TW202000696A - Anti-dengue virus antibodies and applications thereof - Google Patents

Abstract

The present invention relates to anti-virus dengue virus antibodies and applications thereof. Specifically, the anti-virus dengue virus antibodies of the present invention are serotype specific and useful for specific detection and differentiation of various dengue virus serotypes in a biological sample. The present invention also provides methods and kits for detecting dengue virus and methods and compositions for use in diagnosis and treatment of dengue virus disease using the anti-virus dengue virus antibodies as described herein.

Description

Anti-dengue virus antibody and its application

The invention relates to an anti-dengue virus antibody and its application. Specifically, the antiviral dengue virus antibody of the present invention is serotype specific, and can be used for the specific detection and differentiation of various dengue virus serotypes in biological samples. The invention also provides a method and kit for detecting dengue virus, as well as a method and composition for diagnosing and treating dengue virus disease using the antiviral dengue virus antibody as described herein.

Today, approximately 3.9 billion people in the world live in areas at risk of dengue fever transmission. It is estimated that there are 100 million cases of dengue fever infection each year, including 250,000-500,000 severe cases and about 25,000 deaths. Dengue fever is a viral disease transmitted by mosquitoes, caused by the spread of any one of the four dengue virus serotypes (DENV1, DENV2, DENV3, and DENV4). Globally, dengue fever is mostly transmitted by two major mosquitoes: Aedes aegypti (adapted to the city) and Aedes albopictus ( Aedes albopictus ). The rapid spread of dengue fever is attributed to the urbanization of tropical and subtropical countries, increased travel within and between countries where dengue is endemic, and ineffective vector control strategies. In fact, the disease and economic burden of dengue fever has become a global public health problem.

To date, there are no effective antiviral drugs available for the treatment of dengue infection, and the vaccine is still in the evaluation stage. In addition, infection with one dengue virus serotype confers lifelong immunity to the same serotype, but when infected with another serotype, the risk of severe dengue fever increases. Therefore, early diagnosis of DENV is very important for disease management. Early diagnosis of dengue virus infection will interfere with treatment of patients and control of epidemics. Humans infected with dengue virus show a wide range of clinical manifestations, from asymptomatic infection to severe dengue fever, making accurate diagnosis difficult. Current laboratory diagnostic tests for dengue fever include virus isolation, viral RNA detection, antigen detection, and serological methods. Virus isolation and viral RNA detection are effective within the first five (5) days of the disease; both methods can identify the dengue virus serotype. However, virus isolation is time-consuming, and virus RNA detection requires special equipment and training personnel. Anti-DENV IgM or IgG antibodies only appear in the blood 5 days after the onset of the disease and may cross-react with other flaviviruses; therefore, detection of anti-DENV IgM or IgG within the first 5 days of symptoms does not confirm the presence of the virus, and False positive results may often occur.

In the acute phase of the disease (ie, between day 1 and day 7 after the onset of the disease), dengue virus nonstructural protein 1 (NS1), a 50 kDa glycoprotein, is secreted in the patient's serum and Accumulate to high concentration. The presence of NS1 in human serum can be confirmed within 1 to 9 days after infection. In addition, when viral RNA cannot be detected by RT-PCR and before the appearance of IgM antibody, NS1 protein can be detected. NS1 protein is a good target for early diagnosis markers. NS1 protein antigen detection test can be simple, low cost, processing a large number of samples, and a rapid way for early diagnosis of DENV. However, although there are several commercially available NS1 tests that can be used to diagnose dengue fever in the early stages of the disease, none of them can distinguish between various dengue serotypes.

There is still a need to develop diagnostic methods that can detect and differentiate various dengue serotypes.

The present invention is based on the identification of many isolated antibodies that unexpectedly exhibit excellent specificity for each dengue virus serotype. Each serotype specific antibody of the present invention can detect a specific serotype of dengue virus without substantially cross-reacting with other serotypes. The present invention provides such antibodies and antigen-binding fragments thereof, as well as compositions and kits containing them, and methods of using them. The invention can be used to specifically detect one or more dengue virus serotypes in a sample, specifically a sample from a patient suspected of being infected by a virus. The present invention also provides cross-reactive antibodies specific for dengue virus (all serotypes), which can be paired with any serotype specific antibodies described herein and used in immunoassays to detect dengue virus in a sample.

In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof, wherein the isolated anti-system is selected from the group consisting of: (i) specific for the first of dengue virus serotype 1 (DENV1) Antibody, comprising (a) heavy chain variable region (V _H ), including heavy chain complementarity determining region 1 (HC CDR1) as shown in SEQ ID NO: 2, heavy chain complementarity determining as shown in SEQ ID NO: 4 Region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 6; and (b) a light chain variable region (V _L ), including SEQ ID NO: 9 Light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 11, and light chain complementarity determining region 3 (LC CDR3) shown in SEQ ID NO: 13 ; (Ii) a secondary antibody specific for dengue virus serotype 2 (DENV2), including (a) heavy chain variable region (V _H ), including the heavy chain complementarity determining region shown in SEQ ID NO: 16 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 18, and heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 20; and (b) light The chain variable region (V _L ) includes the light chain complementarity determining region 1 (LC CDR1) shown in SEQ ID NO: 23, the light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 25, and SEQ ID NO: 27 Light chain complementarity determining region 3 (LC CDR3); (iii) A third antibody specific for Dengue virus serotype 3 (DENV3), including (a) heavy chain variable region (V _H ), including the heavy chain complementarity determining region 1 (HC CDR1) shown in SEQ ID NO: 30, the heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 32, and the SEQ ID NO: 34 the heavy chain complementarity determining region 3 (HC CDR3); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 37 the heavy chain complementarity determining region 1 (LC CDR1), SEQ ID NO: Light chain complementarity determining region 2 (LC CDR2) shown in 39, and light chain complementarity determining region 3 (LC CDR3) shown in SEQ ID NO: 41; (iv) Specific for dengue virus serotype 4 (DENV4 )'S fourth antibody, comprising (a) the heavy chain variable region (V _H ), including the heavy chain complementarity determining region 1 (HC CDR1) shown in SEQ ID NO: 44 and SEQ ID NO: 46 The heavy chain complementarity determining region 2 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 48; and (b) the light chain variable region (V _L ), including SEQ ID NO: 51 indicates the light chain complementarity determining region 1 (LC CDR1), SEQ ID NO: 53 indicates the light chain complementarity determining region 2 (LC CDR2), and SEQ ID NO: 55 indicates the light chain complementarity determining region 3 ( LC CDR3); (v) Fifth antibody that cross-reacts with dengue virus serotypes type 1, type 2, type 3, and type 4 (DENV1, DENV2, DENV3, and DENV4), including (a) The heavy chain variable region (V _H ), such as the heavy chain complementarity determining region 1 (HC CDR1) shown in SEQ ID NO: 58 and the heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 60, and SEQ ID NO: 62 shown in the heavy chain complementarity determining region 3 (HC CDR3); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 65, respectively of the light chain complementarity determining region 1 ( LC CDR1), light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 67, and light chain complementarity determining region 3 (LC CDR3) shown in SEQ ID NO: 69; and (vi) any (i ) To (v).

In certain embodiments, the first antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 71 and a light chain variable region (V _L ) comprising SEQ ID NO: 72.

In certain specific embodiments, the second antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 73 and a light chain variable region (V _L ) comprising SEQ ID NO: 74.

In certain specific embodiments, the third antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 75 and a light chain variable region (V _L ) comprising SEQ ID NO: 76.

In certain specific embodiments, the fourth antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 77 and a light chain variable region (V _L ) comprising SEQ ID NO: 78.

In certain specific embodiments, the fifth antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 79 and a light chain variable region (V _L ) comprising SEQ ID NO: 80.

The dengue virus specific antibody of the present invention may be a full-length antibody. The antigen-binding fragment of the antibody of the present invention may be scFv, (scFv)2, Fab, Fab', F(ab') ² .

In another aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding an antibody heavy chain variable region (V _H ) or an antibody light chain variable region (V _L ) or both, wherein the V _H and V _{L is} as described herein.

The invention also provides vectors (eg, expression vectors) containing any of the nucleic acids described herein and host cells containing such vectors.

The present invention further provides a method for preparing a specific antibody against dengue virus, comprising (i) cultivating a host cell as described herein under conditions allowing the expression of the antibody, and optionally (ii) harvesting the antibody from cell culture .

In another aspect, the invention provides a composition comprising (a) any antibody specific for dengue virus described herein, any nucleic acid as described herein, or any vector described herein; and (b ) Carrier, for example, a pharmaceutically acceptable carrier.

In certain embodiments, the composition of the present invention is a pharmaceutical composition for treating dengue virus diseases.

In certain embodiments, the composition of the present invention is a diagnostic composition for diagnosing dengue virus diseases.

In another aspect, the present invention provides a method for detecting dengue virus in a sample suspected of containing the dengue virus, comprising combining the sample with an isolated antibody or antigen thereof specific for dengue virus as described herein The fragments are contacted, and the binding of the antibody to the sample is determined. The results of this combination are used to determine the presence of each dengue virus serotype in the sample. Specifically, (i) the binding of the first antibody to the sample indicates the presence of DENV1 in the sample; (ii) the binding of the second antibody to the sample indicates the presence of DENV2 in the sample; (iii) the third The binding of the antibody to the sample indicates the presence of DENV3 in the sample; and/or (iv) the binding of the fourth antibody to the sample indicates the presence of DENV4 in the sample. In certain embodiments, the corresponding serotype-specific antibodies of the invention are paired with partner antibodies for sandwich assays.

In another aspect, the present invention provides a kit for detecting the presence of one or more dengue virus serotypes in a sample, comprising one or more dengue virus serotype specific antibodies described herein and optional Partner antibody. The partner antibody may be a dengue virus serotype cross-reactive antibody, for example, a fifth antibody as described herein.

In some embodiments, at least one antibody in the kit contains a detectable label.

Examples of such detectable labels include, but are not limited to, enzyme labels, fluorescent labels, metal labels, and radioactive labels.

In certain embodiments, the kit is an immunoassay kit.

Examples of the immunoassay include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay (FIA) luminescence immunoassay, LIA), or ILMA immunofluorescence.

In some specific embodiments, the immunoassay is performed in a lateral flow assay format.

Specifically, the immunoassay is a sandwich assay.

The details of one or more specific embodiments of the invention are set forth in the following description. Other features or advantages of the present invention will become apparent from the following detailed description of specific embodiments and the scope of the attached patent application.

The present invention relates to serotype specific antiviral dengue virus antibodies. The present invention provides such antibodies and antigen-binding fragments thereof, which can be used to specifically detect and differentiate various dengue virus serotypes in biological samples. The present invention further provides a method and carrier for preparing antibodies or antigen-binding fragments thereof, as well as kits and compositions containing them, as well as methods for using them to detect dengue virus and to diagnose and treat dengue virus disease. The invention further provides cross-reactive antibodies specific for dengue virus, which can be paired with any serotype-specific antibodies described herein and used in immunoassays to detect dengue virus in a sample.

The following description is only intended to illustrate various specific embodiments of the present invention. Therefore, the specific embodiments or modifications discussed herein should not be construed as limiting the scope of the invention. It is obvious to those skilled in the art that various changes or equivalents can be made without departing from the scope of the present invention.

I. definition

In order to provide a clear and quick understanding of the present invention, certain terms are first defined. Additional definitions are elaborated throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

As used herein, the singular forms "a", "an" and "the" include plural indicators unless the context clearly dictates otherwise. Thus, for example, reference to "a component" includes a plurality of such components and equivalents known to those skilled in the art.

The words "include (verb)" or "include (verb noun)" are usually used in the meaning of including (verb)/including (verb noun), which means that one or more features, ingredients or components are allowed to exist. The term "contains (verb)" or "contains (verb noun)" includes the term "consisting" or "consisting of".

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues connected by peptide bonds. The term "protein" usually refers to a relatively large polypeptide. The term "peptide" generally refers to a relatively short polypeptide (eg, containing up to 100, 90, 70, 50, 30, or 20 amino acid residues).

As used herein, words such as "about" or "approximately" refer to acceptable degrees of deviation that will be understood by those of ordinary skill in the art, and they may vary to a certain extent, depending on the context in which they are used. Generally, "about" or "approximately" may mean a value within a range of ±10% around the quoted value.

As used herein, the term "substantially the same" means that the two sequences have 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more Of homology.

As used herein, the term "antibody" (interchangeably used in plural) refers to an immunoglobulin molecule that has the ability to specifically bind a specific target antigen. As used herein, the term "antibody" includes not only complete (ie, full-length) antibody molecules, but also antigen-binding fragments that retain antigen-binding ability, such as Fab, Fab', F(ab')2, and Fv. Such fragments are also well known in the art and are generally used in vitro and in vivo. The term "antibody" also includes humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (eg, bispecific antibodies), and any other modified configuration of immunoglobulin molecules , Which contains antigen recognition sites with the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

A complete or complete antibody contains two heavy chains and two light chains. Each heavy chain contains a variable region (V _H ) and first, second and third constant regions (C _H 1, C _H 2 and C _H 3); each light chain contains a variable region (V _L ) And a constant region ( _CL ). The antibody has a "Y" shape. The stem of Y is composed of the second and third constant regions of two heavy chains joined together by disulfide bonds. Each arm of Y includes a variable region and a first constant region of a single heavy chain combined with a variable region and a constant region of a single light chain. The variable region of the light chain and the variable region of the heavy chain are responsible for antigen binding. The variable regions in the two chains usually contain three highly variable regions, called complementarity determining regions (CDRs); that is, the light (L) chain CDRs include LC CDR1, LC CDR2, and LC CDR3, And heavy (H) chain CDRs include HC CDR1, HC CDR2, and HC CDR3. The three CDRs are marked by framework regions (FR1, FR2, FR3, and FR4), which are more highly conserved than the CDRs and form a scaffold that supports a highly variable region. The constant regions of the heavy and light chains are not responsible for antigen binding, but are involved in the functions of various effectors. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

As used herein, the terms "antigen-binding domain" or "antigen-binding fragment" refer to the part or region of an intact antibody molecule that is responsible for antigen binding. The antigen-binding fragment can bind the same antigen to which the parent antibody binds. Examples of antigen-binding fragments include, but are not limited to: (i) Fab fragments, which may be a V _H -C _H 1 by a chain and V _L - C _L chain monovalent fragment consisting of; (ii) F (ab ' ) 2 fragments , which may be a bivalent fragment through the hinge region disulfide bond two Fab fragments linked composition; (iii) Fv fragments, V _H and V transmission joined by non-covalent interactions with the structure of the antibody molecule _L Domain composition; (iv) single-chain Fv (scFv), which can be a single multi-peptide chain consisting of a V _H domain and a V _L domain through a peptide linker; (v) (scFv) ₂ , which can comprising two V _H domains are connected ligated, and two V _L domains through a peptide which is coupled to two V _H domains via disulfide bonds.

As used herein, the term "chimeric antibody" refers to an antibody that contains multiple peptides from different sources (eg, different species). In some specific embodiments, in these chimeric antibodies, the variable regions of the light chain and the heavy chain can mimic antibodies derived from a mammal (eg, non-human mammals such as mice, rabbits, and rats) And the constant part may be homologous to the sequence in an antibody derived from another mammal, such as a human.

As used herein, the term "humanized antibody" refers to an antibody that includes a framework region from a human antibody and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin.

As used herein, the term "human antibody" refers to an antibody in which substantially the entire sequence of light and heavy chain sequences (including complementarity determining regions (CDRs)) are derived from human genes. The human antibody may include one or more amino acid residues not encoded by the human germline immunoglobulin sequence, for example, through mutations in one or more CDRs, or mutations in one or more FRs, for example Decrease possible immunogenicity, increase affinity, eliminate cysteine which may cause undesired folding, etc.

As used herein, "isolated" material means that it has been changed from its natural state through human hands. In certain embodiments, if the multiple peptides (eg, antibodies) or nucleic acids of the present invention are substantially free of cellular material or chemical precursors or other chemicals/components that may be involved in the peptide/nucleic acid preparation process, It can be said that they are "isolated" or "purified". It should be understood that the terms "isolated" or "purified" do not necessarily reflect the degree to which the peptide is "absolutely" isolated or purified, for example, by removing all other substances (eg, impurities or cellular components). In some cases, for example, isolated or purified polypeptides include those containing less than 50%, 40%, 30%, 20%, or 10% (by weight) of other proteins (eg, cellular proteins) Polypeptide preparations with less than 50%, 40%, 30%, 20% or 10% (by volume) medium, or with less than 50%, 40%, 30%, 20% or 10% ( By weight) chemical precursors or other chemicals/components involved in the synthesis process.

As used herein, the terms "specific binding" or "specific binding" refer to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope of its target antigen. Antibody that "specifically binds" to a target antigen or epitope is a term well known in the art, and methods to determine such specific binding are also well known in the art. A molecule is said to exhibit "specificity" if it reacts or binds more frequently, faster, longer and/or with a higher affinity for a specific target antigen or epitope than other targets/epitopes Combine". If the antibody binds with greater affinity, retention, and/or retention for a longer time than other substances, then the antibody "specifically binds" to the target antigen. In other words, it can also be understood by reading the definition that, for example, an antibody that specifically binds to the first target antigen may or may not specifically or preferentially bind to the second target antigen. Therefore, "specific combination" or "preferred combination" does not necessarily require (although it can include) exclusive combination. Usually, but not necessarily, reference to bonding means specific/preferred bonding. The binding affinity is defined according to the dissociation constant (K _D ). Typically, when using an antibody that specifically binds may refer specifically binds (identification) which target antibody with a KD value of less than about 10 ^{~ 7} M, for example, about 10 ^{~ 8} M or less, for example about 10 ^{~ 9} M or less, about 10 ^{to 10} M or less, about 10 ^{to n} M or less, about 10 ^{to 12} M or less, or even lower, to bind specific targets with affinity corresponding to K _D than to non-specific antigens ( (Eg BSA or casein) binding affinity is at least 10 times lower, for example at least 100 times lower, for example at least 1,000 times lower, for example at least 10,000 times lower.

As used herein, the term "cross-reactivity" can refer to the ability of an antibody to react with similar antigen sites on different proteins. For example, cross-reactive antibodies to dengue virus serotypes can mean that such antibodies can be combined with more than one serotype of dengue virus (eg, all four serotypes of dengue virus) or even other flaviviruses such as Zika virus and And/or Japanese encephalitis virus cross-reaction (ie, antibody binding) rather than specific reaction with dengue virus of a specific serotype.

Dengue virus is a single-stranded RNA virus that is a member of the Flaviviridae family, Flavivirus. As used herein, dengue virus refers to any serotype of dengue virus, including dengue virus serotype 1 (DENV1), dengue virus serotype 2 (DENV2), dengue virus serotype 3 (DENV3), and dengue virus serotype 4 (DENV4). Those different dengue virus serotypes are genetically related, but have different antigenicities. The immunity is serotype specific, and there is no cross-protection between serotypes, that is, infection of one serotype cannot protect humans from other serotypes. Serotypes can be determined by conventional methods known in the art, such as RT-PCR using specific primer sets (TS1, TS2, TS3, or TS4, together with D1) to amplify capsid and membrane proteins from dengue virus Serotype-specific fragments of the region, as described by Konogoi et al. in Virology Journal (2016) 13:182. Typical strains of each dengue virus serotype are available in this technology, such as DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241.

As used herein, the terms "dengue virus disease" or "dengue fever" refer to diseases caused by dengue virus infection, usually transmitted by mosquitoes. Humans with dengue virus infection may show no signs or have mild to severe symptoms. When symptoms appear, they usually start 3 to 14 days after infection. Typical dengue fever (DF) often presents with fever, headache, severe muscle and joint pain, nausea, and rash. The severe form (DHF/DSS) is characterized by all symptoms of DF, accompanied by bleeding manifestations (gingival or nose, Urine, fecal or subcutaneous bleeding), severe abdominal pain, persistent vomiting, fatigue, irritability or restlessness, which can be life-threatening if left untreated.

The terms "NS1", "NS1 polypeptide" and "NS1 protein" are used interchangeably herein. NS1 refers to a non-structural glycoprotein encoded by dengue virus, which binds to the membrane on the cell surface and is released into the systemic circulation as early as 1 day after the onset of symptoms, and can be detected at least about 9 days after infection . Therefore, NS1 has become a good marker for the diagnosis of dengue fever infection, especially in the early stages. The mature form of NS1 contains 352 amino acid residues and has a molecular weight of about 40 kDa or higher, depending on the degree of glycosylation. NS1 is serotype-specific and the amino acid sequence of NS1 from different dengue virus serotypes is known and available in the art, for example, SEQ ID NO: 91 of NS1 from DENV1 (DENV1 NS1), from SEQ ID NO: 92 of NS1 of DENV2 (DENV2 NS1), SEQ ID NO: 93 of NS1 from DENV3 (DENV3 NS1), and SEQ ID NO: 94 of NS1 from DENV4 (DENV4 NS1). NS1 can be prepared by recombinant methods known in the art, or can be prepared and harvested from the culture supernatant of dengue virus, for example, DENV1 Hawaii is used to produce DENV1 NS1, DENV2 16681 is used to produce DENV2 NS1, DENV3 H87 is used to DENV3 NS1 is produced, and DENV4 H241 is used to produce DENV4 NS1.

Table 1 shows exemplary amino acid sequences for NS1 of DENV1, DENV2, DENV3, and DENV4.

The terms "nucleic acid" or "polynucleotide" may refer to polymers composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs, including nucleic acid analogs with non-naturally occurring nucleotides. The polynucleotide can be synthesized, for example, using an automatic DNA synthesizer. It should be understood that when the nucleotide sequence is represented by a DNA sequence (ie A, T, G, C), this also includes RNA in which "U" is substituted for "U" (ie A, U, G, C) sequence. The term "cDNA" refers to DNA that is complementary or identical to mRNA in single-stranded or double-stranded form.

The term "complementary" refers to the topological compatibility or matching of the interaction surfaces of two polynucleotides. Therefore, the two molecules can be described as complementary, in addition the contact surface features are complementary to each other. When the nucleotide sequence of the first polynucleotide is the same as the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, the first polynucleotide is complementary to the second polynucleotide. Therefore, the polynucleotide of sequence 5'-TATAG-3' is complementary to the polynucleotide of sequence 5'-CTATA-3'.

The term "coding" refers to the inherent characteristics of a specific nucleotide sequence (eg, gene, cDNA, or mRNA) in a polynucleotide to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have A given nucleotide sequence (ie, rRNA, tRNA, and mRNA) or a given amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, the gene encodes the protein. Those skilled in the art should understand that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. It should also be understood that the skilled person can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide described there to reflect the password used by any particular host organism in which the polypeptide is represented child. Therefore, unless otherwise stated, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate from each other and encode the same amino acid sequence.

The term "recombinant nucleic acid" refers to a polynucleotide or nucleic acid having sequences that are not naturally linked together. The recombinant nucleic acid may exist in the form of a vector. The "vector" may contain a given target nucleotide sequence and regulatory sequence. Vectors can be used to express a given nucleotide sequence (expression vector) or to maintain a given nucleotide sequence so that it replicates, manipulates, or transfers between different locations (eg, between different organisms). For the above purposes, the vector can be introduced into a suitable host cell. "Recombinant cell" refers to a host cell into which recombinant nucleic acid has been introduced. "Transformed cell" refers to a cell into which a DNA molecule encoding a target protein is introduced by recombinant DNA technology.

Vectors can be of various types, including plastids, slime bodies, fosmids, episomes, artificial chromosomes, bacteriophages, viral vectors, etc. Generally, in a vector, a given nucleotide sequence is operably linked to a regulatory sequence so that when the vector is introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. Regulatory sequences may include, for example, but not limited to, promoter sequences (eg, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene ( AOX1 ) promoter), start codon, origin of replication, enhancer, secretion signal sequence (for example, an α-mating factor signal), stop codon, and other control sequences (for example, Shine-Dalgarno sequence and termination sequence ). Preferably, the vector may further contain a marker sequence (eg, an antibiotic resistance marker sequence) for subsequent screening/selection procedures. For the purpose of protein production, in a vector, a given target nucleotide sequence may be linked to another nucleotide sequence in addition to the above regulatory sequence, thereby generating a fusion polypeptide and benefiting the subsequent purification process. The fusion polypeptide includes a tag for purification, for example, His-tag.

The terms "individual" or "subject" as used herein include human and non-human animals, such as companion animals (such as dogs, cats, and the like), and farm animals (such as cows, sheep, pigs, horses, and the like) ), or laboratory animals (such as rats, mice, guinea pigs and the like).

The term "treatment" as used herein refers to the administration or administration of a composition comprising one or more active agents to an individual suffering from a disease, symptoms or conditions of the disease, or disease progression, for the purpose of treating, curing, alleviating, relieving, Change, remedy, improve, promote, or affect a disease, a symptom or condition of a disease, a disability caused by a disease, or the progression of a disease or its symptoms or conditions.

The term "effective amount" as used herein refers to the amount of active ingredient that imparts the desired biological effect in the treated individual or cell. The effective amount can vary depending on various reasons, such as the route and frequency of administration, the weight and type of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage in each case based on the disclosure herein, established methods, and their own experience.

II. Antibodies specific for dengue virus

The invention is based on the identification of many isolated antibodies that have serotype specificity for dengue virus, including monoclonal antibody 12-4.1 (specifically for DENV1), monoclonal antibody 33-7.1 (specifically for DENV2), and monoclonal antibody 43-1.3 (Specialized in DENV3), and monoclonal antibody 22-1.5 (Specialized in DENV4). It was found that these anti-dengue virus antibodies can recognize a specific serotype of dengue virus without cross-reacting with other serotypes. More specifically, these anti-dengue virus antibodies are specific for dengue virus NS1 protein of different serotypes; that is, monoclonal antibody 12-4.1 is specific for NS1 polypeptide derived from DENV1, and monoclonal antibody 33-7.1 is specific For NS1 polypeptide derived from DENV2, monoclonal antibody 43-1.3 is specific for NS1 polypeptide derived from DENV3, while monoclonal antibody 22-1.5 is specific for NS1 polypeptide derived from DENV4. It was found that these anti-dengue virus antibodies are effective and feasible in various forms of immunoassays for detecting dengue virus in samples, with excellent sensitivity and specificity. The present invention is also based on the identification of specific cross-reactive antibodies against dengue virus (monoclonal antibody 82-1.1), which cross-react with various serotypes of dengue virus, including DENV1, DENV2, DENV3, and DENV4, and can interact with Any serotype-specific antibodies described herein are paired, and the serotype-specific antibodies are used in immunoassays to detect dengue virus in a sample.

Therefore, this article describes serotype-specific antibodies against dengue virus, including monoclonal antibodies 12-4.1 (specifically DENV1), monoclonal antibodies 33-7.1 (specifically DENV2), monoclonal antibodies 43-1.3 (specifically DENV3), and monoclonal antibody 22-1.5 (specifically DENV4), and functional variants. Also described herein are cross-reactive antibodies specific for dengue virus, including monoclonal antibodies 82-1.1 (cross-reactive with DENV1, DENV2, DENV3, and DENV4) and functional variants thereof. The reactivity of these monoclonal antibodies with DENV1-4 was determined using ELISA and Western blot analysis. The characteristics of these monoclonal antibodies are shown in Table 2. The heavy chain variable region (V _H ) and light chain of each of monoclonal antibody 12-4.1, monoclonal antibody 33-7.1, monoclonal antibody 43-1.3, monoclonal antibody 22-1.5, and monoclonal antibody 82-1.1 variable region (V _L) and complementarity determining regions (CDR1, CDR2, CDR3) of the amino acid sequence shown in table 3 below.

The functional variant of the monoclonal antibody 12-4.1 (specifically for DENV1) (also known as "first antibody specific for DENV1", as described herein) may include: (a) heavy chain variable region (V _H ), It includes the heavy chain complementarity determining region (HC CDR1) of SEQ ID NO: 2, the heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 4, and the heavy chain complementarity determining region 3 (HC of SEQ ID NO: 6 CDR3); and (b) light chain variable region (V _L ), which comprises the light chain complementarity determining region 1 (LC CDR1) of SEQ ID NO: 9, the light chain complementarity determining region 2 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 13, or an antigen-binding fragment thereof.

In certain specific embodiments, the first antibody comprises a VH containing SEQ ID NO: 71 or an amino acid sequence substantially the same as it and a _VH containing SEQ ID NO: 72 or an amino acid sequence substantially identical to it V _L. In particular, the first antibody includes at least 80% (eg 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96) of SEQ ID NO: 71. %, 98%, or 99%) V _{H of} an amino acid sequence that is identical, and contains at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%) having SEQ ID NO: 72 , 92%, 94%, 95%, 96%, 98% or 99%) the identity of the amino acid sequence V _L. The first antibody further comprises an antibody produced by any recombinant polynucleotide sequences encoding the V _H or V _L related to the amino acid sequence of encoding the herein (engineered) derived.

Functional variants of monoclonal antibodies 33-7.1 (specifically DENV2) (also called "secondary antibodies specific to DENV2", as described herein) may contain: (a) heavy chain variable region (V _H ), It contains the heavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO: 16, the heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 18, and the heavy chain complementarity determining region 3 of SEQ ID NO: 20 ( HC CDR3); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 23 light chain complementarity determining region of 1 (LC CDR1), SEQ ID NO: 25 light chain complementarity determining region 2 ( LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 27.

In certain specific embodiments, the second antibody comprises a _VH containing the amino acid sequence of SEQ ID NO: 73 or substantially the same as it and an amino acid sequence containing SEQ ID NO: 74 or substantially the same as it V _L. Specifically, the second antibody includes at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96) of SEQ ID NO: 73. %, 98%, or 99%) V _{H of} an amino acid sequence that is identical, and contains at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%) of SEQ ID NO: 74 , 92%, 94%, 95%, 96%, 98% or 99%) the identity of the amino acid sequence V _L. The second antibody also includes an antibody of any recombinant polynucleotide sequences encoding the V _H or V _L related to the amino acid sequence of encoding the herein (engineered) derived.

The functional variant of monoclonal antibody 43-1.3 (specifically DENV3) (also known as "third antibody specific to DENV3", as described herein) may include: (a) heavy chain variable region (V _H ), It includes the heavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO: 30, the heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 32, and the heavy chain complementarity determining region 3 of SEQ ID NO: 34 ( HC CDR3); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 37 light chain complementarity determining region of 1 (LC CDR1), SEQ ID NO: 39 light chain complementarity determining region 2 ( LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 41.

In certain embodiments, the third antibody comprises SEQ ID NO: V _H 75 or a substantially identical amino acid sequence, and comprising SEQ ID NO: 76 amino acid sequence substantially identical thereto, or the V _L. Specifically, the third antibody includes at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96) of SEQ ID NO: 75. %, 98%, or 99%) V _{H of} an amino acid sequence that is identical, and contains at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%) that has at least 80% of SEQ ID NO: 76 , 92%, 94%, 95%, 96%, 98% or 99%) the identity of the amino acid sequence V _L. The third antibody further comprises an antibody produced by any recombinant polynucleotide sequences encoding the V _H or V _L related to the amino acid sequence of encoding the herein (engineered) derived.

The functional variant of the monoclonal antibody 22-1.5 (specifically for DENV4) (also known as "the fourth antibody specific for DENV4", as described herein) may include: (a) heavy chain variable region (V _H ), It includes the heavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO: 44, the heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 46, and the heavy chain complementarity determining region 3 of SEQ ID NO: 48 ( HC CDR3); and (b) light chain variable region (V _L ), which comprises the light chain complementarity determining region 1 (LC CDR1) of SEQ ID NO: 51, the light chain complementarity determining region 2 of SEQ ID NO: 53 ( LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 55.

In certain specific embodiments, the fourth antibody comprises a VH containing SEQ ID NO: 77 or an amino acid sequence that is substantially the same as it and a _VH containing SEQ ID NO: 78 or an amino acid sequence that is substantially the same as it V _L. Specifically, the fourth antibody includes at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96) of SEQ ID NO: 77. %, 98%, or 99%) V _{H of} an amino acid sequence that is identical, and contains at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%) having SEQ ID NO: 78 , 92%, 94%, 95%, 96%, 98% or 99%) the identity of the amino acid sequence V _L. The fourth antibodies also include antibodies produced by any recombinant polynucleotide sequences encoding the V _H or V _L related to the amino acid sequence of encoding the herein (engineered) derived.

The functional variant of monoclonal antibody 82-1.1 (also called "the fifth antibody specific for DENV1, DENV2, DENV3, and DENV4", as described herein) may contain: (a) heavy chain variable region (V _H ), which comprises the heavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO: 58, the heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 60, and the heavy chain complementarity determining region of SEQ ID NO: 62 3 (HC CDR3); and (b) the light chain variable region (V _L ), which comprises the light chain complementarity determining region 1 (LC CDR1) of SEQ ID NO: 65, the light chain complementarity determining region of SEQ ID NO: 67 2 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 69.

In certain embodiments, the fifth antibody comprises a VH containing SEQ ID NO: 79 or an amino acid sequence substantially the same as it and a _VH containing SEQ ID NO: 80 or an amino acid sequence substantially identical to it V _L. Specifically, the fifth antibody includes at least 80% (e.g., 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96) of SEQ ID NO: 79. %, 98%, or 99%) V _{H of} an amino acid sequence that is identical, and contains at least 80% (eg, 82%, 84%, 85%, 86%, 88%, 90%) having SEQ ID NO: 80 , 92%, 94%, 95%, 96%, 98% or 99%) the identity of the amino acid sequence V _L. The fifth antibodies also include recombinant antibodies from any polynucleotide sequence encoding a V _H or V _L related to the amino acid sequence of encoding the herein (engineered) derived.

"Substantially the same" words can be related to the amino acid sequence B means a variant thereof (e.g., in the FRs, CDRs, V _H or V _L) is not substantially different as compared to the reference antibody, such that the variant has substantially similar For the binding activity (eg, affinity, specificity, or both) and biological activity of the reference antibody. Such variants may include minor amino acid changes. It is understood that the polypeptide can have a limited number of changes or modifications that can be made within a certain portion of the polypeptide that is not related to its activity or function, and still produce an equivalent or similar organism with an acceptable level Active or functional variants. In some examples, the change of the amino acid residue is a conservative amino acid substitution, which refers to the amino acid residue of a chemical structure similar to another amino acid residue and the function, activity, or other of the peptide Biological effects have little or no effect on the nature. Generally, relatively more substitutions can be made in the FR region compared to the CDR region, as long as they do not adversely affect the binding function and biological activity of the antibody (eg, reduce the binding affinity by more than 50% compared to the original antibody ). In some embodiments, the sequence identity between the reference antibody and the variant may be about 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95 %, 96%, 98% or 99%, or higher. Variants can be prepared according to methods known to those of ordinary skill in the art to change the polypeptide sequence, such as those found in references following these methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al. Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, conservative substitutions of amino acids include between amino acids in the following groups: (i) A, G; (ii) S, T; (iii) Q, N; (iv) E, D; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.

The antibodies described herein may be animal antibodies (eg, mouse-derived antibodies), chimeric antibodies (eg, mouse-human chimeric antibodies), humanized antibodies, or human antibodies. The antibodies described herein may be monoclonal antibodies or multiple antibodies. "Single antibody" refers to the group of homologous antibodies, and "multiple antibody" refers to the group of heterologous antibodies. The antibodies described herein may also include antigen-binding fragments thereof, for example, Fab fragments, F(ab')2 fragments, Fv fragments, single chain Fv (scFv) and (scFv) ₂ . Antibodies or antigen-binding fragments thereof can be prepared by methods known in the art.

III. Preparation of antibodies

Many methods conventional in the art can be used to obtain antibodies or antigen-binding fragments thereof.

In certain embodiments, the antibodies provided herein can be prepared by conventional fusion tumor technology. Typically, the target antigen, for example, dengue virus NS1 protein, is optionally coupled to a carrier protein, for example, keyhole limpet hemocyanin (KLH) and/or mixed with an adjuvant, such as complete Freund's adjuvant , Can be used to immunize host animals to produce antibodies that bind to this antigen. Lymphocytes secreting monoclonal antibodies are harvested and fused with myeloma cells to produce fusion tumors. The fusion tumor cell lines formed in this way are then screened to identify and select those cell lines that secrete the desired monoclonal antibodies.

In certain embodiments, the antibodies provided herein can be prepared by recombinant technology. In related aspects, isolated nucleic acids encoding the disclosed amino acid sequences, as well as vectors containing such nucleic acids and host cells transformed or transfected with the nucleic acids are also provided.

For example, nucleic acids containing nucleotide sequences encoding the heavy and light chain variable regions of such antibodies can be cloned into expression vectors (eg, bacterial vectors such as E. coli vectors, yeast vectors, viral vectors, or mammals) Vector), by conventional techniques, any vector can be introduced into a suitable cell (eg, bacterial cell, yeast cell, plant cell, or mammalian cell) to express the antibody. Examples of nucleotide sequences encoding the heavy and light chain variable regions of antibodies as described herein are shown in Table 3 below. Examples of mammalian host cell lines are human embryonic kidney cell lines (293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (VERO cells), and human hepatocytes (Hep G2 cells). Recombinant vectors used to express the antibodies described herein generally contain a nucleic acid encoding the amino acid sequence of the antibody, and the nucleic acid is operably linked to a promoter, whether constitutive or inducible. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of nucleic acids encoding antibodies. The vector optionally contains prokaryotic and eukaryotic system selection markers. In some examples, both heavy chain and light chain coding sequences are included in the same expression vector. In other examples, each heavy and light chain of the antibody is cloned into a single vector and produced separately, and then the antibody assembly can be cultured under appropriate conditions.

Recombinant vectors used to express the antibodies described herein generally contain a nucleic acid encoding the amino acid sequence of the antibody, and the nucleic acid is operably linked to a promoter, whether constitutive or inducible. Recombinant antibodies can be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, insects, and mammalian cells. A typical vector contains transcription and translation terminators, an initiation sequence, and a promoter that can be used to regulate the expression of the nucleic acid encoding the antibody. The vector optionally contains prokaryotic and eukaryotic system selection markers. The antibody protein produced can be further isolated or purified to obtain a substantially homogeneous preparation for further determination and application. Suitable purification methods, for example, may include fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), High-performance liquid chromatography (high-performance liquid chromatography, HPLC), ammonium sulfate precipitation, and gel filtration.

When the required length antibody, any coding sequences V _H and V _L chains of the coding sequences described herein may be coupled to an immunoglobulin Fc region, and the resulting full-length antibody genes encoding the heavy and light chains may be suitable Host cells, such as plant cells, mammalian cells, yeast cells, or insect cells.

Antigen-binding fragments can be prepared by conventional methods. For example, F(ab') ₂ fragments can be produced by pepsin digestion of full-length antibody molecules, and Fab fragments can be prepared by reducing the disulfide bonds of F(ab') ₂ fragments. Alternatively, such fragments can be prepared by recombinant techniques by expressing heavy and light chain fragments in a suitable host cell and assembling them in vivo or in vitro to form the desired antigen-binding fragments. By linking the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region, single-chain antibodies can be prepared by recombinant technology. Preferably, a flexible linker is introduced between the two variable regions.

IV. Antibody application

The serotype-specific antibodies of the present invention are specific to each dengue virus serotype, and each serotype does not react with any other closely related flaviviruses, such as Japanese encephalitis (JE) virus and Zika virus (ZIKV) ). Therefore, the present invention provides a method using any disclosed antibody or any combination thereof, which can be effectively used to detect dengue virus in a sample.

Generally, the method of the invention involves contacting the sample with any exposed antibody or any combination thereof, and measuring the binding of the antibody to the sample. Specifically, the binding of the antibody to the sample includes (i) the binding of the first antibody to DENV1 NS1 and forming the first antibody-DENV1 NS1 complex, and (ii) the binding of the second antibody to DENV2 NS1 and forming the second antibody- DENV2 NS1 complex, (iii) the third antibody binds to DENV3 NS1 and forms a third antibody-DENV3 NS1 complex, and (iv) the fourth antibody binds to DENV4 NS1 and forms a fourth antibody-DENV4 NS1 complex. The method of the present invention also includes determining whether a specific serotype of dengue virus is present based on the binding of the antibody to the sample, wherein (i) the binding of the first antibody to DENV1 NS1 and forming the first antibody-DENV1 NS1 complex indicates the presence of DENV1 in the sample , (Ii) the second antibody binds to DENV2 NS1 and forms a second antibody-DENV2 NS1 complex indicating that DENV2 is present in the sample, (iii) the third antibody binds to DENV3 NS1 and forms a third antibody-DENV3 NS1 complex Indicates that DENV3 is present in the sample, and (iv) the fourth antibody binds to DENV4 NS1 and forms a fourth antibody-DENV4 NS1 complex indicates that DENV4 is present in the sample.

Those of ordinary skill in the art are aware of various assay formats that use antibodies to detect antigens or pathogens in a sample. These assays using antibodies specific to the target antigen/pathogen are commonly referred to as immunoassays. Examples of immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), luminescence immunoassay (LIA), or ILMA immunofluorescence assay. These assays can be used to detect the presence of dengue virus in biological samples, including blood, serum, plasma, saliva, cerebrospinal fluid, urine, and other tissue samples.

In certain specific embodiments, the assay is a sandwich assay.

In some embodiments, the measurement is performed by first fixing the capture antibody on a solid support. The immobilized antibody is then incubated with the biological sample, and the dengue virus or its target antigen, for example, binds NS1 polypeptide (if present in the sample) to the antibody to form an antibody-virus/antigen complex or conjugate Compound. The unbound sample can then be removed by washing the solid support with a suitable buffer, such as PBS containing 0.05% Tween, and adding a detection antibody to the solid support, the detection antibody can bind the immobilized The antibody-virus/antigen complex also contains a detectable label. Specifically, the capture antibody and the detection antibody need to bind the virus/antigen at different epitopes, so that the virus/antigen can be "sandwiched" between these two antibodies. Preferred detectable labels include enzyme labels (eg horseradish peroxidase), fluorescent labels, metal labels, and radioactive labels. Some specific examples of detectable marks include gold nanoparticles, colored latex beads, magnetic particles, carbon nanoparticles, and selenium nanoparticles. The detection antibody is incubated with the immobilized antibody-virus/antigen complex for a period of time sufficient to detect bound virus/antigen. The unbound detection antibody is then removed, and the bound detection antibody is detected based on the detectable label. For example, enzyme labeling can usually be detected by adding a matrix and then performing spectroscopic or other analysis on the reaction product.

To determine the presence or absence of the target dengue virus in the sample, the signal detected from the detectable label bound to the solid support is usually compared with the signal corresponding to the cutoff value. The cut-off value is usually the average signal obtained when the immobilized antibody is incubated with a sample from an uninfected individual. Generally, a sample that produces a signal higher than the cutoff value is considered a positive reaction to the presence of dengue virus in the sample.

The serotype specific antibody of the present invention can be used as a capture or detection antibody, paired with a partner antibody to form an antibody pair and can be sandwiched for determination. The partner antibody as described herein also needs to be capable of binding the target virus/antigen, but binds an epitope that is different from any serotype-specific antibodies of the invention that are used for pairing in sandwich assays. In some embodiments, the partner antibody used for the sandwich assay may be an antibody with a different "serotype specificity" to the dengue virus described herein (ie, different from the first antibody, The second antibody, the third antibody and the fourth antibody), the partner antibody can also bind to the corresponding serotype of dengue virus but bind to different epitopes. In some embodiments, the partner antibody may be a "serotype cross-reactive" antibody, which is a multi-strain or single-strain antibody that can recognize all dengue virus serotypes but is different from any serotype of the present invention Epitopes of specific antibodies. A specific example of such a cross-reactive antibody is a fifth antibody (eg, monoclonal antibody 82-1.1), which cross-reacts with at least four dengue virus serotypes and recognizes different from any serotype-specific antibodies of the present invention Separate the epitope to form a pair with it for sandwich measurement.

In certain embodiments, the method of the invention for detecting dengue virus in a sample comprises (i) contacting the sample with a cross-reactive antibody specific for dengue NS1 polypeptide (for the four serotypes) (For example, a fifth antibody specific to DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1), as a capture antibody, to form a cross-reactive antibody-NS1 (any serotype) complex; (ii) make the cross-reactive antibody-NS1 more The peptide (any serotype) complex is contacted with a serotype-specific monoclonal antibody as described herein (eg, a first antibody specific for DENV1 NS1, a second antibody specific for DENV2 NS1, a specific antibody specific for DENV3 NS1 Triple antibody, or fourth antibody specific for DENV4 NS1), as a detection antibody to form a cross-reactive antibody-NS1 (any serotype)-serotype-specific monoclonal antibody complex; and (iii) detection of cross-reactive antibody -NS1 (any serotype)-The presence of a serotype-specific monoclonal antibody complex to detect the presence of dengue virus of each serotype in the sample.

In certain embodiments, the method of the invention for detecting dengue virus in a sample comprises (i) contacting the sample with a serotype specific monoclonal antibody described herein (eg, specific for DENV1 NS1 The first antibody, the second antibody specific for DENV2 NS1, the third antibody specific for DENV3 NS1, or the fourth antibody specific for DENV4 NS1) as capture antibodies to form a monoclonal antibody-serotype-specific NS1 complex (Ii) contacting the monoclonal antibody-respective serotype dengue virus complex with a cross-reactive antibody specific for dengue NS1 polypeptide (for the four serotypes) (for example, for DENV1 NS1, DENV2 NS1 DENV3 NS1 and DENV4 NS1 specific fifth antibody) as detection antibodies to form monoclonal antibody-serotype specific NS1-cross-reactive antibody complex; and (iii) detection of the serotype specific monoclonal antibody-NS1 -The presence of cross-reactive antibody complexes, thereby detecting the presence of dengue virus of the respective serotype in the sample.

In one example, the method of the present invention is performed in an ELISA sandwich assay. In this assay, the capture antibody is coated on an ELISA plate. After blocking, the ELISA plate is incubated with the biological sample, washed, and then incubated with the detection antibody. For example, the capture antibody is a multiple or single cross-reactive antibody specific for dengue NS1 polypeptide (any serotype), and the detection antibody is a first antibody specific for DENV1 NS1, a second antibody specific for DENV2 NS1, A third antibody specific for DENV3 NS1, or a fourth antibody specific for DENV4 NS1, or any combination thereof, as described herein, and using an ELISA colorimetric TMB reagent for color development of the ELISA plate.

In another example, the method of the present invention is carried out in flow-through or side-stream form. In this assay, a detection antibody with a detectable label, such as a colorimetric label (eg, colloidal gold), is fixed on a membrane, such as a nitrocellulose membrane (as a band). A biological sample suspected of containing the dengue virus is applied to the membrane where the detection antibody is present. The biological sample migrates along the membrane through the area containing the detection antibody, wherein if the dengue virus is present in the biological sample, the detection antibody will bind to the NS1 of the dengue virus. Then, the complex of the detection antibody and the antigen to which it binds migrates to the test area where the capture antibody is immobilized, and the capture antibody binds to NS1 of the dengue virus, thereby forming a sandwich of detection antibody, antigen, and capture antibody. The concentration/aggregation of the detection antibody at the test (capture) area indicates the presence of specific dengue NS1 in the sample. Such tests can usually be performed using very small amounts of biological samples.

In a related aspect, the invention also provides a kit for carrying out the method of the invention, which comprises any antibody or combination thereof as described herein. The kit may further include instructions for using the kit to detect dengue virus in a sample.

In certain embodiments, the immunoassay is in the form of a sandwich.

Specifically, the kit includes a pair of dengue virus serotype-specific antibodies selected from the group consisting of a first antibody specific for DENV1 NS1, a second antibody specific for DENV2 NS1, and a third antibody specific for DENV3 NS1, and A fourth antibody specific to DENV4 NS1, and any combination of these groups, each antibody is paired with a partner antibody for sandwich determination.

In some embodiments, at least one serotype-specific antibody serves as a capture antibody and is paired with a partner antibody that serves as a detection antibody.

In other specific embodiments, at least one serotype-specific antibody serves as a detection antibody and is paired with a partner antibody that serves as a capture antibody.

As a detection antibody, the antibody may contain detectable labels, such as enzyme labels, fluorescent labels, metal labels, and radioactive labels.

In some examples, in a lateral flow format, the kit contains a measurement band (eg, nitrocellulose membrane); the detection antibody can bind to the reaction zone of the band, and the capture antibody can bind to the Strip test area. The strip also contains a sample pad in which a sample of body fluid is placed, and then the sample migrates to the opposite end of the strip by capillary action, whereby the sample first binds to the detection antibody in the reaction zone, where the The antigen binds to the detection antibody to form an antigen-detection antibody complex, and then the antigen-detection antibody complex binds to the capture antibody in the test (capture) area. Colloidal gold is used as a detectable label for the detection antibody. For example, the concentration/aggregation of the detection label in the test (capture) area is displayed in red, indicating that a specific antigen is present in the sample. Alternatively, the test zone may have a chromophoric matrix, and when the antigen-detection antibody complex binds to the capture antibody, the chromophoric matrix is converted into a visible colored product.

In some examples, in the ELISA format, the kit includes a microtiter plate with a well to which the capture antibody is fixed; a solution containing the detection antibody; and a chromogenic developer.

In particular, the kit may further include additional reagents or buffers, medical devices for collecting biological samples from individuals, and/or containers for holding and/or storing samples.

In a further specific embodiment, the present invention provides a composition comprising one or more antibodies as described herein and a pharmaceutically acceptable carrier.

In certain embodiments, the composition of the present invention is a pharmaceutical composition for treating dengue virus diseases.

In certain embodiments, the composition of the present invention is a diagnostic composition for diagnosing dengue virus diseases.

As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize the active ingredient and be safe for the individual receiving treatment. The carrier can be a diluent, carrier, excipient or base for the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, sterile water, syrup and methyl cellulose. The composition may additionally contain lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoate; sweeteners; and flavoring agents . The composition of the present invention can provide a rapid, sustained or delayed release effect of an active ingredient after administration to a patient.

According to the invention, the composition can be in the form of tablets, pills, powders, lozenges, sachets, tablets, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injections And packaging powder.

The compositions of the present invention can be delivered by any physiologically acceptable route, such as oral, parenteral (eg, intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. For parenteral administration, it is preferably used in the form of a sterile aqueous solution, which may contain other substances sufficient to make the solution isotonic with blood, such as salt or glucose. If necessary, the aqueous solution can be buffered appropriately (preferably pH value is 3 to 9). The preparation of suitable parenteral compositions under sterile conditions can be accomplished using standard pharmacological techniques well known to those skilled in the art, and requires no additional creative work.

The present invention is further illustrated by the following examples, which are provided for illustration rather than limitation. Based on the disclosure of the present invention, those skilled in the art should understand that, without departing from the spirit and scope of the present invention, many changes can be made to the specific embodiments disclosed and still obtain the same or similar results.

Examples

1.1 Virus strain

Dengue virus strains DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241 were propagated in C6/36 Alternaria mosquitoes in RPMI 1640 medium supplemented with 2% FBS, and cultured at 28°C until cytopathic effects were observed . As a control group, Japanese encephalitis virus strain JEV SA14-14-2 was propagated in BHK-21 cells in RPMI 1640 medium containing 2% FBS. The number of each virus was determined by plaque assay inoculated with BHK-21 cells.

1.2 NS1 Preparation of protein

The cell culture supernatant of Vero cells infected with viruses (DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241) was harvested after 5 days and was not activated by UV irradiation. In order to purify the soluble form of NS1 protein in the cell culture supernatant, we performed column immunoaffinity chromatography according to the manufacturer's instructions. To this end, anti-NS1 monoclonal antibodies (for all serotypes) that have been immobilized on HiTrap ^™ NHS-activated HP columns (GE Healthcare, Uppsala, Sweden) were used to purify NS1 protein from each dengue serotype. Equilibrate the column with 5 volumes of PBS, pass the viral cell culture supernatant through the immunoaffinity column, wash out unbound protein with 10 volumes of PBS, and elute the binding with a glycine solution at pH 2.8 The NS1 is then buffered to neutral pH. The purified DENV NS1 protein was then detected by Western blot analysis, and the NS1 protein concentration was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific, Illinois, USA).

1.3 Focus on DENV1-4 NS1 Serotype-specific monoclonal antibody production and characterization

All experiments used BALB/c mice purchased from the National Laboratory Animal Center and raised in the animal feeding facility of the Institute of Preventive Medicine. Experiments using animals are approved by the International Association of Laboratory Animal Management Evaluation and Certification (IACUC number AN-104-12, AN-105-05). Four-week-old BALB/c mice were immunized intraperitoneally with 15 μg of immunoaffinity-purified NS1 protein in complete Freund's adjuvant for the first inoculation, and then inoculated with incomplete Freund's adjuvant. 15 μg NS1 protein was used to immunize mice against their respective serotypes for subsequent booster immunization. DENV-specific antiserum was obtained from mice after continuous challenge. Briefly, the spleens of immunized mice were removed. Spleen cells were fused with NSI/1-Ab4-1 myeloma cells to produce fused tumor cells, which were selected according to standard procedures (Kohler and Milstein, 1975). The fused cells were washed twice with RPMI, then mixed in a 15 ml conical tube, and 1 ml of 50% (w/v) PEG 1500 (Roche, Penzberg, Germany) was added within 1 minute with gentle stirring. The mixture was diluted twice by slowly adding (1 minute) 1 ml RPMI, then slowly adding (2 minutes) 8 ml serum-free RPMI. The mixture was then centrifuged at 400 xg for 5 minutes. The fused cell pellet was resuspended in RP medium supplemented with 20% FBS in HAT medium (Life technologies, Burlington, Ontario, Canada) and HFCS solution (Roche, Mannhein, Germany). Next, dispense the resuspended mixture in 96-well plates in an amount of 200 μl per well. Fusion tumor cell lines secreting specific antibodies against NS1 were identified by indirect ELISA (using purified DENV NS1 as the coating antigen for each serotype). Individual cells are produced by limiting dilution. Western blot analysis of lysates from C6/36 cells infected with DENV was performed to determine (a) the specificity of anti-NS1 monoclonal antibody, and (b) whether the epitope recognized by the antibody was conformational or linear. Monoclonal antibodies were isotyped using commercially available mouse monoclonal antibody isotype kits (IsoStrip ^™ , Roche, Mannheim, Germany). Fused tumor cells were injected into pristane-induced BALB/c mice to produce ascites. Monoclonal antibodies were then purified from ascites fluid using protein G-sepharose columns (HiTrap protein G, GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions.

1.4 HRP Combine

In order to conjugate monoclonal antibodies to HRP (Innova Biosciences, Cambridge, UK), 100 μg of HRP and 20 μl aliquots of modification reagent were mixed with 200 μl of 1 mg/ml monoclonal antibody. After incubating the mixture at room temperature (20-25°C) for 3 hours, the reaction was terminated with an aliquot of 20 μl of quencher. After another 30 minutes at room temperature, 260 μl of glycerin was added, and the final solution was stored at -20°C. The final concentration of monoclonal antibody-HRP is 400 μg/ml.

1.5 Development of four serotype specificities NS1 capture ELISA

Several serotype specific and highly reactive monoclonal antibodies were selected based on the characteristics of anti-NS1 monoclonal antibodies. The serotype-specific antibodies of the four serotype-specific monoclonal antibodies were tested by their compatibility with serotype-cross-reactive monoclonal antibodies. In order to determine the best combination of capture ELISA with regard to assay sensitivity, serotype-specific monoclonal antibodies and serotype-cross-reactive monoclonal antibodies were used as capture or detection antibodies. Specifically, select the serotype-cross-reactive monoclonal antibody as the capture antibody, and determine the specific serotype specific monoclonal antibodies corresponding to the detection antibodies (monoclonal antibody 12-4.1, monoclonal antibody 33-7.1) , The best pair of monoclonal antibodies 43-1.3 and monoclonal antibodies 22-1.5), assembling the ELISA of four dengue serotypes NS1, as shown in Figure 1A, Figure 1B, Figure 2A and Figure 2B. The most suitable experimental conditions are determined by checkerboard titration, such as coating concentration and dilution of monoclonal antibody-HRP. Microplates (Costar Corning, Corning, New York) were coated with 100 μl of 10 μg/ml capture cross-reactive monoclonal antibody and incubated overnight at 4°C. The wells were then blocked with blocking buffer (PBS, 0.05% Tween, 5% skimmed milk powder) at 37°C for 1 hour and washed with washing buffer (PBS, 0.05% Tween). Then, the virus culture supernatant or NS1 protein was serially diluted in blocking buffer and incubated at 37°C for 1 hour. After washing the plate, 100 μl of 0.8 μg/ml serotype specific monoclonal antibody-HRP was added, and the plate was incubated at 37°C for 1 hour. Then, the well plate was washed again, 100 μl of TMB reagent was added, and the well plate was incubated at room temperature for 10 minutes. Then the reaction was stopped with 1N sulfuric acid, and the absorbance was read at 450 nm using a microplate reader.

1.6 Clinical samples

A total of 146 clinical serum samples were used in this study; 85 samples included confirmed dengue fever cases reported to the Centers for Disease Control of the Taipei City Health Department, and 61 samples were collected from three hospitals during 2016-2017 (National Army Kaohsiung General Hospital , Zuoying Branch of the National Kaohsiung General Hospital, and Okayama Branch of the National Kaohsiung General Hospital). Collect all clinical serum samples in the acute phase (1-7 days after disease onset), and use serotype-specific one-step SYBR green I real-time RT-PCR, dengue virus-specific IgM/IgG capture ELISA and commercially available Platelia Dengue NS1 The Ag ELISA kit (Bio-Rad, Marnes-la-Coquette, France) was tested. The research method was approved by the Institutional Review Board of the National Military Kaohsiung General Hospital (IRB No. KAFGH 104-048).

1.7 Use serotype specificity NS1 capture ELISA Testing NS1 Clinical serum

The microplate was coated with 100 μl of 10 μg/ml cross-reactive monoclonal antibody and incubated overnight at 4°C. Subsequently, the wells were blocked with blocking buffer (PBS, 0.05% Tween, 5% skimmed milk powder), and incubated with 50 μl of serum diluted 1:1 in blocking buffer at 37°C for 1 hour. Then, the well plate was washed four times with 0.05% PBS/T at 37°C with 100 μl of 0.8 μg/ml serotype specific monoclonal antibody-HRP (monoclonal antibody 12-4.1, monoclonal antibody 33-1.7 , Monoclonal antibody 43-1.3 and monoclonal antibody 22-1.5) were incubated together for 1 hour, and washed four times again. The subsequent steps involved in clinical serum testing are described in the previous section. Normal human serum samples (Sigma-Aldrich, Saint Louis, USA) served as negative control groups. For each serotype, samples that exceeded the reference cut-off value (calculated as the double average of the negative control) were considered positive in the NS1 capture ELISA.

1.8 Through commercially available Platelia Dengue NS1 Ag ELISA Set detection NS1 antigen (Bio-Rad the company, Marnes-la-Coquette ,France )

According to the manufacturer's instructions, a commercially available Platelia Dengue NS1 Ag ELISA kit (Bio-Rad, Marnes-la-Coquette, France) was used to detect DENV NS1 protein in clinical serum samples. Briefly, the positive, negative, calibration and sample dilutions were incubated on well plates with HRP-conjugated monoclonal antibodies at 37°C for 90 minutes. After washing the well plate six times, 160 μl TMB was added to each well, and the well plate was incubated at room temperature away from light for 30 minutes. The enzyme reaction was stopped by adding 100 μl of stop solution, and the optical density was measured at the wavelength of OD 450/620 nm.

1.9 Reproducibility

On the other day, a total of 58 DENV positive serum samples (16 DENV1 serum samples, 17 DENV2 serum samples, 16 DENV3 serum samples, and 9 DENV4 serum samples) were tested by a second operator in a four-serotype NS1 capture ELISA And 50 negative serum samples.

1.10 Statistical Analysis

The diagnostic accuracy, sensitivity, specificity, and corresponding 95% confidence interval (CI95) of each ELISA were evaluated using GraphPad Prism version 6.0 (GraphPad Software Corporation, San Diego, California), and the significance standard was set to P value<0.05.

1.11 Colloidal gold probe Preparation

35±5 nm colloidal gold (TANBead NanoGold-40, Taiwan Advanced Nanotech) was used for IgG conjugation. The pH of the colloidal gold solution (1% w/v) was adjusted with 0.2N NaOH, and the anti-dengue NS1 monoclonal antibody 82-1.1 (in PBS, pH7.4) was added to the pH-adjusted colloidal gold solution. The most suitable pH value for conjugation of each serotype: D1 band, pH 7.8; D2, D3, and D4 bands, pH 7.4. The optimized antibody concentration for conjugation is 1 μg/band. The antibody/colloidal gold mixture was gently mixed for 90 minutes, blocked with 2% BSA solution for 30 minutes, and centrifuged at 7000 rpm for 15 minutes. After centrifugation and washing once with 2% BSA (20 mM Tris/HCl buffer [pH7.2] containing 2% [w/v] BSA), the gold particles were suspended in 2% BSA. The anti-dengue NS1 monoclonal antibody 82-1.1 coated colloidal gold probe was placed in a pad to dry, and then stored at 4°C overnight.

1.12 Lateral flow test strip Preparation

Control lines were prepared as follows: 0.2 (for D1, D3, and D4) to 0.32 (for D2) μg/band goat anti-mouse IgG (Jackson ImmunoResearch, Pennsylvania, USA). The test line (capture antibody) was prepared as follows: monoclonal antibody 12-4.1, monoclonal antibody 43-1.3, monoclonal antibody 22-1.5 with an action strength of 0.75 μg/band (for D1, D3, D4 bands) in PBS ( pH 7.4) and monoclonal antibody 33-7.1 with an action strength of 0.8 μg/band (for D2 band) in PBS (pH 7.4), respectively applied to cellulose acetate loaded nitrocellulose membrane bands (pore size : 12 μm diameter) near the top, and dried at room temperature for 1 hour. After treatment of the components, the dengue fever serotype NS1 lateral flow test device was assembled.

1.13 Strip on NS1 Antigen detection and detection sensitivity

The determination was made by applying the appropriate test DENV NS1 protein solution of the sample (50 μl) and 50 μl running buffer (0.1% Tween-20/0.85% NaCl) to the device's sample pad. Testing the combined solution of DENV NS1 and the detection reagent raised the membrane, and after 20 minutes at room temperature, the colloidal gold was deposited at the site of the solid phase antibody.

1.14 DENV Serotype NS1 Cross-reactivity

Cell culture supernatants of Vero cells infected with viruses (DENV1 Hawaii, DENV2 16681, DENV3 H87, DENV4 H241, Japanese encephalitis virus, and Zika virus) were measured with DENV serotype NS1 bands to assess cross-reactivity.

1.15 Sequence analysis of variable regions of monoclonal antibodies

Fusion tumor cells from fusion tumors 12-4.1, 33-7.1, 43-1.3, 22-1.5 and 82-1.1. Total RNA was isolated from fusion tumor cell lines using the RNeasy mini kit (Qiagen Corporation, Valencia, California, USA) according to the manufacturer's method, and then reverse transcription was performed using oligo dT primers to generate cDNA. PCR was used to amplify the heavy chain and light chain variable chains of the antibody, and confirmed that the sequence was a functional variable region. For the V _H and V _L genes of the four fusion tumor cells, see the sequence in the attachment.

2. result

2.1 against DENV And characterization of monoclonal antibodies

NS1 proteins from various serotypes of DENV were prepared and injected into BALB/c mice for immunization. The spleen was removed from the immunized mice, and the spleen cells were isolated and fused with myeloma cells to produce fusion tumor cells. Fused tumor cell lines secreting specific antibodies against NS1 were identified by ELISA using purified DENV NS1 as the coating antigen for each serotype. In addition, Western blot analysis of lysates from C6/36 cells infected with DENV was performed to determine (a) specificity of anti-NS1 monoclonal antibody and (b) whether the epitope recognized by the antibody was conformational or linear. In addition, commercial kits were used to isotype monoclonal antibodies.

^a 以不同登革病毒血清型感染的C6/36細胞裂解物以SDS-PAGE樣本緩衝液處理，並以每種單株抗體進行墨點分析。^b 從以不同血清型DENV感染的Vero細胞的細胞培養物上清液中免疫親和純化不同的NS1抗原。以專一性NS1抗原包覆微孔板並與每種單株抗體反應。Table 2. Characterization of monoclonal antibodies reacting with NS1 protein of DENV serotype (D1-4)

^a C6/36 cell lysates infected with different dengue virus serotypes were treated with SDS-PAGE sample buffer and subjected to dot blot analysis with each monoclonal antibody. ^b Immunoaffinity purification of different NS1 antigens from the cell culture supernatant of Vero cells infected with different serotypes DENV. The microplate was coated with specific NS1 antigen and reacted with each monoclonal antibody.

As shown in Table 2, the first to fourth clones 12-4.1, 33-7.1, 43-1.3 and 22-1.5 are DENV1, DENV2, DENV3 and DENV4 specific serotypes, respectively, while the fifth clone, Monoclonal antibody 82-1.1 cross-reacts with DENV1, DENV2, DENV3 and DENV4.

Amino acid sequences of four serotype-specific monoclonal antibodies (12-4.1, 33-7.1, 43-1.3 and 22-1.5) and cross-reactive monoclonal antibodies (82-1.1), including the complementarity determining region 1- 3 and the framework regions 1-4 (FW1-4) and corresponding nucleic acid sequences (1-3 Determination of CDR V _H and V _L domains), and is provided in table 3 below.

Table 3. Amino acid sequences and corresponding nucleic acids of four serotype-specific monoclonal antibodies (12-4.1, 33-7.1, 43-1.3, and 22-1.5) and cross-reactive monoclonal antibodies (82-1.1) sequence.

2.2 Sandwich ( capture ) ELISA

2.2.1 Use serotype-specific monoclonal antibodies as capture antibodies

The four serotype-specific monoclonal antibodies of the present invention are used as capture antibodies in a sandwich (capture) ELISA. Figure 1A shows the basic design of a sandwich (capture) ELISA using serotype-specific monoclonal antibodies of the invention as capture antibodies, which are paired with cross-reactive monoclonal antibodies as detection antibodies.

The microplate was coated with the serotype-specific monoclonal antibody of the present invention as a capture antibody, and cultured at 4°C overnight. The wells are then blocked and washed. Cell culture supernatants of Vero cells infected with different serotypes of DENV (DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241) or epidemic Japanese encephalitis (JE) virus or cell cultures of uninfected Vero cells The supernatant (negative control group) was added separately to the wells and incubated at 37°C for 1 hour. After washing, cross-reactive monoclonal antibodies labeled with horseradish peroxidase (HRP) were added to the wells and incubated at 37°C for 1 hour. The wells were washed again, and 3,3',5,5'-tetramethylbenzidine (TMB) was added to the wells as a matrix for HRP. Read the absorbance at 450 nm. As shown in FIG. 1B, it was proved that the serotype-specific monoclonal antibody of the present invention was successfully used as a capture antibody in a sandwich (capture) ELISA, exhibiting excellent serotype-specificity without being associated with other flaviviruses (Japanese encephalitis (JE ) Virus) Cross reaction.

2.2.2 Use serotype-specific monoclonal antibodies as detection antibodies

The four serotype-specific monoclonal antibodies of the present invention are used as detection antibodies in sandwich (capture) ELISA. 2A shows the basic design of a sandwich (capture) ELISA using the serotype-specific monoclonal antibodies of the present invention as detection antibodies, which is paired with cross-reactive monoclonal antibodies as capture antibodies.

The microplates were coated with cross-reactive monoclonal antibodies as capture antibodies and incubated overnight at 4°C. The wells are then blocked and washed. Cell culture supernatants of Vero cells infected with different serotypes DENV (DENV1 Hawaii, DENV2 16681, DENV3 H87, and DENV4 H241) or epidemic Japanese encephalitis (JE) virus or cell cultures of uninfected Vero cells The clear solution (negative control group) was added separately to the wells and incubated at 37°C for 1 hour. After washing, the serotype-specific monoclonal antibodies of the present invention labeled with horseradish peroxidase (HRP) were added to the wells and incubated at 37°C for 1 hour. The wells were washed again, and 3,3',5,5'-tetramethylbenzidine (TMB) was added to the wells as a matrix for HRP. Read the absorbance at 450 nm. As shown in FIG. 2B, the serotype-specific monoclonal antibody of the present invention was successfully used as a detection antibody in a sandwich (capture) ELISA, exhibiting excellent serotype-specificity without being associated with other flaviviruses (Japanese encephalitis (JE ) Virus) Cross reaction.

2.2.3 Detection limit

Serially diluted and purified NS1 protein of each DENV serotype was used for capture ELISA to determine the detection limit of the serotype specific monoclonal antibody of the present invention. Table 4 and Figure 3 show the results.

Table 4. Detection limits of the four serotype-specific NS1 capture ELISAs of the present invention and the commercially available Platelia Dengue NS1 AG ELISA

The results show that the capture ELISA using the serotype-specific monoclonal antibody of the present invention achieves excellent sensitivity, ranging from 1 to 4 ng/ml of DENV1-4 NS1 protein, and the commercially available DENV ELISA kit (Pleterlia NS1 Ag ELISA, Bio-Rad) is comparable, although the commercially available kit cannot distinguish serotypes.

2.3 Bands

The four serotype-specific monoclonal antibodies of the present invention are also used to develop bands for DENV detection. Figure 4 shows the basic design of serotyping NS1 bands using the serotype-specific monoclonal antibodies of the invention as capture antibodies (top panel) or detection antibodies (detection group) paired with cross-reactive monoclonal antibodies.

As shown in FIG. 5, the serotype-specific monoclonal antibodies of the present invention (12-4.1, 33-7.1, 43-1.3, and 22-1.5) proved to be successful as capture antibodies, cross-reactive monoclonal antibodies of the present invention (82.1.1) Pairing as a detection antibody, in the serotyping NS1 band, showed excellent serotype specificity for DENV1, DENV2, DENV3, and DENV4 (from top to bottom), but not with other flaviviruses (Japanese encephalitis (JE) virus and Zika virus (ZIKV)) cross reaction.

In addition, the detection limit of the serotype-specific monoclonal antibody of the present invention in the band was determined. Table 5 shows the results.

Table 5 The detection limits of four serotype-specific NS1 bands, the rapid detection of Dengue NS1 AG by Bio-Rad, and the rapid detection of SD dengue NS1 Ag by SD

The results show that the NS1 band using the serotype-specific monoclonal antibody of the present invention achieves excellent sensitivity, ranging from 62.5 to 125 ng/ml of DENV1-4 NS1 protein, which is comparable to the commercially available DENV band set, and These commercially available kits cannot distinguish serotypes.

2.4 Clinical Trials

A total of 146 clinical serum samples were collected to determine the sensitivity and specificity of the sandwich (capture) ELISA using the four serotype specific monoclonal antibodies of the present invention.

Collect 146 clinical serum samples between one (1) and seven (7) days after the onset of the disease, and through routine serotyping RT-PCR, dengue specific IgM/IgG capture ELISA, commercially available dengue NS1 Ag ELISA method and The serotype-specific NS1 capture ELISA of the present invention was analyzed. Table 6 shows the results. Figure 6 shows the specificity of the sandwich (capture) ELISA of the present invention for testing the serum of patients with acute fever.

Table 6. Analysis of acute clinical sera by RT-PCR, IgM/IgG, Platelia Dengue NS1 AG ELISA and the four serotype-specific NS1 capture ELISAs of the present invention

The results showed that 146 clinical serum samples were confirmed to include seventeen (17) DENV1 serotype samples, nineteen (19) DENV2 serotype samples, sixteen (16) DENV3 serotype samples, and nine (9) DENV4 serotype samples; five (5) samples were negative by RT-PCR, but dengue virus-specific IgM/IgG capture ELISA was positive; fifty-five (55) samples were positive by Platelia NS1 Ag ELISA; eight Ten (80) samples were determined to be negative by all test methods.

The results show that the sandwich (capture) ELISA using the four serotype-specific monoclonal antibodies of the present invention shows excellent sensitivity and specificity, in which the D1 ELISA (monoclonal antibody 12-4.1) can detect 15 samples from 17 DENV1 samples. DENV1 samples, D2 ELISA (monoclonal antibody 33-7) can detect 18 DENV2 samples from 19 DENV2 samples, D3 ELISA (monoclonal antibody 43-1.3) can detect 12 DENV3 samples from 16 DENV3 samples, And D4 ELISA (monoclonal antibody 22-1.5) capable of detecting 6 DENV4 samples from 9 DENV1 samples; five (5) samples not detected by serotype RT-PCR were determined to be positive and identified as DENV2; and All eighty (80) negative samples were also determined to be negative without false positive results. 6A to 6F show the specificity of the sandwich (capture) ELISA of the present invention for testing serum of patients with acute fever.

Tables 8.1 and 8.2 summarize the results of the sensitivity and specificity of the sandwich (capture) ELISA using the four serotype specific monoclonal antibodies of the present invention.

Table 8.1

Table 8.2

The results show that the sandwich (capture) ELISA based on the four serotype-specific monoclonal antibodies of the present invention can detect and distinguish the four dengue virus serotypes in serum samples, even after five (5) days after the onset of the disease. Show overall 100% specificity and 84.8% sensitivity.

no

When read in conjunction with the accompanying drawings, the foregoing summary of the invention and the following detailed description of the invention will be better understood. For the purpose of illustrating the present invention, currently preferred specific embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown.

In the drawings:

Figure 1A shows the basics of a sandwich (capture) ELISA using the serotype-specific monoclonal antibodies (mAbs) of the present invention as capture antibodies and paired with a serotype cross-reactive antibody of dengue virus as a detection antibody design.

Fig. 1B shows the use of the serotype-specific monoclonal antibodies (mAbs) of the present invention, 12-4.1, 33-7.1, 43-1.3, and 22-1.5 as capture antibodies, and a dengue as a detection antibody The results of the sandwich (capture) ELISA of virus serotype cross-reactive antibody pairing. In this assay, the cell culture supernatant from Vero cells infected with DENV1, DENV2, DENV3, DENV4, or JEV was used as the source of the NS1 antigen, and the supernatant from uninfected Vero cells was used as the negative control group.

Figure 2A shows the basics of a sandwich (capture) ELISA using the serotype-specific monoclonal antibody (mAbs) of the present invention as a detection antibody and paired with a serotype cross-reactive antibody of dengue virus as a capture antibody design.

Figure 2B shows the use of the serotype specific monoclonal antibodies (mAbs) of the present invention, 12-4.1, 33-7.1, 43-1.3, and 22-1.5 as detection antibodies, and a dengue as a capture antibody The results of the sandwich (capture) ELISA of virus serotype cross-reactive antibody pairing. In this assay, the cell culture supernatant from Vero cells infected with DENV1, DENV2, DENV3, DENV4, or JEV was used as the source of the NS1 antigen, and the supernatant from uninfected Vero cells was used as the negative control group.

Figure 3 shows the use of serotype-specific monoclonal antibodies (mAbs) of the present invention, 12-4.1, 33-7.1, 43-1.3, and 22-1.5 as detection antibodies, and a dengue as a capture antibody Sensitivity in sandwich (capture) ELISA of virus serotype cross-reactive antibody pairing. Serially diluted and purified NS1 protein of each DENV serotype was used for capture ELISA to determine the detection limit of the serotype specific monoclonal antibody (mAbs) of the present invention. Bovine serum albumin (BSA) was used to establish a baseline. Each data point represents the mean ± standard deviation (SD) of three repeated tests.

Fig. 4 shows the use of the serotype-specific monoclonal antibodies (mAbs) of the present invention as capture antibodies (upper panel) or detection antibodies (lower panel), and cross-reactive antibodies with a dengue virus serotype (monoclonal) Antibody 82-1.1) Basic design of paired sandwich bands.

Figure 5 shows the characteristics of the sandwich strip of the present invention. Pair serotype-specific monoclonal antibodies (mAbs) 12-4.1, 33-7.1, 43-1.3, and 22-1.5 with dengue virus serotype cross-reactive antibodies (monoclonal antibody 82-1.1) as capture and Detect antibodies and then test for binding specificity. In this assay, the cell culture supernatant from Vero cells infected with DENV1, DENV2, DENV3, DENV4, or JEV was used as the source of NS1 antigen, and the supernatant from uninfected Vero cells was used as the negative control group.

6A to 6F include graphs showing the specificity of the sandwich (capture) ELISA of the present invention for testing serum of patients with acute fever. Figure 6A shows the results of ELISA tests on the serum of 17 patients infected with DENV1. Figure 6B shows the results of ELISA tests on the serum of 19 patients infected with DENV2. Figure 6C shows the results of ELISA tests on the serum of 16 patients infected with DENV3. Figure 6D shows the results of ELISA tests on the serum of 9 patients infected with DENV4. Figure 6E shows the results of ELISA tests on the serum of five patients infected with DENVs. Figure 6F shows the results of ELISA tests on the serum of 80 other fever patients. The T/N ratio is calculated by dividing the OD450 value of the patient's serum by the average of OD450 of all negative healthy normal serum samples (ie, the patient's serum OD450/average OD450 of negative healthy normal serum). If the OD450 value of the sample is at least twice the average value of the negative control group, the sample is considered positive.

Fig. 7 shows the CDR sequences of the heavy chain and light chain of the antibody of the present invention.

Figure 8 shows the heavy and light chain sequences of the antibody of the present invention.

no

Claims (21)

An isolated antibody or antigen-binding fragment specific for dengue virus, wherein the isolated anti-system is selected from the group consisting of: (i) a first antibody, which is specific for dengue virus serotype 1 (DENV1), including (a) heavy chain variable region (V _H ), including heavy chain complementarity determining region 1 (HC CDR1) as shown in SEQ ID NO: 2, heavy chain as shown in SEQ ID NO: 4 Complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 6; and (b) light chain variable region (V _L ), including as SEQ ID NO: 9, the light chain complementarity determining region 1 (LC CDR1), the light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 11, and the light chain complementarity determining region shown in SEQ ID NO: 13 3 (LC CDR3); (ii) The second antibody, which is specific to Dengue virus serotype 2 (DENV2), contains (a) heavy chain variable region (V _H ), and contains SEQ ID NO: 16 The heavy chain complementarity determining region 1 (HC CDR1) shown, the heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 18, and the heavy chain complementarity determining region 3 shown in SEQ ID NO: 20 (HC CDR3); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 23 is shown the light chain complementarity determining region 1 (LC CDR1), such as SEQ ID NO: 25 the light chain shown in FIG. Complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 27; (iii) a third antibody, which is specific for Dengue virus serotype 3 ( DENV3), including (a) heavy chain variable region (V _H ), including heavy chain complementarity determining region 1 (HC CDR1) as shown in SEQ ID NO: 30, heavy chain complementarity as shown in SEQ ID NO: 32 determining region 2 (HC CDR2), and as SEQ ID NO: complementarity determining region of the heavy chain shown in FIG. 34 3 (HC CDR3); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 37 [Suo The light chain complementarity determining region 1 (LC CDR1) shown, the light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 39, and the light chain complementarity determining region 3 shown in SEQ ID NO: 41 ( LC CDR3); (iv) Fourth antibody, which is specific for Dengue virus serotype 4 (DENV4) and contains (a) heavy chain variable region (V _H ), which is shown in SEQ ID NO: 44 Heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) as shown in SEQ ID NO: 46, and heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 48; and (b) a light chain variable region (V _L), comprising SEQ ID NO: 51 of the light shown in chain complementarity determining region 1 (LC CDR1), such as SEQ ID NO: 53 shown in the light chain complementarity determining region 2 (LC CDR2), And the light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 55; (v) the fifth antibody, which is related to the dengue virus serotypes type 1, type 2, type 3, and type Type 4 (DENV1, DENV2, DENV3, and DENV4) cross-react, including (a) heavy chain variable region (V _H ), including heavy chain complementarity determining region 1 (HC CDR1) shown in SEQ ID NO: 58, The heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 60, and the heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 62; and (b) the light chain variable region (V _L ), including the light chain complementarity determining region 1 (LC CDR1) shown in SEQ ID NO: 65, the light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 67, and as SEQ ID NO : Light chain complementarity determining region 3 (LC CDR3) shown in 69; and (vi) any combination of (i) to (v). The isolated antibody or antigen-binding fragment of claim 1, wherein (i) the first antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 71 and a light chain variable comprising SEQ ID NO: 72 Region (V _L ); (ii) The second antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 73 and a light chain variable region (V _L ) comprising SEQ ID NO: 74; (iii ) The third antibody comprises a heavy chain variable region (V _H ) comprising SEQ ID NO: 75 and a light chain variable region (V _L ) comprising SEQ ID NO: 76; (iv) the fourth antibody comprises SEQ ID NO: 77 heavy chain variable region (V _H ) and light chain variable region (V _L ) comprising SEQ ID NO: 78; and/or (v) the fifth antibody comprises a SEQ ID NO: 79 The heavy chain variable region (V _H ) and the light chain variable region (V _L ) comprising SEQ ID NO: 80. The isolated antibody or antigen-binding fragment of claim 1, wherein the antigen-binding fragment is selected from scFv, (scFv)2, Fab, Fab', and F(ab ')2. A composition comprising the isolated antibody or antigen-binding fragment thereof according to claim 1. The composition according to claim 4, which is a pharmaceutical or diagnostic composition, used to treat or diagnose a dengue virus disease. The composition of claim 4, which contains a pharmaceutically acceptable carrier. A method for detecting dengue virus in a sample suspected of containing dengue virus, comprising contacting the sample with an isolated antibody or antigen-binding fragment specific for dengue virus, and measuring the binding of the antibody to the sample, wherein the The isolated anti-system is selected from the group consisting of: (i) The first antibody, which is specific for Dengue virus serotype 1 (DENV1), which contains the heavy chain variable region (V _H ), which The chain variable region (V _H ) includes a heavy chain complementarity determining region 1 (HC CDR1) as shown in SEQ ID NO: 2, a heavy chain complementarity determining region 2 (HC CDR2) as shown in SEQ ID NO: 4, and The heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 6; and the light chain variable region (V _L ), the light chain variable region (V _L ) comprising the SEQ ID NO: 9 Light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) shown in SEQ ID NO: 11, and light chain complementarity determining region 3 (LC CDR3) shown in SEQ ID NO: 13 ); (ii) a second antibody, which is specific to Dengue virus serotype 2 (DENV2), which contains a heavy chain variable region (V _H ), the heavy chain variable region (V _H ) contains, for example, SEQ Heavy chain complementarity determining region 1 (HC CDR1) shown in ID NO: 16, heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 18, and heavy chain shown in SEQ ID NO: 20 Complementarity determining region 3 (HC CDR3); and light chain variable region (V _L ), the light chain variable region (V _L ) comprises SEQ ID NO: 23 shown in light chain complementarity determining region 1 (LC CDR1), The light chain complementarity determining region 2 (LC CDR2) as shown in SEQ ID NO: 25, and the light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 27; (iii) a third antibody, which is specific for dengue virus of serotype 3 (DENV3), comprising a heavy chain variable region (V _H), the heavy chain variable region (V _H) comprising the SEQ ID NO: 30 shown in the heavy chain complementarity determining Region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) as shown in SEQ ID NO: 32, and heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 34; and light Chain variable region (V _L ), the light chain variable region (V _L ) comprises the light chain complementarity determining region 1 (LC CDR1) shown in SEQ ID NO: 37, and the light chain shown in SEQ ID NO: 39 Complementarity determining region 2 (LC CDR2), and the light chain shown in SEQ ID NO: 41 Complementarity determining region 3 (LC CDR3); (iv) Fourth antibody, which is specifically for Dengue virus serotype 4 (DENV4), which contains a heavy chain variable region (V _H ), the heavy chain variable region (V _H ) contains the heavy chain complementarity determining region 1 (HC CDR1) as shown in SEQ ID NO: 44, the heavy chain complementarity determining region 2 (HC CDR2) as shown in SEQ ID NO: 46, and as SEQ ID NO : Heavy chain complementarity determining region 3 (HC CDR3) shown in 48; and light chain variable region (V _L ), the light chain variable region (V _L ) comprising the light chain complementarity decision shown in SEQ ID NO: 51 Region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) as shown in SEQ ID NO: 53, and light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 55; and ( v) Any combination of (i) to (iv). The method of claim 7, wherein each of the first antibody, the second antibody, the third antibody, and the fourth antibody is paired with a partner antibody. The method according to claim 8, wherein the partner antibody is a serotype cross-reactive antibody of dengue virus, which comprises (a) a heavy chain variable region (V _H ), which comprises a heavy chain as shown in SEQ ID NO: 58 Chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 60, and heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 62 ; And (b) light chain variable region (V _L ), including light chain complementarity determining region 1 (LC CDR1) shown in SEQ ID NO: 65, light chain complementarity determining region 2 shown in SEQ ID NO: 67 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 69. The method of claim 7, wherein (i) the binding of the first antibody to the sample indicates the presence of DENV1 in the sample; (ii) the binding of the second antibody to the dengue virus indicates the presence of DENV2 in the sample; (iii) Binding of the third antibody to the dengue virus indicates the presence of DENV3 in the sample; and/or (iv) Binding of the fourth antibody to the dengue virus indicates the presence of DENV4 in the sample. A kit for detecting the presence of dengue virus in a sample, wherein the kit contains one or more antibodies specific for dengue virus, selected from the group consisting of: (i) the first antibody, which is specific for the first dengue virus serotype 1 (DENV1), comprising a heavy chain variable region (V _H), the heavy chain variable region (V _H) comprising the SEQ ID NO: heavy chain CDRs of Figure 2 Region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) as shown in SEQ ID NO: 4, and heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 6; and light Chain variable region (V _L ), the light chain variable region (V _L ) comprising the light chain complementarity determining region 1 (LC CDR1) shown in SEQ ID NO: 9, the light chain shown in SEQ ID NO: 11 Complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 13; (ii) Second antibody, which is specific for Dengue virus serotype 2 ( DENV2), comprising a heavy chain variable region (V _H), the heavy chain variable region (V _H) comprising the SEQ ID NO: 16 the heavy chain complementarity determining region of FIG 1 (HC CDR1), SEQ ID NO as : Heavy chain complementarity determining region 2 (HC CDR2) shown in 18, and heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 20; and light chain variable region (V _L ), the light chain variable region (V _L) comprises SEQ ID NO: 23 shown in the light chain complementarity determining region 1 (LC CDR1), such as SEQ ID NO: 25 the heavy chain complementarity determining region 2 (LC CDR2), as well as SEQ ID NO: 27 shows the light chain complementarity determining region 3 (LC CDR3); (iii) a third antibody, which is specifically for dengue virus serotype 3 (DENV3), which contains the heavy chain variable region ( V _H ), the heavy chain variable region (V _H ) includes a heavy chain complementarity determining region 1 (HC CDR1) as shown in SEQ ID NO: 30, a heavy chain complementarity determining region 2 as shown in SEQ ID NO: 32 (HC CDR2), and the heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 34; and the light chain variable region (V _L ), the light chain variable region (V _L ) comprising SEQ ID NO: 37, light chain complementarity determining region 1 (LC CDR1), SEQ ID NO: 39, light chain complementarity determining region 2 (LC CDR2), and SEQ ID NO: 41, light chain complementarity Decision region 3 (LC CDR3); (iv) Fourth antibody, which is specific for dengue Serotypes Type 4 (DENV4), comprising a heavy chain variable region (V _H), the heavy chain variable region (V _H) comprising the SEQ ID NO: heavy chain complementarity determining region 44 of FIG 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) as shown in SEQ ID NO: 46, and heavy chain complementarity determining region 3 (HC CDR3) as shown in SEQ ID NO: 48; and light chain variable region (V _L ), the light chain variable region (V _L ) includes the light chain complementarity determining region 1 (LC CDR1) shown in SEQ ID NO: 51, and the light chain complementarity determining region 2 shown in SEQ ID NO: 53 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) as shown in SEQ ID NO: 55; (v) any combination of (i) to (iv); and (vi) partner antibody as required . As in the set of claim 11, wherein the partner antibody is a dengue virus serotype cross-reactive antibody, which comprises (a) a heavy chain variable region (V _H ), which comprises SEQ ID NO: 58 Heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) shown in SEQ ID NO: 60, and heavy chain complementarity determining region 3 (HC CDR3) shown in SEQ ID NO: 62 ); and (b) a light chain variable region (V _L), comprising SEQ ID NO: 65 of the light shown in chain complementarity determining region 1 (LC CDR1), such as SEQ ID NO: 67 the light chain shown in complementarity determining regions 2 (LC CDR2), and the light chain complementarity determining region 3 (LC CDR3) shown in SEQ ID NO: 69. The kit of claim 11, wherein at least one of the serotype-specific antibodies or the partner antibody contains a detectable label. The kit according to claim 13, wherein the detectable label is selected from the group consisting of an enzyme label, a fluorescent label, a metal label, and a radioactive label. The kit of claim 13, wherein the detectable label is selected from the group consisting of gold nanoparticles, colored latex beads, magnetic particles, carbon nanoparticles, and selenium nanoparticles. The kit according to claim 11, wherein the kit is an immunoassay kit. As set forth in claim 16, wherein the immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), A group consisting of luminescence immunoassay (LIA), or ILMA immunofluorescence assay. The kit according to claim 16, wherein the immunoassay is performed as a lateral flow assay. The kit of claim 16, wherein the immunoassay is a sandwich assay. A nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region (V _H ), a light chain variable region (V _L ), or both, wherein the heavy chain variable region (V _H ) and light chain variable region (V _L ) is as described in claim 1. A host cell comprising the nucleic acid according to claim 20.

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