0.479	0.043

DF-5M32RMb4

2.013

1.487

0.923

0.631

0.482

0.032

Embodiment 3 Stability assessment

The monoclonal antibody prepared in Example 1 was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, and samples were taken for state observation at 7 days, 14 days, and 21 days, and The activity test was carried out on the samples which had been placed for 21 days. For the activity test method, refer to Example 2 (the activity of the sample was evaluated by the OD result of enzyme immunoassay).

The results of the stability test of DF-5M32RMb1 are shown in Table 3. The results showed that the antibodies from DF-5M32RMb1 to DF-5M32RMb4 had no obvious change in protein status after being placed for 21 days under the three assessment conditions, and the activity did not increase with the increase of assessment temperature. The downward trend shows that the self-produced antibody prepared by the present disclosure has good stability.

table 3

Sample concentration (ng/mL)	6.25	3.125	0
4°C, 21 days sample	1.421	0.897	0.041
-80℃, 21 days sample	1.469	0.811	0.033
37°C, 21 days sample	1.395	0.803	0.039

Embodiment 4 performance evaluation

The antibody prepared in the above Example 1 and the control antibody were used as labeled antibodies, respectively, and used together with another anti-NS1 antibody (obtained from Feipeng Biotechnology, which can also be obtained by immunization with NS1 immunogen) as a coating antibody, to detect Dengue I Type, type II, type III and type IV antigens (purchased from Faipeng biology), compare the performance difference between the antibody prepared in example 1 and the control antibody on the colloidal gold platform, the antibody prepared in example 1 shows a higher than control antibody A better level of performance.

Table 4

The above-mentioned embodiment is a preferred implementation mode of the present disclosure, but the implementation mode of the present disclosure is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, All simplifications should be equivalent replacement methods, and all are included in the protection scope of the present disclosure.

Industrial Applicability

The anti-dengue virus antibody provided by the disclosure has good affinity to the NS1 protein of the dengue virus, and the detection of the dengue virus by using the antibody has good sensitivity and specificity. This disclosure provides a more excellent antibody selection for the detection of dengue virus, therefore, the anti-dengue virus antibody, dengue virus detection reagent and kit provided by the disclosure have excellent practical performance and broad market application prospects .

Claims (18)

1. An antibody or an antigen-binding fragment, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that, the HCDR1-HCDR3 comprise any one of SEQ ID NO: 15, SEQ ID NO: 21-23. The amino acid sequence consistent with HCDR1-HCDR3 of the chain variable region; the LCDR1-LCDR3 includes an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.
2. The antibody or antigen-binding fragment according to claim 1, wherein the CDRs are defined by the Kabat, Chothia, IMGT, Lesk, AbM or Contact numbering system;

   Optionally, the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.
3. The antibody or antigen-binding fragment according to claim 1, wherein said antibody or antigen-binding fragment comprises the following CDRs:

   HCDR1, which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;

   HCDR2, which comprises the amino acid sequence shown in SEQ ID NO.2, or consists of it;

   HCDR3, which comprises the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19, or consists of the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.19;

   LCDR1, which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;

   LCDR2, which comprises the amino acid sequence shown in SEQ ID NO.5, or consists of it;

   LCDR3, which comprises the amino acid sequence shown in SEQ ID NO.6, or consists of it.
4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, said antibody or antigen-binding fragment further comprising the framework regions HFR1, HFR2, HFR3 and HFR4 of the heavy chain variable region, and the light chain variable region The framework regions LFR1, LFR2, LFR3 and LFR4, the

   HFR1 comprises any amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:20, having more than 90% homology with SEQ ID NO:7 or having more than 80% homology with SEQ ID NO:20;

   HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having more than 80% homology with SEQ ID NO: 8;

   HFR3 comprises SEQ ID NO: 9 or an amino acid sequence having more than 80% homology with SEQ ID NO: 9;

   HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having more than 80% homology with SEQ ID NO: 10; and

   LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having more than 80% homology with SEQ ID NO: 11;

   LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having more than 80% homology with SEQ ID NO: 12;

   LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having more than 80% homology with SEQ ID NO: 13;

   LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having more than 80% homology with SEQ ID NO: 14.
5. An antibody or antigen-binding fragment, characterized in that, the antibody or antigen-binding fragment comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region comprises any one of claims 1 to 3 The HCDR1-3 and the HFR1-4 of claim 4, the light chain variable region comprises the LCDR1-3 of any one of claims 1 to 3 and the LFR1-4 of claim 4;

   Optionally, the heavy chain variable region comprises an amino acid sequence selected from any of SEQ ID NO: 15, SEQ ID NO: 21-23, or consists of it;

   The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, or consists of it.
6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, further comprising a constant region;

   Optionally, said constant region comprises a heavy chain constant region and/or a light chain constant region;

   Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region ;

   Optionally, the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose , turkey, fighting cock or human; optionally, the species source of the constant region is mouse;

   Optionally, the heavy chain constant region is SEQ ID NO: 24 or an amino acid sequence having more than 80% homology with SEQ ID NO: 24; the light chain constant region is SEQ ID NO: 25 or an amino acid sequence with SEQ ID NO: 24 NO:25 amino acid sequence with more than 80% homology.
7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antigen-binding fragment is selected from any of F(ab')2, Fab', Fab, Fv and scFv of the antibody A sort of.
8. An antibody or an antigen-binding fragment, comprising a heavy chain and/or a light chain, wherein the heavy chain comprises the heavy chain variable region of claim 5 and the heavy chain constant region of claim 6; Said light chain comprises the light chain variable region according to claim 5 and the light chain constant region according to claim 6;

   Optionally, the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 17, 26, 27, 28; the amino acid sequence of the light chain is shown in SEQ ID NO: 18.
9. A nucleic acid, characterized in that the nucleic acid encodes the antibody or antigen-binding fragment of any one of claims 1-8.
10. A cell, characterized in that the cell comprises the nucleic acid of claim 9.
11. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 8, characterized in that the method comprises culturing the cell according to claim 10.
12. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody or antigen-binding fragment of any one of claims 1 to 8 and a coupling moiety coupled thereto;

    Optionally, the coupling moiety is selected from a purification label or a detectable label, such as colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioactive isotope , fluorophore, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, one or more of glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labels;

    Optionally, the coupling moiety is selected from magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
13. A kit or diagnostic reagent, characterized in that the kit or diagnostic reagent comprises the antibody or antigen-binding fragment of any one of claims 1 to 8 or the antibody conjugate of claim 12;

    Optionally, the kit or diagnostic reagent further comprises an antibody or antigen-binding fragment, antibody conjugate, or fusion protein that binds to an antigen fragment other than dengue NS1 protein.
14. Use of the antibody or antigen-binding fragment of any one of claims 1 to 8 or the antibody conjugate of claim 12 in the preparation of kits or diagnostic reagents;

    Optionally, the reagent or kit is used for detecting dengue virus or dengue NS1 protein, diagnosing dengue virus infection or diseases related to dengue virus infection.
15. The antibody or antigen-binding fragment described in any one of claims 1 to 8, the antibody conjugate described in claim 12, or the kit or diagnostic reagent described in claim 13 is used to detect dengue virus or dengue NS1 protein, Use in diagnosing dengue virus infection or a disease associated with dengue virus infection.
16. The use according to claim 14 or 15, wherein the diseases associated with dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.
17. A method of diagnosing a subject being infected with dengue virus or a disease associated with dengue virus infection, comprising:

    A) making the antibody or functional fragment thereof according to any one of claims 1 to 8, or the antibody conjugate according to claim 12, or the reagent according to claim 13 under conditions sufficient for a binding reaction to occur or the kit is contacted with a sample from said subject for a binding reaction; and

    B) detecting the immune complex produced by the binding reaction;

    Optionally, the diseases associated with dengue virus infection include dengue fever, dengue hemorrhagic fever or dengue shock syndrome.
18. A method of detecting dengue virus or dengue NS1 protein in a test sample comprising:

    a) under conditions sufficient for an antibody/antigen binding reaction to occur, the dengue virus or dengue NS1 protein antigen in the test sample is combined with the antibody or a functional fragment thereof according to any one of claims 1 to 8, or the antibody conjugate of claim 12, or the reagent or kit of claim 13 contacted to form an immune complex; and

    b) detecting the presence of said immune complex indicating the presence of said antigen in said test sample;

    Optionally, in step a), the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment;

    Optionally, in step a), the immune complex further includes a second antibody that binds to the dengue virus or dengue NS1 protein antigen.

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