CN117551189A

CN117551189A - anti-Zika virus monoclonal antibody and application thereof

Info

Publication number: CN117551189A
Application number: CN202311338200.3A
Authority: CN
Inventors: 林育佳; 陈立; 张荣华; 袁克湖
Original assignee: Zhuhai Heavy Chain Biotechnology Co ltd
Current assignee: Zhuhai Heavy Chain Biotechnology Co ltd
Priority date: 2023-10-16
Filing date: 2023-10-16
Publication date: 2024-02-13

Abstract

The invention relates to a novel anti-Zika virus monoclonal antibody, a kit comprising the antibody and related application of the antibody in immunodetection. The anti-Zika virus monoclonal antibody can specifically bind to the Zika virus NS1 protein, and shows high sensitivity and high specificity of targeting Zika virus, so that the anti-Zika virus monoclonal antibody can be used for clinically detecting the Zika virus.

Description

anti-Zika virus monoclonal antibody and application thereof

Technical Field

The invention relates to the field of immunodetection, in particular to a monoclonal antibody for detecting Zika virus, a preparation method thereof and related application of the monoclonal antibody in immunodetection.

Background

ZiKV is a virus transmitted by mosquito, sexually transmitted, and by mother and infant, and belongs to the flaviviridae genus of flaviviridae, together with dengue virus (DNEV), tick-borne encephalitis virus TBEV), yellow Fever Virus (YFV), japanese Encephalitis Virus (JEV), and West Nile Virus (WNV). Infection with Zika virus causes rash, fever, conjunctivitis, arthralgia, myalgia, etc., but there are also infected persons without obvious clinical symptoms, probably due to the difference in the major vectors of viral transmission. With the intensive research, researchers found that neonatal small head deformity, guillain-barre syndrome of adults, cardiovascular complications, acute liver injury and blood coagulation dysfunction are all related to zika virus infection. The continued transmission and infection of Zika virus has become a global health problem, and the World Health Organization (WHO) announced Zika virus infection as an international focus on health events in 2016. Therefore, it is important to accurately diagnose whether or not the Zika virus is infected.

The main detection methods for diagnosing Zika virus infection at present are etiology detection and serology detection. The etiology detection is mainly nucleic acid detection (such as RT-qPCR, LAMP), and has the advantages of high accuracy and short detection time. However, the equipment used in the method is expensive, false positives are easy to occur, and the virus is difficult to accurately detect the card-like virus due to the short window period of the virus. Serological assays include: ELISA (ELISA) and indirect fluorescent antibody assay (IFA) for detecting serum-specific IgM antibodies and plaque reduction neutralization assay (PRNT) for detecting neutralizing antibodies. Serological tests have the advantage of being simple and rapid, but IgM/IgG is usually detected 7 days after onset and is affected by the onset of symptoms in patients with different infections.

The Zika virus is a single-stranded positive-strand RNA virus with a diameter of 40-70 nm and envelope, and RNA is translated into a long protein chain consisting of 3 structural proteins (capsid protein C, precursor membrane protein PRM and envelope protein E) and 7 non-structural proteins (NS 1, NS2A, NS2B, NS, NS4A, NS4B and NS 5). Among them, the NS1 protein is an important protein secreted exclusively by viruses and interacting with hosts, plays an important role in viral infection, replication and immune escape processes, and can activate TLRs pathway and inhibit complement system. Thus, the NS1 protein, one of the major antigens of viruses, is an important biological target for early diagnosis of the infection by the zika virus.

However, there is a wide range of similarity between the NS1 proteins of the flaviviridae viruses, and the high degree of homology of this viral protein results in cross-reactivity of the produced antibodies between the different viruses, as reported by the cross-talk of dengue virus NS1 standard diagnostic reagent with the zika virus. Second, when a patient is previously infected with other flaviviridae viruses, the body will produce a large amount of antibodies against the primary infection, thereby making serological testing susceptible to false positive results.

Therefore, there is a need in the art for a detection reagent and related method for the Zika virus that has high specificity, high sensitivity, and no or little cross-reaction with other yellow fever viruses, thereby achieving simple, rapid, sensitive and specific detection of the Zika virus.

Disclosure of Invention

The invention discloses a method for preparing a recombinant anti-Zika virus monoclonal antibody, which comprises the steps of immunizing a mouse by using NS1 protein of Zika virus, fusing spleen cells of the mouse with myeloma cells, screening hybridoma cell strains which are specifically combined with the NS1 protein of Zika virus and do not cross react with other virus NS1 proteins of flaviviridae by an ELISA method, and constructing the recombinant antibody. Thus, the present invention has been achieved.

Thus, in a first aspect, the present invention provides an anti-Zika virus monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising a heavy chain complementarity determining region V represented by SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3, and a light chain variable region _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region including light chain complementarity determining region V represented by SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6 _L CDR1、V _L CDR2 and V _L CDR3。

In a second aspect, the invention provides a nucleic acid molecule encoding the anti-zika virus monoclonal antibody or antigen-binding fragment thereof of the first aspect.

In a third aspect, the invention provides a vector comprising the nucleic acid molecule of the second aspect.

In a fourth aspect, the invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

In a fifth aspect, the present invention provides the use of an anti-zika virus monoclonal antibody or antigen-binding fragment thereof of the first aspect in the preparation of a reagent for detecting zika virus.

In a sixth aspect, the present invention provides a method of detecting a zika virus for non-diagnostic or diagnostic purposes comprising the step of using an anti-zika virus monoclonal antibody or antigen binding fragment thereof of the first aspect.

In a seventh aspect, the invention provides a kit for detecting a zika virus comprising the anti-zika virus monoclonal antibody or antigen-binding fragment thereof of the first aspect and instructions for use.

In summary, the invention provides a novel anti-Zika virus monoclonal antibody or antigen binding fragment thereof, which shows high sensitivity and high specificity of binding to the Zika virus NS1 protein, can be suitable for various immunochromatography detection, can be further used for detecting Zika viruses with nanogram-level and picogram-level concentration, and greatly improves the sensitivity of Zika virus detection and analysis, thereby meeting the requirements of clinical detection of the Zika viruses.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the results of the binding reaction of monoclonal antibody Z1 of the present invention with the Zika virus NS1 protein and dengue virus type 1 (as a control, square).

FIG. 2 shows the results of the binding reaction of the recombinant antibody Z1' of the present invention with the Zika virus NS1 protein (circle) and dengue virus type 1 (square as control).

FIG. 3 shows the results of the binding reaction of the recombinant antibodies Z2'-Z12' of the invention with the Zika virus NS1 protein and dengue virus type 1 (DENV 1 as control).

FIG. 4 shows the binding reaction results of recombinant antibodies Z1' of the present invention to the NS1 proteins of Zika virus, dengue virus types 1-4 (DENV 1, DENV2, DENV3, DENV 4), yellow Fever Virus (YFV), west Nile Virus (WNV), japanese Encephalitis Virus (JEV) and tick-borne encephalitis virus (TBEV).

Detailed Description

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It is to be understood that the following description is intended to illustrate the invention by way of example only, and is not intended to limit the scope of the invention as defined by the appended claims. And, it is understood by those skilled in the art that modifications may be made to the technical scheme of the present invention without departing from the spirit and gist of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

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 the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided to better understand the present invention.

Where a range of values is provided, such as a range of concentrations, a range of percentages, or a range of ratios, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range, and any other stated or intervening value in that stated range, is encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.

In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially/mainly of … …". The expression "comprising," "including," or "consisting essentially of … …" is generally understood to mean an open-ended expression that includes not only the individual elements, components, assemblies, method steps, etc., specifically listed thereafter, but also other elements, components, assemblies, method steps. In addition, the expression "comprising," "including," or "consisting essentially of … …" is also to be understood in some instances as a closed-form expression, meaning that only the elements, components, assemblies, and method steps specifically listed thereafter are included, and no other elements, components, assemblies, and method steps are included. At this time, the expression is equivalent to the expression "consisting of … …".

For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers expressing quantities, percentages or proportions used in the specification and claims, and other numerical values, are to be understood as being modified in all instances by the term "about" unless otherwise indicated. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies can be defined accordingly as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V _H ) And heavy chainConstant region (C) _H ) Composition is prepared. The heavy chain constant region consists of 3 domains (C _H1 、C _H2 And C _H3 ) Composition is prepared. Each light chain consists of a light chain variable region (V _L ) And a light chain constant region (C _L ) Composition is prepared. The light chain constant region consists of one domain C _L Composition is prepared. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). V (V) _H And V _L The region can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). For each heavy or light chain, its variable region comprises three CDRs, CDR1, CDR2 and CDR3, respectively. Thus, each V _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable region (V _H And V _L ) Respectively forming antigen binding sites.

The rules of allocation of amino acids to regions or domains give the relevant definitions in several documents: kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda m.d. (1987 and 1991)); chothia & Lesk j.mol.biol.1987;196:901-917; chothia et al, nature 1989;342:878-883; ehrenmann, francois, quentin Kaas, and Marie-Paule Lefranc. "IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell acceptors, MHC, igSF and MhcSF." Nucleic acids research 2009;38 (suppl_1): D301-D307.

The exact boundaries of CDRs have been defined differently from system to system, and the Kabat system provides not only a clear residue numbering system applicable to any variable region of an antibody, but also precise residue boundaries defining 3 CDRs, referred to as Kabat CDRs; chothia found that some subfractions within the CDRs of the Kabat system, which are termed Chothia CDRs with boundaries overlapping the Kabat CDRs, have almost identical peptide backbone conformations, despite great diversity at the amino acid sequence level. The weight mentioned above The boundaries of the stack, in turn, are described by Padlan and MacCallum, and CDR boundary definitions may not strictly adhere to the above-described system, such as the AbM definition. In this context, the CDRs may be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al to define the CDRs. According to the Chothia numbering system, V of antibodies _H CDR1 is located at positions 26 to 32, V _H CDR2 at positions 52 to 57, V _H CDR3 is located at positions 99 to 108 and V _L CDR1 is located at positions 24 to 39, V _L CDR2 is located at positions 55 to 61 and V _L CDR3 is located at positions 94 to 102.

As used herein, the term "mab" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule may be an immunoglobulin, whether it be a natural immunoglobulin or an immunoglobulin obtained partially or wholly by synthetic means. The antibody molecules may also include all polypeptides or proteins having antibody binding domains, antibody fragments having antibody binding domains are molecules such as Fab, scFv, fv, dAb, fd, and bifunctional antibodies. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtainable by the hybridoma technique first reported by Kohler et al G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J]Natural, 1975;256 495) but may also be obtained using recombinant DNA techniques (see, e.g., U.S. patent 4,816,567). As used herein, the terms "monoclonal antibody" and "mab" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaningAnd are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine may be represented by Ala or A, glycine may be represented by Gly or G, valine may be represented by Val or V, leucine may be represented by Leu or L, isoleucine may be represented by Ile or I, proline may be represented by Pro or P, phenylalanine may be represented by Phe or F, tyrosine may be represented by Tyr or Y, tryptophan may be represented by Trp or W, serine may be represented by Ser or S, threonine may be represented by Thr or T, cysteine may be represented by Cys or C, methionine may be represented by Met or M, asparagine may be represented by Asn or N, glutamine may be represented by Gln or Q, aspartic acid may be represented by Asp or D, glutamic acid may be represented by Glu or E, lysine may be represented by Lys or K, arginine may be represented by Arg or R, and histidine may be represented by His or H.

As used herein, the term "recombinant antibody" refers to an antibody that is expressed by cloning an antibody gene into an expression vector by molecular biological techniques and then transfecting the expression vector into a suitable host cell line. The recombinant antibody-encoding gene may or may not be identical to the naturally derived antibody-encoding gene. For example, the whole coding gene of an antibody obtained by immunizing an animal may be cloned into an expression vector to express, thereby obtaining an antibody identical to the antibody obtained by immunizing an animal, or a gene coding for a variable region (including a heavy chain variable region and a light chain variable region) of an antibody obtained by immunizing an animal may be cloned into an expression vector together with a gene coding for a constant region of an antibody derived from another species (e.g., human), thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having mouse heavy chain and light chain variable regions linked to human constant regions. Such antibodies are commonly referred to in the art as "chimeric antibodies".

As used herein, the term "antigen-binding fragment" refers to fragments and antibody analogs from antibodies capable of binding an antigen, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antigen binding fragments retain at least some of the binding activity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen binding fragment retains at least 10% of the parent binding activity. In particular, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to: fab, fab ', F (ab') ₂ Fv fragments, linear antibodies (linear antibodies), single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Wherein the "Fab fragment" consists of a light chain, a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. "Fab' fragments" contain portions of one light chain and one heavy chain (including VH domains, CH1 domains, and regions between CH1 and CH2 domains); whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to form F (ab') ₂ A molecule. The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.

In the present invention, PCR amplification of the nucleotide sequence encoding the antibody may also be performed using primer pairs. In the primer sequence, some of the sites involve only a single base, such as any of adenine (A), guanine (G), cytosine (C) and thymine (T), while some involve a combination of two, three or four bases, in which case these bases are referred to as degenerate bases to each other, which is determined primarily based on the degeneracy of the codons. Degenerate bases may be represented by the letter R, Y, M, K, S, W, H, B, V, D, N, where R represents A/G, Y represents C/T, M represents A/C, K represents G/T, S represents C/G, W represents A/T, H represents A/T/C, B represents G/T/C, V represents G/A/C, D represents G/A/T, and N represents A/T/C/G.

The terms "sequence identity", "identity" or "homology" as used herein have art-recognized meanings and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W, ed., academic Press, new York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M, and Griffin, h.g., eds., humana Press, new Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G, academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J, eds., M stock Press, new York, 1991). Although there are many ways to measure identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled person to be suitable for conservative amino acid substitutions in peptides or proteins and can generally be performed without altering the biological activity of the resulting molecule. In general, one skilled in the art recognizes that single amino acid substitutions in the non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings pub. Co., p. 224).

As described above, the present invention aims to provide an anti-Zika virus monoclonal antibody having high sensitivity and high specificity. The inventor uses the Zika virus NS1 protein to immunize a mouse, takes spleen cells of the mouse to fuse with myeloma cells, screens out hybridoma cell strains capable of specifically combining with the Zika virus NS1 protein by an ELISA method, thereby completing the invention.

Thus, in a first aspect, the present invention provides an anti-Zika virus monoclonal antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising a heavy chain complementarity determining region V represented by SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3, and a light chain variable region _H CDR1、V _H CDR2 and V _H CDR3, said light chain variable region comprising a sequence represented by SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6Light chain complementarity determining region V _L CDR1、V _L CDR2 and V _L CDR3。

The sequences of SEQ ID NOs 1 to 6 are as follows:

SEQ ID NO:1：GYSFTX ₁ y, wherein X1 is S or N;

SEQ ID NO:2：DPX ₂ DSE, wherein X ₂ Is S or T;

SEQ ID NO:3：RSSWYFDX ₃ wherein X3 is V or A;

SEQ ID NO:4：GASESVGX ₄ YGNSFLH, wherein X ₄ Is N or S;

SEQ ID NO:5：LX ₅ SNLX ₆ s, wherein X ₅ Is A or G, X ₆ Q or E;

SEQ ID NO:6：QQNNEX ₇ PST, wherein X ₇ D or E.

In a specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 16 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 18 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, said light chain variable region comprising an amino acid sequence represented by SEQ ID NO. 13, SEQ ID NO. 17 and SEQ ID NO. 20, respectivelyLight chain complementarity determining region V _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 17 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 15 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain having an amino acid sequence set forth in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 11, respectively Complement determination region V _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 16 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 18 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 17 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 17 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In yet another specific embodiment, the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In a specific embodiment, the antibody is an intact antibody comprising a variable region and a constant region. For the antibodies of the invention, any Framework Region (FR) as well as any constant region may be used. The amino acid sequence of the FR or constant region used in the antibody of the invention may be the amino acid sequence of the original FR or constant region from which it is derived, or may be a different amino acid sequence obtained by substituting, deleting, adding and/or inserting 1 or more amino acids into the amino acid sequence of the original FR or constant region. The structure used to support the CDRs or sets of CDRs of the invention typically belong to the antibody heavy or light chain sequence or a major portion thereof, wherein the CDRs or sets of CDRs are located in naturally occurring V encoded by rearranged immunoglobulin genes _H And V _L The CDRs or sets of CDRs of the antibody variable domains are located at corresponding positions.

As an example, each Framework Region (FR) may have the following sequence:

HFR1 (heavy chain framework region 1): QVQLQQPGAELVKPGASVKLSCKAS (SEQ ID NO: 21);

HFR2 (heavy chain framework region 2): LMHWVKQRPGHGLEWIGEV (SEQ ID NO: 22);

HFR3 (heavy chain framework region 3): TNYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR (SEQ ID NO: 23);

HFR4 (heavy chain framework region 4): WGQGTTLTVSS (SEQ ID NO: 24);

LFR1 (light chain framework region 1): DIVLTQSPASLAVSLGQRATISC (SEQ ID NO: 25);

LFR2 (light chain framework region 2): WYQQKPGQPPKLLIY (SEQ ID NO: 26);

LFR3 (light chain framework region 3): GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO: 27);

LFR4 (light chain framework region 4): FGGGTKLEIK (SEQ ID NO: 28).

In a specific embodiment, the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 having the sequences set forth in SEQ ID NOS.21-24, respectively, or having 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, respectively, of the sequences set forth in SEQ ID NOS.21-24Or sequences with greater than 99.9% identity. Those skilled in the art will appreciate that heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 are complementary to heavy chain complementarity determining region V _H CDR1、V _H CDR2 and V _H CDR3 from amino terminus to carboxy terminus was as defined for HFR1, V _H CDR1、HFR2、V _H CDR2、HFR3、V _H The sequence of CDR3 and HFR4 is arranged to form a heavy chain variable region sequence.

In yet another specific embodiment, the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4 having the sequence represented by SEQ ID NOs 25-28 or sequences having 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more identity thereto, respectively. Those skilled in the art will appreciate that the light chain framework regions LFR1, LFR2, LFR3 and LFR4 and light chain complementarity determining region V _L CDR1、V _L CDR2 and V _L CDR3 from amino terminal to carboxy terminal following LFR1, V _L CDR1、LFR2、V _L CDR2、LFR3、V _L CDR3 and LFR4 are arranged in sequence to form a light chain variable region sequence.

In a specific embodiment, the antibody further comprises a constant region sequence, such as, but not limited to, a constant region sequence selected from any one of IgG, lgA, igM, igE and IgD, which is not particularly limited herein, and can be selected by one of skill in the art based on the need.

In yet another specific embodiment, the constant region sequence may be derived from a rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose, or human, but is not limited thereto.

In a specific embodiment, the constant regions of the antibodies of the invention are derived from mice.

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

It will be apparent to those skilled in the art that the determination of the nucleic acid coding sequence is well within the ability of a protein, such as the amino acid sequence of the anti-Zika virus monoclonal antibody of the present invention. In addition, in order to obtain monoclonal antibodies by recombinant means, the nucleic acid molecules may be cloned into a vector and the vector further introduced into an expression cell to express the antibody protein using the expression cell.

In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

In a specific embodiment, the vector may be a pTOPO vector.

In yet another specific embodiment, the vector may be a eukaryotic expression vector.

In a preferred embodiment, the pcDNA vector may be pcDNA3.1.

The expression cells are prepared by introducing the above-described nucleic acid molecules or the above-described vectors into host cells by molecular biological methods well known to those skilled in the art.

As described above, the present inventors immunized mice with the Zika virus NS1 protein, fused the spleen cells of the mice with myeloma cells, and screened out hybridoma cell lines capable of specifically binding to the Zika virus NS1 protein by ELISA, designated as Z1. After screening a monoclonal cell line secreting the antibody of interest, heavy and light chain variable region cdnas may be recovered from the cell line by RT-PCR, and appropriate immunoglobulin constant regions (e.g., human constant regions) selected, and then the heavy and light chain variable region cdnas and constant region cdnas are transferred into host cells such as COS or CHO cells, thereby obtaining the expression cells expressing the antibody of interest of the present invention. Other antibodies or chimeric molecules that retain the specificity of the original antibody can be produced using monoclonal antibodies and other antibodies and recombinant DNA techniques that can include introducing DNA encoding the immunoglobulin variable or Complementarity Determining Regions (CDRs) of the antibodies into the constant regions or constant region plus framework regions of different immunoglobulins.

In a specific embodiment, the expression cell may be a mammalian cell, such as a chinese hamster ovary cell, a little hamster kidney cell, a monkey kidney cell, a mouse thymoma cell, a human embryonic kidney cell. In a more specific embodiment, the expression cell may be, for example, a monkey kidney cell transformed with SV40 (COS-7, ATCC CRL 1651), a human embryonic kidney cell (HEK 293 or HEK293 cell subcloned for growth in suspension culture, graham et al, 1977,J.Gen Virol.36:59), baby hamster kidney cell (BHK, ATCC CCL 10), chinese hamster ovary cell/-DHFR 1 (CHO, urlaub et al, 1980, proc.Natl. Acad.sci.usa77:4216; such as DG 44), mouse thymoma cells (NSO), mouse testis support cells (TM 4, mather,1980, biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG 2, HB 8065), mouse mammary tumors (MMT 060562, ATCC CCL 51), TR1 cells (Mather et al, 1982,Annals N.Y.Acad.Sci.383:44-68), MRC5 cells, FS4 cells, etc., but are not limited thereto.

In a specific embodiment, the Zika virus is detected by specific binding of the anti-Zika virus monoclonal antibody or antigen binding fragment thereof of the present invention to the NS1 protein of the Zika virus.

In a specific embodiment, the detection is performed by any one or more of immunochromatography, enzyme-labeled antibody (ELISA), chemiluminescence, and electrochemiluminescence.

In yet another specific embodiment, the detection may be direct, indirect, sandwich, and competition.

In a preferred embodiment, the immunochromatography includes, but is not limited to, fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography.

In the present invention, the anti-Zika virus monoclonal antibody may be used as a coating antibody. For example, anti-Zika virus monoclonal antibodies are bound to a solid phase such as a solid support. The solid support used in the detection method of the present invention is not particularly limited, and may be a porous or nonporous material such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, or the like.

Without wishing to be bound by theory, the anti-zika virus monoclonal antibodies of the invention may also be used as labeled antibodies. For example, the anti-zika virus monoclonal antibody may be conjugated to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, and the like.

For example, when performing a colloidal gold immunochromatographic test, the anti-Zika virus monoclonal antibody of the present invention may be labeled with colloidal gold, a nitrocellulose membrane (NC membrane) may be coated with another anti-Zika virus antibody, and a detection line (T line) may be obtained by drawing the membrane. And assembling according to the preparation mode of the immune test strip to obtain the colloidal gold test paper. During detection, an analyte in a positive sample is combined with an anti-Zika virus antibody marked by colloidal gold to form a complex, the complex is combined with a coating antibody at a T line to form a sandwich complex, and the colloidal gold is aggregated and precipitated to display red, so that the positive sample is indicated.

For another example, when performing immunochromatographic tests based on colored latex microspheres, the immunochromatographic rapid test card can be assembled in a conventional manner using the latex microspheres labeled with the anti-Zika virus monoclonal antibody of the present invention, a nitrocellulose membrane (NC membrane) coated with another anti-Zika virus antibody, a sample pad, a water-absorbent paper, a polyester sheet, and the like. In detection, the analyte in the positive sample is combined with the latex microsphere marked with the monoclonal anti-Zika virus antibody, and agglutination reaction occurs at room temperature, so that after a period of time, the result can be observed and judged by naked eyes.

For another example, when performing a fluorescent microsphere immunochromatographic test, time-resolved fluorescent microspheres can be labeled with the anti-Zika virus monoclonal antibody of the present invention, a nitrocellulose membrane (NC membrane) can be coated with another anti-Zika virus antibody, and assembled with a sample pad or the like to form an immunochromatographic rapid test card. When the detection is carried out, the to-be-detected object in the sample is combined with the fluorescent microsphere marked antibody in the combination pad and is subjected to forward chromatography through capillary action, and after the to-be-detected area is reached, the to-be-detected object is further combined with another monoclonal anti-Zika virus antibody fixed on the T line of the detection line to form a double-antibody sandwich. After chromatography is finished, the immunofluorescence instrument is used for reading the fluorescence intensities of the T line and the C line, calculating the T/C value, and the content of the to-be-detected object in the sample can be calculated through a standard curve built in the instrument.

The present inventors have found that the anti-zika virus monoclonal antibody of the present invention exhibits extremely high detection sensitivity, for example, the sensitivity of detection of zika virus by colloidal gold immunochromatography can reach a concentration of 1ng/mL or less, and the sensitivity of detection of zika virus by latex chromatography can reach a concentration of 200pg/mL or less, both as a coated antibody and a labeled antibody.

The inventors have additionally found that in the assays of the invention the labelled antibody and the coated antibody may be the same or different, and preferably are different. That is, when the anti-Zika virus monoclonal antibody of the present invention such as Z10 '(or Z11') is used for immunochromatography to detect Zika virus, it can be used as a labeling antibody to label magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, colloidal gold, etc., as well as a coating antibody to coat a solid support such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, etc.

In yet another specific embodiment, the detection may be a quantitative detection.

In yet another specific embodiment, the detection of the Zika virus NS1 protein may be accomplished by labeling a displayable signaling indicator.

In a further specific embodiment, the displayable signaling indicator may be selected from any one of, but not limited to, colloidal gold, a fluorescent substance, a radioisotope, an enzyme catalyzing the development of a substrate, and a chemiluminescent reagent.

In a preferred embodiment, the enzyme catalyzing the development of a substrate may be selected from, but is not limited to, any of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase.

In a preferred embodiment, the fluorescent substance may be selected from any one of, but not limited to, umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.

In a preferred embodiment, the chemiluminescent reagent may be an aminobenzene dihydrazide, but is not limited thereto.

In a preferred embodiment, the radioisotope may be selected from, but is not limited to ³ H、 ¹⁴ C、 ³⁵ S、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ I、 ¹⁷⁷ Lu、 ¹⁶⁶ Ho sum ¹⁵³ Sm.

In a sixth aspect, the present invention provides a method of detecting ZIKA virus (ZIKA) for non-diagnostic or diagnostic purposes comprising the step of using an anti-ZIKA virus monoclonal antibody of the first aspect or an antigen binding fragment thereof.

In the context of the present invention, the detection is performed by immunochromatography, enzyme-linked antibody (ELISA), chemiluminescence, electrochemiluminescence. The immunochromatography may include, but is not limited to, fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, quantum dot immunochromatography, and the like. The detection can be selected by those skilled in the art as necessary, and the present invention is not particularly limited. In addition, the detection may be a direct method, an indirect method, a sandwich method, and a competition method.

As described above, the anti-Zika virus monoclonal antibody of the present invention may be used as a coating antibody or as a labeled antibody. It is noted that the anti-Zika virus monoclonal antibody of the present invention can detect Zika virus with high sensitivity and high specificity when used in combination with another anti-Zika virus antibody, whether it is used as a coating antibody or a labeling antibody. Thus, in the assays of the invention, the labeled antibody and the coated antibody may be the same or different, and preferably are different. That is, when the anti-Zika virus monoclonal antibody of the present invention is used for immunochromatography to detect Zika virus, it can be used as a labeling antibody to label magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, colloidal gold, etc., and also as a coating antibody to coat a solid support such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, etc.

Also without wishing to be bound by theory, one skilled in the art will appreciate that the detection methods of the present invention for non-diagnostic purposes may include, but are not limited to, scientific research, quarantine inspection, and the like.

The detection method of the present invention may be used in point of care testing (POCT) or electrochemical immunoassay systems. The detection method according to the invention or any exemplary form thereof can be used in automated and semi-automated systems and is optimized for the specific case.

In a specific embodiment, the detection is performed by using the specific binding of the anti-zika virus monoclonal antibody or antigen binding fragment thereof of the invention to the NS1 protein of zika virus.

Kits according to the invention and any of the exemplary forms thereof may be used in automated and semi-automated systems and are optimized.

Examples

In the following examples, the methods of preparation of the antibodies of the invention and characterization of relevant properties are shown. Unless otherwise indicated, all test procedures used herein were conventional, and all test materials used in the examples described below were purchased from a conventional reagent store, unless otherwise indicated. 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 invention belongs.

It should be noted that the terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The above summary, detailed description, and examples that follow are merely for the purpose of illustrating the invention and are not intended to limit the invention in any way. The scope of the invention is determined by the appended claims without departing from the spirit and scope of the invention.

Example 1: preparation of monoclonal antibody against NS1 protein of Zika virus

Immunization: the Zika virus NS1 protein (heavy chain organism accession number HP 816-1) was used as an immunogen for immunization of mice. Specifically, female BALB/c mice of 6-8 weeks of age were selected. Mice were immunized 4 times, each time at 2 week intervals, with 100 μg of immunizing agent per mouse. The first immunization was performed by mixing the NS1 protein with equal volumes of Freund's complete adjuvant (Sigma-Aldrich Co.), subcutaneously injecting via the back, and the second three immunization was performed by mixing the NS1 protein with equal volumes of Freund's incomplete adjuvant (Sigma-Aldrich Co.), and injecting via the abdominal cavity. 7 days after the fourth immunization, the tail of the mice is cut, the serum is separated, and the antibody titer level of the antiserum of the immunized mice is detected by adopting an indirect ELISA method so as to observe the immune response effect. Serum antibody titers above 1 were selected: 10000 mice were subjected to a cell fusion experiment and were intraperitoneally injected with the NS1 protein without adjuvant 3 days before the cell fusion experiment for booster immunization (100 μg /).

Hybridoma finenessCell establishment: on the day of fusion, the spleen of immunized mice was removed under sterile conditions and the organs were made into single cell suspensions. Mouse myeloma cells (SP 2/0) were taken with the immunized BALB/c mouse spleen cells described above at a ratio of 1:5, and washing the cells twice before fusing with PEG. The cells were washed with pre-warmed PEG1500, gently shaken, and pre-warmed serum-free RPMI-1640 medium, and resuspended in HAT selective medium. The cell suspension was plated at 200. Mu.L/well into 96-well plates and at 37℃with 5% CO ₂ The cells are cultured under conditions. After 4 to 7 days of culture, the culture was changed to HT medium, and when the fused cells grew to 1/10-1/5 of the bottom area of the wells of the 96-well plate, the supernatant was taken for antibody detection.

Screening of positive hybridoma cells: the NS1 protein was diluted with coating buffer (0.05 mol/L, pH9.6, PBS) to a final concentration of 1. Mu.g/mL, added to a 96-well plate at 100. Mu.L/well, and coated overnight at 4 ℃; discarding the coating liquid, washing 3 times by using Phosphate Buffer Solution (PBST), and beating to dry; blocking with 2% BSA in PBST, 150. Mu.L/well, incubating at 37℃for 2h, washing with PBST 3 times, and drying by pipetting; the supernatant of the fusion cells, the positive serum of the immunized mice diluted by 1:1000 (as positive control) and the negative serum of the mice diluted by 1:1000 (as negative control) are respectively added into the corresponding holes at 100 mu L/hole, incubated for 1h at 37 ℃, washed 3 times by PBST and patted dry; adding 1:4000 dilution of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (purchased from Sigma), 100 μl/well, incubated for 1h at 37 ℃, washed 3 times with PBST, and patted dry; adding tetramethyl benzidine (3, 3', 5' -tetramethyl benzidine, TMB) substrate, and developing at room temperature and in dark place for 10min at 100 μl/hole; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid per well.

OD detection of all wells with an ELISA apparatus at 450nm wavelength _450nm Values. OD when negative serum _450nm Less than or equal to 0.1 to determine the absorbance OD of the wells (NS 1) _450nm The value is the OD of the negative well _450nm More than 2.1 times of the value is positive as a judgment standard. Positive hybridoma cells were screened for further cloning.

Cloning of positive cell lines: counting positive cell wells secreting antibodiesAfter dilution to 100 cells/10 mL medium, the diluted cell suspension was plated at 100. Mu.L/well to 96 well cell culture plates, placed at 37℃in 5% CO ₂ Culturing in a cell culture incubator. After 6-7 days, the formation of cloned cells was observed under a microscope, a single gram of Long Sheng long holes were marked, and the cell supernatant was taken out, and ELISA detection (the same as the fusion detection described above) was performed to select positive monoclonal cells. Limiting dilution is carried out on positive hole cells, ELISA values are measured 5-6 days after each limiting dilution, and OD obtained by ELISA detection is selected _450nm The monoclonal well with higher positive value is subjected to limiting dilution until the result of ELISA measurement of the whole 96-well plate is positive. And selecting the monoclonal stable strain with high positive value. Finally, a cell strain which stably secretes the anti-NS 1 antibody is obtained and is named as hybridoma cell strain Z1.

Preparation and purification of cell-on-list antibody: the hybridoma cell line was cultured in a 10cm dish based on a 15% serum-containing RPMI-1640 culture, and expanded to about 4X 10 ⁷ At each dish, the cells were centrifuged at 800rpm for 5min, the supernatant was discarded and the cells were transferred to a 2L roller bottle and serum-free medium was added to give a cell density of about 3X 10 ⁵ Each culture was performed in a flask. After further culturing for 1 to 2 weeks, when the cell death rate reaches 80% -90% (at this time, the cell density is about 1X 10) ⁶ -2×10 ⁶ At a rate of 6000rpm for 20min, collecting the cell suspension, collecting the supernatant, and purifying the supernatant by Protein A immunochromatography.

Monoclonal antibodies prepared from hybridoma cells were designated as Z1 antibodies. The purified monoclonal antibody was subjected to split charging (100. Mu.L/tube, 1mg/mL concentration) and stored at 4℃to 8 ℃.

The monoclonal antibodies described above were identified by SDS-PAGE electrophoresis, the antibodies having an antibody heavy chain band of about 51KD and an antibody light chain band of about 26 KD.

And (3) purity detection: the monoclonal antibody is analyzed by size exclusion chromatography (SEC-HPLC), and under the condition that all components in the sample to be detected are ensured to have peaks, the purity percentage of the main peak is calculated by using a peak area normalization method, and the purity is more than 98%.

Example 2: binding ability of antibodies to NS1 protein

The NS1 protein was diluted to a concentration of 1. Mu.g/mL with 0.05mol/L carbonate buffer at pH 9.6, added to a 96-well ELISA plate at 100. Mu.L/well, coated overnight at 4℃and washed 3 times with PBST on an automatic plate washer, and patted dry. Blocking with 2% BSA in PBST, 150. Mu.L/well, incubation at 37℃for 2h, washing 3 times with PBST, and drying. anti-NS 1 protein monoclonal antibody Z1 is subjected to gradient dilution by using PBS buffer solution with pH of 7.4 and 0.02M, the initial concentration of the antibody is 5 mug/mL, and the gradient dilution is sequentially carried out according to three times, so as to obtain a series of monoclonal antibody samples with different concentrations. The diluted monoclonal antibody sample was incubated at 37℃for 1h at 100. Mu.L/Kong Jiaru on the above ELISA plate, washed 3 times and dried by pipetting. Adding 1:4000 dilution of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (purchased from Sigma), 100. Mu.L/well, incubated for 1h at 37℃and washed 3 times with PBST and patted dry. TMB substrate was added at 100. Mu.L/well and developed for 10min at room temperature in the dark. The reaction was terminated by adding 2mol/L sulfuric acid at 50. Mu.L/well. OD determination Using an enzyme-labeled Instrument _450nm The value was analyzed by software to obtain the EC of monoclonal antibody Z1 ₅₀ Values.

The results of the binding reaction of the Z1 antibody with the Zika virus NS1 protein and the dengue virus type 1 NS1 protein (as controls) are shown in FIG. 1. As can be seen from fig. 1, the monoclonal antibody Z1 is capable of binding specifically to the zika virus NS1 (circle), but not to the dengue virus type 1 NS1 (square), showing good specificity. The ELISA results are further analyzed by software to obtain the EC of the monoclonal antibody Z1 and the Zika virus NS1 protein ₅₀ The value was 0.297nM, at nanomolar.

Example 3: cloning and sequencing of antibody variable region sequences

Total RNA was isolated from the hybridoma cell line, cDNA was prepared by reverse transcription, immunoglobulin sequences were cloned from the hybridoma cell line, and the variable region sequences of the hybridoma cell line antibodies were determined.

Extraction of RNA: the total RNA was extracted and immediately reverse transcribed from the hybridoma cell line described above, with reference to the instructions of the total RNA M5 extraction kit (from Beijing polymeric Biotech Co., ltd.).

Reverse transcription of RNA into cDNA: the total RNA extracted in the previous step was reverse transcribed with reference to M5 First Strand cDNA Synthesis Kit Polymei (available from Beijing polymeric Biotechnology Co., ltd.) to prepare cDNA, which was frozen at-20℃for use.

c. PCR amplification and recovery of variable region sequences: the cDNA obtained in the previous step is used as a template, and a universal heavy chain primer Mu Ig V is used _H 5' -A and Mu IgG V _H 3' -2, amplifying immunoglobulin heavy chain (IgH) cDNA by PCR; similarly, the light chain primer Mu Ig kappa V was used _L 5' -A and Mu Ig kappa V _L 3' -1, immunoglobulin light chain (IgK) cDNA was amplified by PCR. Then, the PCR product is recovered; the PCR reaction used a thermostable pfu dna polymerase throughout.

d. Cloning and sequencing of variable region sequences: according to the specification of cloning vector pTOPO-Blunt Cloning kit (purchased from Beijing polymeric Biotechnology Co., ltd.), the heavy chain and light chain variable region genes were respectively linked to pTOPO vector, E.coli DH 5. Alpha. Was transformed, positive clones were picked up, and submitted to Beijing Rui Biotechnology Co., ltd for sequencing.

The heavy chain variable region gene sequence and the light chain variable region gene sequence of the antibody of the hybridoma cell line obtained by the sequencing were analyzed, and the complementarity determining region sequences of the heavy chain and the light chain are shown in the following table 1 (according to Chothia numbering system).

Table 1: complementarity determining region sequences of heavy chain and light chain of antibody

Example 4: preparation and purification of recombinant antibodies

Through gene recombination technology, some sites of the heavy chain and light chain complementarity determining region are point mutated to construct a series of recombinant antibodies with point mutation, cell strains for stably expressing the antibodies are prepared through eukaryotic expression, and the cell strains are cultured and purified on a large scale.

For encoding antibody V as listed in Table 2 below _L CDR and V _H CDR genes, binding SEV shown in Q ID NO. 21-24 _L FR1-V _L V shown in FR4 and SEQ ID NOS 25-28 _H FR1-V _H FR4, constructing eukaryotic expression plasmid of recombinant antibody by molecular cloning method. The eukaryotic expression plasmid is electrically transduced into CHO host cells, and after the electric transduction, the eukaryotic expression plasmid is added into a pressure screening culture medium (50 mu M MSX) for culturing for 20 days, and the supernatant is taken for ELISA detection (the ELISA detection is carried out by using goat anti-mouse IgG marked by HRP as a secondary antibody for screening, and the method is the same as the above) to screen out recombinant antibody cell strains with stable expression. Specific sequence correspondences of heavy and light chain complementarity determining regions of the recombinant antibodies are shown in table 2 below.

Table 2: sequences of heavy and light chain complementarity determining regions of recombinant antibodies

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And (3) carrying out large-scale cell culture on the screened stable transgenic cell strain by adopting a cell roller bottle culture technology, and preparing a recombinant antibody. Cells were grown in (0.2-0.3). Times.10 with medium (Vega CHO) ⁶ The cells/ml were inoculated into roller bottles, 1L roller bottles contained 300ml of medium (Vega CHO), the number of inoculated bottles was determined according to the production requirements, and the roller bottles inoculated with cells were placed into a cell-transfer bottle machine and cultured in a cell incubator. The culture conditions were 900 rpm, the temperature was 37℃and the carbon dioxide was 5%. After 7-9 days of culture, observing under a sampling microscope, and centrifuging to collect samples when the cell activity rate is less than 50%. And carrying out affinity purification on the sample by using a protein A affinity chromatographic column to obtain an antibody, namely a recombinant monoclonal antibody.

The binding capacity of recombinant antibody Z1' -Z12' to the Zika virus NS1 was examined according to the method described in example 2, and the binding data of recombinant antibody Z1' to the Zika virus NS1 protein was shown in FIG. 2. As can be seen from FIG. 2, the recombinant antibody Z1' also has strong binding ability to the Zika virus NS1 protein (circles), shows good affinity, and does not bind to dengue virus type 1 NS1 (squares), and shows good specificity. Other recombinant antibodies Z2' -Z12', in addition to Z1', also exhibited good affinity and specificity for the Zika virus NS1 protein as shown in FIG. 3 (with dengue virus NS1 type 1 as control, squares).

Further, the obtained OD _450nm The values were analyzed by software to obtain EC for each recombinant antibody ₅₀ The values are shown in Table 3 below.

Table 3: recombinant monoclonal antibodies of the invention EC of the Zika virus NS1 protein ₅₀ Value (nM)

Antibodies to	ZIKA	Antibodies to	ZIKA
				Z1’	0.307	Z7’	0.488
Z2’	0.477	Z8’	0.187
				Z3’	0.521	Z9’	0.219
Z4’	0.233	Z10’	0.182
				Z5’	0.220	Z11’	0.528
Z6’	0.214	Z12’	0.187

As can be seen from the results given in Table 3, all the recombinantly obtained monoclonal antibodies have affinities on the nM level for the Zika virus NS1 protein, and wherein the recombinantly obtained Z8', Z10' and Z12' exhibit optimal EC ₅₀ Values.

Example 5: binding specificity of recombinant antibodies to NS1 protein

Recombinant antibody Z1' at a concentration of 0.5 μg/mL was tested for cross-reactivity with the common flaviviridae NS1 protein according to the method described in example 2, including dengue virus type 1-4 (DENV 1-4), yellow Fever Virus (YFV), west Nile Virus (WNV), japanese Encephalitis Virus (JEV) and tick-borne encephalitis Virus (TBEV), the results are shown in FIG. 4 and Table 4 below.

Table 4: the recombinant monoclonal antibody Z1' of the invention and OD of each viral NS1 protein ₄₅₀ Value of

DENV1	DENV2	DENV3	DENV4	YFV	WNV	JEV	TBEV	ZIKA
									0.0375	0.0363	0.0358	0.0368	0.0438	0.0421	0.0373	0.0441	1.7812

Like the recombinant antibody Z1', the other recombinant antibodies Z2' -Z12' of the present invention show no cross-reactivity with other viruses of the flaviviridae family, such as encephalitis B virus and hepatitis C virus, nor with other RNA viruses, such as influenza virus, and exhibit good specificity (results not shown).

Example 6: colloidal gold immunochromatographic assay

Preparing colloidal gold: 100mL of ultrapure water was added to the Erlenmeyer flask, heated to boiling, 2mL of 2% chloroauric acid (Sigma-Aldrich, cat# 16961-25-4) solution was added, 1mL of 2% trisodium citrate (Sigma-Aldrich, cat# 6132-04-3) aqueous solution was added immediately after boiling, stirring and boiling were continued for 10 minutes, and natural cooling was performed for use.

Labeling colloidal gold conjugates: 10mL of the gel Jin Fangru beaker was taken, and 120. Mu.L of 0.2: 0.2M K was added with stirring ₂ CO ₃ Adjusting the pH to 7.0, and continuously stirring for 10 seconds; 100 μg of the labeled antibody (antibody A) was added and stirring was continued for 5 minutes; 0.1mL of 10% BSA was added and stirring was continued for 5 minutes; 12000g was centrifuged for 10 minutes, the supernatant was discarded, and the pellet was fixed to 1mL with a gel Jin Xishi solution (10mM PB,150mM NaCl,0.2%BSA,0.1%TritonX-100,3% Sucrose,0.01% Proclin 300) as an anti-Zika NS1 antibody colloidal gold complex.

Preparing a colloidal gold pad: the colloidal gold composite is diluted by 10 times of colloid Jin Xishi liquid respectively, then soaked in glass fiber (Shanghai gold standard company) and freeze-dried, thus obtaining the gold standard pad.

Nitrocellulose membrane (NC membrane) coating: the coated antibody (antibody B) was diluted to 1mg/mL to prepare a detection line working solution, streaked on the corresponding position of a nitrocellulose membrane (Millipore Co., ltd., product No. HF 135002) by a spot film reader, and dried at 50℃for 1 hour for use.

Assembling a colloidal gold immunochromatography test reagent strip: and assembling the gold-labeled pad, the nitrocellulose membrane coated with the antibody, the absorbent paper, the polyester plate and the sample pad into a colloidal gold immunoassay reagent strip.

Sensitivity detection: samples of recombinant antigen at different concentrations were separately tested. Specifically, 80. Mu.L of the sample to be measured was dropped onto the sample pad, left at room temperature for 15-30 minutes, and the result was judged. The activity of antigen binding to antibodies in the sample can be indicated by the shade of the color of the displayed band. The color of the T line developed by the colloidal gold test paper reaction is compared with a standard color card, the closest color is selected, the grade number of the color number corresponding to the color is used for marking the activity of the product, the colloidal gold chromatographic reagent of the Zika virus NS1 antigen of the manufacturer A is used as a control, and the result is shown in the following table 5 (+represents half grade, such as C5+ represents a strip with color development between C5 and C4, and B represents no strip).

Table 5: colloidal gold immunochromatographic test results of antibody combinations of the present invention

The result shows that the minimum detection limit of the colloidal gold chromatographic reagent composed of the antibody of the invention can reach 1ng/mL (positive with the color card grade 8), and compared with the Zika virus NS1 antigen colloidal gold chromatographic reagent of the manufacturer A, the antibody combination of the invention shows higher sensitivity, which is 0.5 to 1 (color card) grade higher than the product sensitivity of the manufacturer A.

And (3) specificity detection: the reagent strip prepared by the antibody of the invention is used for testing serum samples of 500 healthy people, 1 false positive result is detected, and the specificity is 99.8%.

Other combinations: in addition to the results presented in table 5, the present invention further verifies the sensitivity and specificity of the antibodies of the present invention by testing several combinations presented in table 6 below using colloidal gold immunochromatography. The results show that the antibodies of the invention show similar sensitivity and specificity, both as coated antibodies and labeled antibodies, and both monoclonal antibodies and recombinant antibodies, and that the detection sensitivity for recombinant antigens can reach 1ng/mL.

Table 6: other recombinant antibody combinations of the invention

Example 7: latex chromatography test

The anti-Zika virus monoclonal antibody is evaluated by adopting an immunochromatography method based on colored latex microspheres.

Labeling latex microspheres with recombinant antibodies, comprising the steps of:

1. cleaning the microspheres: adding a certain amount of 0.1M 4-morpholinoethanesulfonic acid (MES) buffer solution pH 6.0 into 1mg microsphere, centrifuging at 20000rpm for 20min after uniformly mixing, and removing supernatant;

2. activating: adding a certain volume of MES buffer solution pH 6.0, mixing uniformly by ultrasonic, adding 12 mug of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 12 mug of g N-hydroxysuccinimide (NHS), reacting for 15min at 37 ℃, mixing uniformly once every 5min, centrifuging for 20min at 20000rpm, and discarding the supernatant;

3. Coupling: adding a certain volume of 50mM boric acid buffer solution with pH of 8.0, mixing uniformly by ultrasonic, adding 0.2mg of recombinant antibody A listed in the following table 7 as a labeled antibody, mixing uniformly at 37 ℃ and reacting overnight;

4. closing: 1ml BSA (5%) was added and blocked for 1h, centrifuged at 20000rpm for 20min and the supernatant was discarded.

5. Cleaning: washing with 0.1M Tris-HCl pH8.5 for 2 times, centrifuging at 20000rpm for 20min, and discarding the supernatant;

6. and (3) preserving: 0.5ml of 25mM MES (pH=7.4) was added for storage, and 50-fold dilution with 25mM MES (pH=7.4) was performed with 1%BSA,0.1%Tween 20 followed by lyophilization and sealing for use.

Recombinant antibody coated nitrocellulose membrane (NC membrane): recombinant antibody B (as listed in table 7 below) as a coated antibody was diluted with 10mm pH7.4 pbs, 2% sucrose to a final concentration of 1.0mg/ml, then coated onto NC membrane of cidolis 140 as a detection line (T line), and sealed for later use after overnight at 37 ℃.

Preparing a sample pad: the sample pad was treated with 10mM PBS pH7.4 and lyophilized for use.

Assembly of the immunochromatography system: the red latex microsphere of the marked antibody, NC film coated with the antibody, a sample pad, absorbent paper and a polyester plate are assembled into an immunochromatography rapid test card in a conventional mode.

Immunoassay: samples containing different concentrations (0.1 ng/mL, 0.2ng/mL, 1ng/mL, 10ng/mL, and 100 ng/mL) of recombinant antigen were assayed separately. Specifically, 80. Mu.L of the sample to be tested is dripped into a rapid test card, and after being placed at room temperature for 15-30 minutes, the result is observed and judged by naked eyes. The activity of binding of the antigen and the antibody in the sample can be indicated according to the color depth of the color development band, and the result is shown in Table 7, and the Zika virus NS1 antigen colloidal gold chromatographic reagent of the manufacturer A is used as a control.

Table 7: latex immunochromatographic test results of the antibody combinations of the invention

As shown in Table 7, the minimum detection limit of the antibody combination of the invention on the recombinant antigen can reach 0.2ng/mL (positive with the color card grade 8), and compared with the colloidal gold chromatographic reagent of the Zhai Ka virus NS1 antigen of the manufacturer A, the antibody combination of the invention shows higher sensitivity on a latex chromatographic platform, and the sensitivity of the antibody combination of the invention is 1.5 (color card) grades higher than that of the product of the manufacturer A.

And (3) specificity detection: on a latex chromatographic platform, 500 clinical negative samples were tested using the reagent strips prepared with the antibodies of the invention, and 1 false positive result was detected with a specificity of 99.8%.

Sequence listing

SEQ ID NO:1 (VH CDR 1): GYSFTX1Y, wherein X1 is S or N

SEQ ID NO. 2 (VH CDR 2): DPX2DSE, wherein X2 is S or T

SEQ ID NO:3 (VH CDR 3): RSSWYFDX3, wherein X3 is V or A

SEQ ID NO. 4 (VL CDR 1): GASESVGX4YGNSFLH, wherein X4 is N or S

SEQ ID NO. 5 (VL CDR 2): LX5SNLX6S, wherein X5 is A or G and X6 is Q or E

SEQ ID NO. 6 (VL CDR 3): QQNNEX7PST wherein X7 is D or E

SEQ ID NO:7(VH CDR1)：GYSFTSY

SEQ ID NO:8(VH CDR1)：GYSFTNY

SEQ ID NO:9(VH CDR2)：DPSDSE

SEQ ID NO:10(VH CDR2)：DPTDSE

SEQ ID NO:11(VH CDR3)：RSSWYFDV

SEQ ID NO:12(VH CDR3)：RSSWYFDA

SEQ ID NO:13(VL CDR1)：GASESVGNYGNSFLH

SEQ ID NO:14(VL CDR1)：GASESVGSYGNSFLH

SEQ ID NO:15(VL CDR2)：LASNLES

SEQ ID NO:16(VL CDR2)：LASNLQS

SEQ ID NO:17(VL CDR2)：LGSNLES

SEQ ID NO:18(VL CDR2)：LGSNLQS

SEQ ID NO:19(VL CDR3)：QQNNEDPST

SEQ ID NO:20(VL CDR3)：QQNNEEPST

SEQ ID NO:21(HFR1)：QVQLQQPGAELVKPGASVKLSCKAS

SEQ ID NO:22(HFR2)：LMHWVKQRPGHGLEWIGEV

SEQ ID NO:23(HFR3)：TNYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR

SEQ ID NO:24(HFR4)：WGQGTTLTVSS

SEQ ID NO:25(LFR1)：DIVLTQSPASLAVSLGQRATISC

SEQ ID NO:26(LFR2)：WYQQKPGQPPKLLIY

SEQ ID NO:27(LFR3)：GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC

SEQ ID NO:28(LFR4)：FGGGTKLEIK

Claims

1. An anti-zika virus monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a heavy chain complementarity determining region V represented by SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 3, and a light chain variable region _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region including light chain complementarity determining region V represented by SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6 _L CDR1、V _L CDR2 and V _L CDR3。

2. The anti-zika virus monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein,

1) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 16 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3；

2) The heavy chain variable region comprises amino acid sequences respectively represented by SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 10Heavy chain complementarity determining region V shown in ID No. 11 _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 18 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3；

3) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 17 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3；

4) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3；

5) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3；

6) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 17 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3；

7) The heavy chain variable region comprises amino acid sequences shown by SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 11 respectivelyHeavy chain complementarity determining region V _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 15 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3；

8) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 16 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3；

9) The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences shown by SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 18 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3；

10 The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences represented by SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 13, SEQ ID NO. 17 and SEQ ID NO. 19, respectively _L CDR1、V _L CDR2 and V _L CDR3；

11 The heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences represented by SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 17 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3; or (b)

12 The heavy chain variable region comprises the heavy chain complementarity whose amino acid sequences are shown by SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 12, respectivelyDetermination region V _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising light chain complementarity determining region V having amino acid sequences represented by SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 20, respectively _L CDR1、V _L CDR2 and V _L CDR3。

3. The anti-zika virus monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3 and HFR4, having the sequence represented by SEQ ID NOs 21-24 or sequences having 80% or more, 85% or more, 90% or more, or 95% or more identity to said sequences, respectively; the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3 and LFR4, having the sequences shown by SEQ ID NOs 25-28 or sequences having 80% or more, 85% or more, 90% or more or 95% or more identity to said sequences, respectively.

4. A nucleic acid molecule encoding the anti-zika virus monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3.

5. A vector comprising the nucleic acid molecule of claim 4; preferably, the vector is a plasmid vector, for example, any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT3, pEFBOS, pBV, pJV and pBJ.

6. An expression cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5, preferably the expression cell is a mammalian cell, e.g. selected from chinese hamster ovary cells, little hamster kidney cells, monkey kidney cells, mouse thymoma cells, human embryonic kidney cells.

7. Use of an anti-zika virus monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of a reagent for detecting zika virus.

8. A method of detecting ZIKA virus (ZIKA) for non-diagnostic purposes comprising the step of using the anti-ZIKA virus monoclonal antibody or antigen binding fragment thereof of any one of claims 1-3.

9. The use according to claim 7 or the method according to claim 8, wherein the detection is performed by immunochromatography, enzyme-linked antibody (ELISA), chemiluminescence, electrochemiluminescence; preferably, the immunochromatography includes fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography; ELISA such as direct method, indirect method, sandwich method and competition method.

10. A kit for detecting a zika virus comprising the anti-zika virus monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-3 and instructions for use.