Detection of antibodies to fever with thrombocytopenia syndrome virus antigen, test strip and preparation method and application thereof

Abstract

The application provides a test strip for detecting antibodies of a fever with thrombocytopenia syndrome virus antigen, a preparation method and application thereof. A pair of antibodies against the fever with thrombocytopenia syndrome virus antigen are screened, and a test strip is prepared based on the pair of antibodies, wherein the detection limit of the test strip is as low as 0.025 ng / mL, and the test strip has high specificity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to antibodies, test strips, preparation methods, and applications for detecting viral antigens of fever with thrombocytopenia syndrome. Background Technology

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne disease caused by Dabie bandavirus (DBV). This virus belongs to the genus Bandavirus in the order Bunyavirales and has been officially named by the International Committee on Taxonomy of Viruses. First discovered in central China in 2010, SFTS is primarily transmitted to humans through the bite of Haemaphysalis longhorn ticks. Clinical manifestations of SFTS include acute fever, thrombocytopenia, leukopenia, gastrointestinal symptoms, and multiple organ dysfunction. Some patients may experience bleeding tendencies and neurological symptoms. The mortality rate is as high as 30%, rising to 54% in elderly patients or those with underlying chronic diseases. Currently, DBV has spread to more than 20 provinces and cities in China, forming high-incidence areas in Jiangsu, Anhui, Henan, Hubei, and Guangdong provinces, with an average of over 200 reported cases annually. In recent years, the number of reported cases in Southeast Asian countries such as South Korea and Japan has increased by 17% annually, leading to a sharp increase in the risk of regional epidemics.

[0003] Currently, there are no specific antiviral treatments or preventative vaccines for SFTS; treatment primarily involves symptomatic and supportive care. Rapid and accurate diagnosis of the pathogen at the earliest possible time is crucial for SFTS control. Traditional detection methods mainly include virus isolation and culture, nucleic acid amplification techniques (such as PCR), and enzyme-linked immunosorbent assay (ELISA). While these methods have a certain degree of accuracy, they suffer from drawbacks such as requiring specialized equipment and technicians, long testing cycles, and high costs. Therefore, there is an urgent need to find a simple, rapid method with low experimental preparation requirements. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof for fever with thrombocytopenia syndrome virus, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2 and light chain CDR3;

[0006] Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are as shown in SEQ ID NO.1, 2, and 3, respectively; and the amino acid sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 are as shown in SEQ ID NO.4, 5, and 6, respectively; or

[0007] The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NO.7, 8 and 9, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.10, 11 and 12, respectively.

[0008] In this invention, the fever with thrombocytopenia syndrome virus is also known as Dabie Bandar virus.

[0009] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment of the fever-associated thrombocytopenia syndrome virus contains the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 90% identity with SEQ ID NO. 13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 90% identity with SEQ ID NO. 14; or

[0010] The heavy chain variable region of the antibody or antigen-binding fragment of the fever with thrombocytopenia syndrome virus contains the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 90% identity with SEQ ID NO. 15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence having at least 90% identity with SEQ ID NO. 16.

[0011] In some embodiments, the heavy chain variable region of the fever with thrombocytopenia syndrome virus antibody or its antigen-binding fragment contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 7, and the light chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 8.

[0012] In one specific embodiment, the heavy chain variable region of the fever with thrombocytopenia syndrome virus antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO.8.

[0013] As used in this invention, the term "CDR," also known as "complementarity-determining region," "CDR region," or "hypervariant region," refers to a region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes.

[0014] Based on the amino acid sequences of the heavy chain variable region and light chain variable region contained in the given fever with thrombocytopenia syndrome virus antibody or its antigen-binding fragment, those skilled in the art can determine the CDRs contained therein using conventional methods in the art. CDR sequences determined by different methods all fall within the scope of protection of this application. For example, the IMGT method, Kabat method, AbM method, Chothia method, or Contact method can be used to define the CDRs in the variable region amino acid sequence.

[0015] In a specific embodiment of the present invention, the IMGT scheme is used to define the CDR in the variable region amino acid sequence.

[0016] When referring to antibodies defined by a specific CDR sequence as described in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). The CDR boundaries of the antibodies of this invention can be determined manually based on any scheme or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing manner.

[0017] In some embodiments, functional variants of the fever-associated thrombocytopenia syndrome virus antibody or its antigen-binding fragment described herein are also included within the scope of protection of this invention. The term "functional variant" refers to a protein having significant or marked sequence identity or similarity to the parent antibody, and retaining the biological activity of the parent antibody. Functional variants encompass, for example, the following variants of the fever-associated thrombocytopenia syndrome virus antibody or its antigen-binding fragment (parent antibody) described herein, which retain the ability to recognize target cells to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the functional variant may, for example, have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the parent antibody in terms of amino acid sequence.

[0018] In some embodiments, the functional variant may, for example, comprise the amino acid sequence of a parent antibody having at least one conserved amino acid substitution. Alternatively or supplementally, the functional variant may comprise the amino acid sequence of a parent antibody having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity of the functional variant compared to the parent antibody.

[0019] In some embodiments, conservative amino acid substitution is known in the art and includes the substitution of one amino acid having a particular physical and / or chemical property with another amino acid having the same or similar chemical or physical property.

[0020] Those skilled in the art can readily mutate the nucleotide sequence corresponding to the antibody described in this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 90% or more homology to the nucleotide sequence corresponding to the fever-associated thrombocytopenia syndrome virus antibody or its antigen-binding fragment described in this invention, as long as they encode the aforementioned fever-associated thrombocytopenia syndrome virus antibody or its antigen-binding fragment, are all derived from and equivalent to the sequence of this invention, and are also included within the scope of protection of this invention.

[0021] In this invention, the antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2, single-chain antibody scFv, and nanobody VHH.

[0022] Fab: Fab is a functional unit of a monoclonal antibody molecule, which contains the heavy chain variable region VH, the heavy chain constant region CH1, the light chain variable region VL, and the light chain constant region CL. It does not contain the Fc fragment and has a molecular weight of approximately 50 kDa.

[0023] scFv: scFv is the variable region of the monoclonal antibody molecule, namely VH+VL, with a molecular weight of approximately 25 kDa.

[0024] VHH: Unlike Fab and scFv, VHH originates from heavy chain antibodies (a type of antibody that naturally lacks a light chain). VHH is the variable region of the heavy chain in a heavy chain antibody, with a molecular weight of approximately 15 kDa. The molecular weight of VHH is only one-tenth that of conventional antibodies, and its size is in the nanometer range, hence it is also called a nanobody (Nano-antibody). Because it has a single structural domain, it is also called a single-domain antibody (SdAbs).

[0025] In some embodiments, the antibody further includes a constant region, and according to the structure of the constant region, the antibody includes IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0026] The second aspect of the present invention provides any of the following products:

[0027] (1) A polynucleotide molecule or a carrier comprising the polynucleotide molecule, said polynucleotide molecule encoding an antibody or antigen-binding fragment thereof of the fever with thrombocytopenia syndrome virus as described in the first aspect of the present invention;

[0028] (2) A modified host cell or a population of host cells comprising thereto, said modified host cell comprising an antibody or antigen-binding fragment of the fever with thrombocytopenia syndrome virus as described in the first aspect of the present invention, or a polynucleotide molecule or a carrier comprising thereto as described in the second aspect of the present invention (1).

[0029] (3) A conjugate of an antibody or antigen-binding fragment of a fever-associated thrombocytopenic purpura virus, said conjugate comprising a complex formed by conjugating the antibody or antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the first aspect of the present invention with a therapeutic agent;

[0030] (4) A pharmaceutical composition for treating a disease or condition caused by fever with thrombocytopenia syndrome virus, said pharmaceutical composition comprising an antibody or antigen-binding fragment of the fever with thrombocytopenia syndrome virus as described in the first aspect of the present invention, or a conjugate of an antibody or antigen-binding fragment of the fever with thrombocytopenia syndrome virus as described in the second aspect (3) of the present invention.

[0031] The term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons.

[0032] In this invention, the polynucleotide molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0033] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0034] In some embodiments, examples of vectors that can be used in this invention include, but are not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors.

[0035] Various vectors known in the art can be used, such as commercially available vectors, and then a polynucleotide encoding the antibody or its antigen-binding fragment can be operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the virus-derived vectors include, but are not limited to: lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, herpesvirus vectors, baculovirus vectors, papillomavirus vectors, and papillomavirus vectors.

[0036] In some embodiments, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0037] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0038] In some embodiments, the vector is selected from plasmid vectors.

[0039] In some embodiments, the plasmid vector is selected from the PTT5 vector.

[0040] In some implementations, the host cell population may also include host cells other than the modified host cells.

[0041] In some implementations, the modified host cells include prokaryotic cells and eukaryotic cells.

[0042] In some implementations, the prokaryotic cells include, but are not limited to, bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsia.

[0043] In some embodiments, the bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus.

[0044] In some embodiments, the eukaryotic cells include, but are not limited to, mammalian cells, insect cells, plant cells, and yeast cells.

[0045] In one specific implementation, the modified host cell is selected from Escherichia coli.

[0046] In some implementations, the therapeutic agents include, but are not limited to, ribavirin, favipiravir, caffeic acid tablets, and amoquine.

[0047] In this invention, the term "treatment" refers to the improvement, prevention, relief, or reversal of a disease or condition or at least one identifiable symptom thereof, including treating patients at risk of or suspected of having the disease, as well as patients who have or have been diagnosed with the disease or condition, and includes suppressing clinical relapse. The term "prevention" as used herein refers to the complete prevention of symptoms of the disease. The term "relief" as used herein refers to a reduction in the severity or duration of symptoms of the disease. Relief includes, but does not require, complete recovery from or complete prevention of the disease or its symptoms.

[0048] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0049] As used herein, the term "pharmaceutically acceptable" means a molecular entity and composition that, when administered to a subject such as a human, does not produce an adverse reaction, allergic reaction, or other adverse reaction, as appropriate. According to this disclosure, the preparation of pharmaceutical compositions comprising antibodies or additional active ingredients is known to those skilled in the art. Furthermore, for administration to a subject (e.g., a human), it should be understood that the pharmaceutical composition should meet standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biostandards.

[0050] In this invention, the subject can be any mammal, including but not limited to: rodent mammals, such as mice and hamsters; and lagomorph mammals, such as rabbits. The mammal can be from the order Carnivora, including Felidae (cats) and Canidae (dogs). The mammal can be from the order Artiodactyla, including Bovidae (cattle) and Suaeda (pigs); or Perissodactylus, including Equidae (horses). The mammal can be from the orders Primates, Cebooids, or Simoids (monkeys); or Anthropoids (humans and apes). In a specific embodiment of this invention, the subject is preferably a human.

[0051] As used herein, "pharmaceutically acceptable carriers and / or excipients" include, but are not limited to, aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral media such as sodium chloride, Ringer's glucose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, adsorption delay agents, salts, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquids, nutritional supplements, and similar materials and combinations thereof, as are known to those skilled in the art. The pH and exact concentration of various components in a pharmaceutical composition are adjusted according to known parameters.

[0052] In some embodiments, the pharmaceutical compositions of the invention are advantageously administered in the form of injectable compositions (as liquid solutions or suspensions); solid forms suitable for reconstitution into solutions or suspensions in liquids prior to injection can also be prepared. These formulations can also be emulsified.

[0053] In some implementations, the disease or condition caused by the fever with thrombocytopenia syndrome virus includes, but is not limited to, one or more of the following: fever, thrombocytopenia, leukopenia, nausea, vomiting, diarrhea, loss of appetite, fatigue, headache, muscle aches, lymphadenopathy, abnormal liver function, kidney damage, myocardial injury, respiratory failure, disseminated intravascular coagulation, and multiple organ dysfunction syndrome.

[0054] A third aspect of the present invention provides an antibody for fever with thrombocytopenia syndrome virus or an antibody derivative thereof, wherein the antibody derivative comprises a complex formed by directly or indirectly conjugating the antibody for fever with thrombocytopenia syndrome virus or an antigen-binding fragment thereof to a detectable marker as described in the first aspect of the present invention.

[0055] In this invention, the term "detectable marker" refers to a reagent that is detectable by means of, for example, spectroscopic, photochemical, biochemical, immunochemical or chemical methods. In some embodiments, the detectable marker can also be observed directly with the naked eye.

[0056] In some implementations, the detectable markers include, but are not limited to, radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, photosensitizing diagnostic agents, and plasma nanomaterials.

[0057] In some embodiments, the radionuclides include, but are not limited to, those mentioned above. 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 124 I, 125 I, 131 I, 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 55 Co、 72 As.

[0058] In some embodiments, the chemiluminescent agent includes, but is not limited to, luminol, isoluminol, aromatic acridine esters, imidazole, acridine salts, and oxalates.

[0059] In some embodiments, the bioluminescent agent includes, but is not limited to, luciferin, luciferase, and jellyfish luminescent protein.

[0060] In some embodiments, the paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

[0061] In some embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0062] In some embodiments, the photosensitive diagnostic agent includes, but is not limited to, dihydroxysilyl phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin.

[0063] In some implementations, the detectable marker is selected from plasma nanomaterials.

[0064] In this invention, the plasma nanomaterials refer to a class of nanoscale materials prepared in a plasma environment or possessing plasma properties.

[0065] In some implementations, the plasma nanomaterials include, but are not limited to, colloidal gold nanoparticles and colloidal silver nanoparticles.

[0066] In some embodiments, the plasma nanomaterial is selected from colloidal gold nanoparticles.

[0067] The fourth aspect of the present invention provides a detection product comprising an antibody or antigen-binding fragment thereof against the fever-associated thrombocytopenic purpura virus as described in the first aspect of the present invention, or an antibody derivative thereof against the fever-associated thrombocytopenic purpura virus as described in the fourth aspect of the present invention.

[0068] In some implementations, the products include, but are not limited to, test strips, reagent kits, reagents, and chips.

[0069] In some implementations, the test strip includes, but is not limited to, test strips, test strip sheets containing test strips, test strip cards containing test strips, and test pens containing test strips.

[0070] In some implementations, the test strip card is formed by inserting a test strip into a plastic casing, wherein the sample application port of the plastic casing is above the sample pad, and the observation window of the plastic casing is above the test line and the control line.

[0071] In some implementations, the test pen is another integrated form, typically formed by placing the test strip in a pen-shaped casing, with the sample application area usually designed at the pen tip.

[0072] In some implementations, the test strip is selected from test strips.

[0073] In some implementations, the test strips include, but are not limited to, immunochromatographic test strips, dry chemistry test strips, and microfluidic test strips.

[0074] In some implementations, the test strip is selected from immunochromatographic test strips.

[0075] In some implementations, the types of kits include, but are not limited to, ELISA kits and immunochromatographic kits.

[0076] In some implementations, the kit may contain, but is not limited to, one or more of the test strips, test strips, reagents, and chips described above.

[0077] In some embodiments, the kit may also include, but is not limited to, a container for holding antibodies or antigen-binding fragments thereof when not in use, instructions for use of the antibodies or antigen-binding fragments thereof, antibodies or antigen-binding fragments thereof attached to a solid support, and one or more known standards.

[0078] The term "known standard" can refer to a known amount or concentration of fever with thrombocytopenia syndrome virus (FPSV), wherein such a solution can be a naturally occurring solution, such as a sample from a patient known to be infected with FPSV; or such a solution can be a synthetic solution, such as a buffer solution in which a known amount of FPSV is diluted. Known standards described herein may include FPSV isolated from subjects, recombinant or purified FPSV proteins.

[0079] In some implementations, the viral protein of the fever with thrombocytopenia syndrome is the viral nucleoprotein, NIH GenBank accession number: ATW63020.1.

[0080] In this invention, the nucleoprotein is a nucleocapsid protein, also known as nucleoprotein, NP protein, or N protein.

[0081] In some embodiments, the immunochromatographic test strip includes a sample pad for adding a sample, a binding pad for immobilizing the detection antibody-detectable marker, a reaction pad with a detection line T and a control line C, and an absorption pad;

[0082] The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the detection antibody are shown in SEQ ID NO.7, 8 and 9, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.10, 11 and 12, respectively.

[0083] The detection line T is coated with a capture antibody. The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the capture antibody are shown in SEQ ID NO.1, 2 and 3, respectively. The amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 of the capture antibody are shown in SEQ ID NO.4, 5 and 6, respectively.

[0084] The quality control line C is coated with anti-IgG antibodies.

[0085] In some embodiments, the heavy chain variable region of the detection antibody contains the amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence having at least 90% identity with SEQ ID NO.15, and the light chain variable region of the detection antibody contains the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence having at least 90% identity with SEQ ID NO.16.

[0086] In some embodiments, the heavy chain variable region of the capture antibody contains the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 90% identity with SEQ ID NO. 13, and the light chain variable region of the capture antibody contains the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 90% identity with SEQ ID NO. 14.

[0087] In some embodiments, the sample pad includes, but is not limited to, a glass fiber membrane, a polyester fiber membrane, or a nonwoven fabric.

[0088] In some implementations, the conjugate pad is used to immobilize a detectable antibody-detectable marker, and the material of the conjugate pad includes, but is not limited to, glass cellulose membrane and polyester cellulose membrane.

[0089] In some embodiments, the reaction pad includes, but is not limited to, a nitrocellulose membrane.

[0090] In some embodiments, the absorbent pad includes, but is not limited to, cellulose filter paper and glass fiber pad.

[0091] In some embodiments, the test strip further includes a backing plate, to which the sample pad, conjugate pad, reaction pad, and absorbent pad are adhered.

[0092] In some implementations, the back panel is a PVC back panel.

[0093] The fifth aspect of the present invention provides a method for preparing the detection product according to the fifth aspect of the present invention, the method comprising: combining a sample pad for adding a sample, a binding pad for immobilizing a detection antibody-detectable marker, a reaction pad with a detection line T and a control line C, and an absorbent pad on a backing plate.

[0094] In some embodiments, the method further includes coating the detection line T of the reaction pad with a capture antibody at a concentration of 1.0-3.0 mg / mL.

[0095] In some embodiments, the method further includes coating the detection line T of the reaction pad with a capture antibody at a concentration of 1.5 mg / mL.

[0096] In some embodiments, the method further includes coating the reaction pad with an anti-IgG antibody at a concentration of 1.0-3.0 mg / mL at control line C.

[0097] In some embodiments, the method further includes coating the reaction pad with an anti-IgG antibody at a concentration of 2.0 mg / mL at control line C.

[0098] In some embodiments, the method further includes a method for preparing a detection antibody-detectable marker, comprising: adjusting the pH of a colloidal gold nanoparticle solution to 7.4-8.0, mixing the colloidal gold nanoparticle solution with the detection antibody to achieve a mass concentration of the detection antibody of 19.5 μg / mL-50.0 μg / mL, and blocking the detection antibody with a blocking agent.

[0099] In some implementations, the mass concentration of the detection antibody is set at 23.4 μg / mL.

[0100] In some embodiments, the method for preparing the detection antibody-detectable marker further includes removing large particulate impurities and resuspending the antibody after blocking.

[0101] In some embodiments, the sealing agent includes, but is not limited to, serum albumin, skim milk, and skim milk powder.

[0102] In some embodiments, the serum albumin includes, but is not limited to, human serum albumin and bovine serum albumin.

[0103] In some embodiments, the blocking agent is selected from bovine serum albumin.

[0104] In some implementations, the conjugate pad is immersed in a treatment solution containing sucrose and serum albumin and left to stand overnight at 37°C.

[0105] In some embodiments, the treatment solution is formulated as 10 mM phosphate buffer PB, pH 7.4, containing 0.1% Tween 20, 5% sucrose and 1% bovine serum albumin.

[0106] In some implementations, a detection antibody-detectable marker is coated onto the binding pad and dried for later use.

[0107] In some embodiments, the method further includes a method for preparing a colloidal gold solution, comprising: diluting a HAuCl4·3H2O suspension, heating, adding sodium citrate solution, continuing to heat for 5-15 minutes after the solution turns a bright wine red color, and removing the heating device to allow it to cool.

[0108] In some implementations, heating continues for 10 minutes after the solution turns a bright wine-red color.

[0109] The sixth aspect of the present invention provides any of the following methods:

[0110] (1) A method for preparing an antibody or antigen-binding fragment of the fever-associated thrombocytopenic syndrome virus as described in the first aspect of the present invention, the method comprising the following steps: culturing the modified host cells or host cell populations containing the modified host cells as described in the second aspect of the present invention (2), and isolating the antibody or antigen-binding fragment of the fever-associated thrombocytopenic syndrome virus as described in the first aspect of the present invention from the culture.

[0111] (2) A method for preparing the modified host cell or host cell population containing the present invention as described in the second aspect (2), the method comprising the steps of: introducing the polynucleotide molecule or the vector containing the present invention as described in the second aspect (1) into the host cell;

[0112] (3) A method for detecting fever with thrombocytopenia syndrome virus in a test sample, the method comprising: contacting the test sample with an antibody or antigen-binding fragment of fever with thrombocytopenia syndrome virus as described in the first aspect of the present invention, an antibody derivative of the antibody or antigen-binding fragment of fever with thrombocytopenia syndrome virus as described in the third aspect of the present invention, and a detection product as described in the fourth aspect of the present invention, and detecting the formation of an immune complex of fever with thrombocytopenia syndrome virus with the antibody or antigen-binding fragment of the present invention.

[0113] In some implementations, the vector is introduced into the host cell by means including but not limited to physical methods, chemical methods, and biological methods.

[0114] In some implementations, the physical methods include, but are not limited to, the use of calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation.

[0115] In some implementations, the chemical method includes, but is not limited to, the use of colloidal dispersion systems and lipid-based systems.

[0116] In some implementations, the biological method includes, but is not limited to, the use of DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0117] In some implementations, the method for detecting fever with thrombocytopenia syndrome virus in the sample is for non-diagnostic and non-therapeutic purposes.

[0118] The seventh aspect of the present invention provides any of the following applications:

[0119] (1) The use of the antibody or antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the first aspect of the present invention, the antibody or antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the third aspect of the present invention, and the detection product described in the fourth aspect of the present invention in the preparation of products for detecting the fever-associated thrombocytopenic purpura virus or fragments thereof;

[0120] (2) The use of the antibody or antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the first aspect of the present invention, the antibody or antibody derivative of the antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the third aspect of the present invention, and the detection product described in the fourth aspect of the present invention in the preparation of products for diagnosing whether a subject suffers from a disease or condition caused by the fever-associated thrombocytopenic purpura virus.

[0121] (3) The use of the antibody or antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the first aspect of the present invention, and the conjugate of the antibody or antigen-binding fragment of the fever-associated thrombocytopenic purpura virus described in the second aspect of the present invention (3) in the preparation of a pharmaceutical composition for treating diseases or symptoms caused by the fever-associated thrombocytopenic purpura virus.

[0122] The eighth aspect of the present invention provides a method for diagnosing whether a subject is infected with fever with thrombocytopenia syndrome virus (FST), or for diagnosing whether a subject suffers from a disease or condition caused by FST, the method comprising contacting a sample of the subject with an antibody or antigen-binding fragment of FST as described in the first aspect of the present invention, an antibody derivative of an antibody or antigen-binding fragment of FST as described in the third aspect of the present invention, and a detection product as described in the fourth aspect of the present invention, and detecting the formation of an immune complex of FST with the antibody or antigen-binding fragment of FST.

[0123] The ninth aspect of the present invention provides a method for treating fever with thrombocytopenia syndrome virus infection, or for treating diseases or symptoms caused by fever with thrombocytopenia syndrome virus, the method comprising administering an antibody or antigen-binding fragment thereof for fever with thrombocytopenia syndrome virus as described in the first aspect of the present invention, a conjugate of an antibody or antigen-binding fragment thereof for fever with thrombocytopenia syndrome virus as described in the second aspect (3) of the present invention, and a pharmaceutical composition for treating diseases or symptoms caused by fever with thrombocytopenia syndrome virus as described in the second aspect (4) of the present invention.

[0124] The advantages and beneficial effects of this invention are as follows:

[0125] This invention provides antibodies, test strips, preparation methods, and applications for detecting SFTS virus antigens. The invention screens for a pair of antibodies against SFTS virus antigens and prepares test strips based on these antibodies. The test strips have a detection limit as low as 0.025 ng / mL and exhibit high specificity. This invention is expected to become a practical tool for early differentiation between acute fever caused by SFTS infection and infectious acute fever caused by other pathogens. It can also be combined with other detection methods and clinical manifestations to provide doctors with preliminary test results to assist in accurate diagnosis and initial treatment. The test strips provided by this invention do not require professional personnel or special instruments and, compared to other methods such as isolation culture and ELISA, have the advantages of being faster and simpler to operate in most public health communities, remote rural areas, and low-income countries and regions. With the global spread of SFTS, most countries urgently need to conduct large-scale screening of potential SFTS patients. In this context, the technical solution provided by this invention is expected to become a more efficient and economical solution to address this epidemic. Attached Figure Description

[0126] Figure 1 This is a schematic diagram of the test strip structure;

[0127] Figure 2 The results of the optimization experiment of the conjugate pad are shown in the figure. (A) The color and morphology of the dried gold nanoparticle-monoclonal antibody complex on the conjugate pad; (B) The detection results of DBV NP antigen standard (1ng / ml) when using glass cellulose membrane Ahlstrom8964 and polyester cellulose membrane MA0800 as conjugate pads respectively.

[0128] Figure 3 This is an SDS-PAGE result image of DBV NP antigen;

[0129] Figure 4 This is a Western blot result of two monoclonal antibodies against recombinant nucleoprotein of fever with thrombocytopenia syndrome virus.

[0130] Figure 5 These are the binding and dissociation curves of two monoclonal antibodies against recombinant nucleoprotein of fever with thrombocytopenia syndrome virus;

[0131] Figure 6 The binding of two antibodies (WT-19 and WR-31) to DBV nucleoprotein (DBV NP) under non-competitive conditions was detected using biolayer interferometry (BLI).

[0132] Figure 7 These are images showing the morphological results of colloidal gold particles: (A) UV / Vis spectrum of gold nanoparticles; (B) TEM morphology characteristics of gold nanoparticles.

[0133] Figure 8 For the interpretation of colloidal gold immunochromatographic test strip results;

[0134] Figure 9 This is a graph showing the results of determining the optimal pH for the gold nanoparticle-labeled antibody WT-31.

[0135] Figure 10 This is a graph showing the optimal amount of labeled antibody for gold nanoparticle-labeled antibody WT-31.

[0136] Figure 11 These are the results of the test strip sensitivity test (from left to right: negative control, 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.1 ng / mL, 0.05 ng / mL, 0.025 ng / mL, 0.01 ng / mL);

[0137] Figure 12 This is a diagram showing the specificity test results of the test strip;

[0138] Figure 13 This is a graph showing the test results of a positive serum sample (A) from a DBV-infected patient and a serum sample (B) from a healthy person. Detailed Implementation

[0139] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0140] Example

[0141] I. Materials and Methods

[0142] 1.1. Materials and Chemical Reagents

[0143] All reagents were of analytical grade and could be used directly in subsequent experiments without further purification. Chloroauric acid (HAuCl4·3H2O, purity ≥47.8%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Trisodium citrate (Na3C6H5O7·2H2O, ≥99.0%), sodium chloride (NaCl, ≥99.5%), and sucrose (C 12 H 22 O 11Potassium carbonate (K2CO3, ≥99.0%) and potassium carbonate were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Tween 20 was provided by Sangon Biotech Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Chemical Company, USA. Sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sample pads (Ahlstrom 8964), nitrocellulose membranes (CN95, Sartorius), absorbent pads, polyvinyl chloride backing plates, and goat anti-human (IgG) were provided by Shanghai Jieyi Biotechnology Co., Ltd. (Shanghai, China). The N protein of fever with thrombocytopenia syndrome virus was cloned and expressed by Jiangsu Provincial Center for Disease Control and Prevention. The ultrapure water used throughout the experiment was prepared by a Millipore Milli-Q ultrapure water system (Billerica, MA, USA), with a resistivity ≥18.25 MΩ·cm. The blocking buffer (PBS solution containing 1% BSA, pH 7.4) was freshly prepared before use. All aqueous solutions were prepared using double-distilled water.

[0144] Serum samples from DBV-infected patients were kindly donated by the Jiangsu Provincial Center for Disease Control and Prevention's Institute of Acute Infectious Disease Control. The nucleoprotein-specific monoclonal antibody was prepared in our laboratory, and its binding specificity to the nucleoprotein (NP) has been verified.

[0145] 1.2 Instruments

[0146] All photographs of the gold nanoparticles and paper lateral flow immunoassay strips were taken using a mobile phone digital camera. Transmission electron microscopy (TEM) images were observed using a JEM-2100 TEM (Nippon Electron Technology Co., Ltd.). All test strip result photographs were acquired using a digital camera. Transmission electron microscopy (TEM) tests were performed using a JEM-2100 TEM (Nippon Electron Technology Co., Ltd.).

[0147] 1.3 Cloning and Expression of DBV Nucleoproteins

[0148] The DBV nucleoprotein gene (GenBank accession number: ATW63020.1) was synthesized and cloned into the pET-28a vector. The recombinant nucleoprotein was expressed in *E. coli* and purified using a nickel column. A simplified procedure is as follows: The pET28a-NP recombinant plasmid was transformed into *E. coli* BL21 competent cells and plated on LB agar containing 50 μg / mL kanamycin for overnight culture. After confirming correct cloning via sequencing, IPTG was added and expression was induced at 20°C for 16-20 hours. The bacterial culture was collected and sonicated (200W for 40 minutes). After centrifugation at 8000 rpm for 20 minutes at 4°C, the supernatant was collected and the target protein was purified using a nickel chelate affinity column. The nucleoprotein was eluted with imidazole-containing elution buffer, and the purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein concentration was determined using a Nanodrop 2000 micro-spectrophotometer (Thermo Fisher Scientific, USA).

[0149] 1.4 Monoclonal Antibody Preparation

[0150] Peripheral blood samples were collected from four DBV recovery patients. Peripheral blood mononuclear cells were isolated using Ficoll-Paque Plus density gradient medium (GE Healthcare, Sweden) according to the manufacturer's instructions. A human scFv phage display library was constructed, and three rounds of biopanning were performed using recombinant DBV NP proteins to ultimately select two scFv gene clones. The heavy and light chain variable region genes were cloned into a PTT5 vector containing the human IgG1 constant region, respectively. The heavy and light chain eukaryotic expression vectors were co-transfected into HEK-293F cells using a polyetherimide transfection method. After transfection, the 293F cell culture supernatant was collected, centrifuged at 10,000×g for 10 min, filtered through a 0.22 μm filter, and purified using a Protein A affinity chromatography column (GE Healthcare, Sweden) via an NGC Quest 10 Plus system (BioRad, USA). Antibody concentration was determined using the BCA Protein Quantitative Kit (Thermo Scientific, USA). The purified antibody was aliquoted and stored at -80°C for later use.

[0151] 1.5 Identification of the binding specificity of humanized monoclonal antibody to DBV-NP

[0152] The binding specificity of humanized monoclonal antibody to NP was identified using indirect ELISA and Western blot (WB). First, NP was coated onto 96-well ELISA plates at 200 ng / well. The purified antibody was serially diluted starting from 1 μg / ml. Primary antibody was incubated at 37 °C for 1 h, washed with PBST, and HRP-labeled anti-human IgG was added. The plates were then incubated at 37 °C for 30 min, followed by TMB color development. The OD450 absorbance was then read after termination. Each sample was repeated three times, and the average value was taken. Next, the target antibody was detected by WB. 10 μg of NP was subjected to SDS-PAGE, and the protein on the PAGE gel was transferred to a PVDF membrane. The membrane was blocked with 3% skim milk and incubated with recombinant NP humanized monoclonal antibody. After washing, HRP-labeled anti-human IgG was added. Finally, the membrane was developed.

[0153] 1.6 Determination of affinity between humanized monoclonal antibodies and NP antigen and competitive binding analysis of antibodies based on BLI technology

[0154] Biolayer interferometry (BLI) was used to detect the affinity and kinetic parameters of humanized monoclonal antibodies binding to NP antigens. Antibodies were immobilized on the surface of a ProA sensor and then reacted with diluted NP antigens. Changes in surface optical interference were analyzed to obtain information about intermolecular interactions. The entire binding and dissociation process was monitored in real time using an OctetRed 96 macromolecular interaction analyzer.

[0155] Antibody competition assays were performed using the Octet R8 system (ForteBio, Molecular Devices) via biolayer interference. Recombinant NP protein was dissolved in PBST buffer at a concentration of 30 µg / mL and immobilized on an Anti-Penta-HIS (HIS1K) biosensor for 0.5 min. First, a first monoclonal antibody was added to NP at a concentration of 15 µg / mL until saturation. When the binding curve flattened, a second monoclonal antibody (15 µg / mL) was immediately added to the biosensor. The area under the curve (AUC) of the second antibody with and without the competing antibody was measured. The AUC values ​​of "second antibody + competing antibody" and "second antibody alone" were standardized with the AUC value of the second antibody alone, and expressed as the percentage of binding by the second antibody alone. The criteria were: a bidirectional binding rate <30% was considered competitive, 30%-70% was partially competitive, and ≥70% was non-competitive.

[0156] 1.7 Synthesis of Colloidal Gold Nanoparticles

[0157] Gold nanoparticles with a diameter of 30 nm were prepared. The simplified steps are as follows: 1 g of chloroauric acid was dissolved in 100 mL of distilled water to prepare a 1% aqueous solution. The solution was placed in a round-bottom flask and heated to 120 °C. While continuously stirring, 1 mL of freshly prepared 1% sodium citrate solution was rapidly added. The solution color gradually changed from pale yellow to wine red. When the solution became a bright wine red, it was boiled for 10 minutes. The heating element was removed, and stirring was continued for 15 minutes until cooling. Finally, the solution was diluted to 100 mL with ultrapure water and stored at 4 °C protected from light. The average particle diameter was confirmed to be 30 nm by spectrophotometry, and the size of the gold nanoparticles was characterized by transmission electron microscopy.

[0158] 1.8 Preparation of Antibody-Colloidal Gold Nanoparticles

[0159] Colloidal gold nanoparticles were conjugated to detection antibodies by optimizing pH and antibody concentration parameters. The preparation method of colloidal gold nanoparticles is as follows: First, the pH of the colloidal gold nanoparticle solution was adjusted to 7.7 using 0.1 mol / L potassium carbonate solution. Then, 23.4 μg of monoclonal antibody was added to 1 mL of the colloidal gold nanoparticle solution. After gentle stirring at room temperature for 20 minutes, 100 μL of 10% (w / v) bovine serum albumin solution was added, and stirring was continued for another 20 minutes for blocking. The resulting product was centrifuged at 1500 rpm for 20 minutes to remove large particulate impurities, followed by another centrifugation at 15000 rpm for 20 minutes. Finally, the precipitate was resuspended in 0.01 mol / L pH 8.0 Tris-HCl buffer containing 1% BSA and 10% sucrose (resuspending volume was one-fifth of the original volume) for subsequent experiments.

[0160] 1.9 Development of Immunochromatographic Test Strips

[0161] See the schematic diagram of the test strip structure. Figure 1 The paper immunochromatographic test strip consists of four parts: a sample pad (10×4 mm), a conjugation pad (8×4 mm), a nitrocellulose membrane (20×4 mm), and an absorbent pad (10×4 mm). The sample pad is used to add DBV nucleoprotein samples.

[0162] The conjugate pad is used to immobilize the bioconjugate. Its primary function is to support the detection antibody-colloidal gold nanoparticles and maintain their functional stability until detection begins. This function is achieved through the composition of the binding buffer, which contains carbohydrates (such as sucrose) that act as a protectant and resolubilizing agent. Two cellulose membranes were used in this study: a glass cellulose membrane (Ahlstrom 8964) and a polyester cellulose membrane (MA0800). Overall, the fluid flow rates of the two membranes were not significantly different. Figure 2(A) shows the color and morphology of dried gold nanoparticles-monoclonal antibodies sprayed on glass cellulose membrane (Ahlstrom 8964) and polyester cellulose membrane (MA0800). The glass cellulose membrane (Ahlstrom 8964) absorbs the gold-labeled antibody more uniformly. Figure 2 (B) Demonstrates the detection performance of antigen standards (1 ng / mL) using glass cellulose membrane (Ahlstrom 8964) and polyester cellulose membrane (MA0800) as conjugate pads, respectively. Before assembling the paper-based lateral flow immunoassay analyzer, the conjugate pads were immersed in a treatment solution (treatment solution formulation: 10 mM phosphate buffer PB, pH 7.4, containing 0.1% Tween 20, 5% sucrose, and 1% bovine serum albumin) and incubated overnight at 37°C. Subsequently, antibody-colloidal gold nanoparticles coated with the detection solution were sprayed onto the conjugate pads at a spray volume of 15-25 μL / cm³. 2 Place in a 37℃ oven and dry for 1 hour.

[0163] A double-ended marker (SJ001, Shenzhen Stationery Store) equipped with an ultra-fine tip (SJ002, 1.97×34 mm, Shenzhen Stationery Store) was used to draw test lines and control lines on a nitrocellulose membrane. The test lines were coated with 1.5 mg / mL DBV monoclonal antibody at a volume of approximately 1.5-2.0 μL / cm, while the control lines were coated with 2.0 mg / mL goat anti-human IgG at a volume of approximately 1.5-2.0 μL / cm. The distance between the two lines was 5 mm. The nitrocellulose membrane was blocked with 1% (w / v) bovine serum albumin solution and then dried at room temperature. All components were fixed to a PVC backing. Finally, the test strips were cut to a width of 4 mm, sealed, and stored at 4°C for later use. The developed test strips can be mass-produced.

[0164] 1.10 Testing Procedure

[0165] When the sample (70 μL) is added to the sample pad of the test strip, it undergoes chromatography towards the absorbent pad under capillary action. The presence of the target protein is determined by the specific colorimetric tracing of gold nanoparticles, and the detection result can be observed visually within 10 minutes. The sensitivity of this immunochromatographic test strip was then determined.

[0166] All animal experiments in this invention were conducted under the guidance of the Animal Welfare Committee of the Jiangsu Provincial Center for Disease Control and Prevention, and the experimental animals were treated with utmost care and respect throughout the process. All experimental procedures involving human specimens were approved by the Ethics Committee of the Jiangsu Provincial Center for Disease Control and Prevention.

[0167] II. Results and Discussion

[0168] 2.1 Expression and purification of recombinant proteins

[0169] To utilize DBV nucleoprotein for antibody library screening, an *E. coli* expression system was employed. DBV nucleoprotein was expressed in *E. coli* BL21. Subsequently, the recombinant protein was purified using a Ni-NTA affinity column combined with a protein purification system, followed by SDS-PAGE analysis. Results ( Figure 3 The results showed that the target protein migrated as a clear band with a molecular weight of approximately 27 kDa, which is consistent with the theoretical molecular weight, indicating that the expression and purification of the DBV nucleic acid polymer were successfully completed.

[0170] 2.2 Preparation and Identification of Monoclonal Antibodies Against DBV Nucleocapsid Protein

[0171] In the Western blot (WB) assay, both antibodies showed a distinct band at a relative molecular mass of 27 kDa. This result further confirms that both obtained human monoclonal antibodies are DBV-NP specific antibodies, and that all antibodies bind to the linear epitopes of the NP antigen, such as... Figure 4 .

[0172] 2.3 Antibody Affinity Assay

[0173] BLI was used to analyze the binding and dissociation kinetics between the two antibodies and NP. BLI results showed that both antibodies immobilized on the sensor could generate clear binding and dissociation curves with serially diluted NP, and the curves exhibited a clear dose-response relationship. Figure 5 By fitting the binding and dissociation constants of the antigen and antibody, the affinity constants of the two antibodies were obtained as 0.136 nmol / L and 0.773 nmol / L, respectively (Table 1). The antibody affinity constants are both in the nmol / L range.

[0174] Table 1. Affinity constants of the two monoclonal antibodies against the recombinant nucleoprotein of fever with thrombocytopenia syndrome virus.

[0175]

[0176] Biolayer interference (BLI) results (see Figure 6) showed that the WT-19 + WT-31 group rapidly increased in the initial stage and continued to rise around 200 seconds. The response reached a plateau at approximately 250 seconds. This result indicates that the combination of WT-19 and WT-31 exhibits a strong binding interaction. The significant increase in interference signal during the second antibody incubation step suggests that the two antibodies bind to different epitopes on the DBV NP, without competitive repulsion, making it suitable for subsequent development of lateral flow immunoassays.

[0177] 2.4 Apparent characteristics, distribution state and particle size analysis of colloidal gold solutions

[0178] The prepared colloidal gold solution was wine-red in color, homogeneous and clear, with no floating debris on the surface and no precipitate aggregation at the bottom of the beaker. The colloidal gold solution was then scanned using a UV spectrophotometer in the wavelength range of 400-800 nm. Generally, a narrower absorption peak indicates better homogeneity and stability of the colloidal gold. Figure 7 As shown in Figure A, when the maximum absorption wavelength of the colloidal gold solution is 525 nm, its average particle size is 30 nm. Finally, characterization by transmission electron microscopy revealed (…). Figure 7 (B) The prepared colloidal gold solution consists of spherical particles, exhibits good dispersibility, and shows no aggregation.

[0179] 2.5 Assembly and Detection Principle of Immunochromatographic Test Strips

[0180] This experiment utilizes the sandwich antigen-antibody reaction principle to construct an antibody-gold-labeled antibody-antigen-antibody complex for DBV detection. The detection procedure for the gold nanoparticle-based immunochromatographic test strip is as follows: An appropriate amount of sample is added to the sample well of the test card, and the sample is chromatographically deposited along the test card under capillary action. If the sample contains DBV antigen, the antigen will bind to the detection monoclonal antibody in the binding pad, and the complex continues to migrate to the capture monoclonal antibody region (detection line) on the membrane, displaying a red band indicating a positive result. Simultaneously, unbound complex continues to migrate forward and binds to the anti-mouse polyclonal antibody on the control line, causing it to turn red. Therefore, the positive or negative result of the test strip depends on whether the detection line turns red. If the control line does not turn red, the test result is considered invalid regardless of whether the detection line turns red. Figure 8 ).

[0181] WT-31 monoclonal antibody was used as the detection antibody, and WT-19 was used as the capture antibody to form an antibody pair for lateral chromatography analysis. The capture antibody WT-19 was immobilized on the detection line, and the detection antibody WT-31 was used to label colloidal gold, thus constructing a sandwich immunoassay system. The sequences of WT-19 and WT-31 antibodies are shown in Table 2.

[0182] Table 2. Amino acid sequences of WT-19 and WT-31 antibodies

[0183]

[0184]

[0185] 2.6 Optimization of Gold Nanoparticle-Monoclonal Antibody Conjugates

[0186] According to the principle of colloidal gold labeling, the most stable gold nanoparticle-monoclonal antibody conjugate can only be formed under specific pH conditions; when the pH value of colloidal gold is close to or slightly higher than the isoelectric point of the protein, the antibody adsorption capacity on the surface of the gold particles is strongest. The effect of pH gradients (6.5, 6.8, 7.1, 7.4, 7.7, 8.0) was investigated by adding different volumes of 0.1 mol / L potassium carbonate solution to 1 mL of gold nanoparticle suspension. An excess antibody group and a salt-free blank control group were included in the experiment. Under the destructive effect of high-concentration salt solution, the gold nanoparticle-antibody solution at pH below 7.4 showed a color change from red to blue due to aggregation. However, the solution color did not change within the pH range of 7.4-8.0, indicating that no aggregation occurred. Figure 9 Finally, the optimal labeling pH was determined to be 7.7.

[0187] Determining the minimum amount of protein required to form a stable probe is another key factor affecting labeling efficacy. Excessive protein labeling leads to probe aggregation, severely impacting label activity because free proteins in the immunogold-colloid complex solution preferentially bind to the labeling site, creating a "blocking effect." Conversely, insufficient protein labeling results in incomplete colloidal gold labeling, reducing detection sensitivity and causing false positives. To obtain the optimal binding antibody concentration, salt tolerance tests were performed by adding different concentrations of monoclonal antibody (0, 0.65, 1.3, 1.95, 2.6, 3.25, 3.9, 4.55, 5.2 μg) to 200 μL of gold nanoparticle solution (pH=7.7). Figure 10 As shown, when the antibody dosage is 0-3.25 μg, high-concentration salt solutions directly induce aggregation, manifested as a change in solution color from red to blue; while the solution of gold nanoparticles containing 3.9 μg of monoclonal antibody remains red in NaCl solution. According to literature reports, the actual antibody dosage usually needs to be increased by 20% from this baseline, so the optimal antibody concentration was finally determined to be 23.4 μg / mL.

[0188] 2.7 Sensitivity detection of immunochromatographic test strips

[0189] To determine the sensitivity of the immunochromatographic test strip, a series of 10-fold serial dilutions were performed using recombinant DBV protein at an initial concentration of 10 μg / mL. After a negative result, a 2-fold serial dilution was performed at the previous concentration, with the lowest concentration at which a positive result was observed serving as the limit of detection (LOD) of the test strip. Experimental results showed that the test strip achieved a detection sensitivity of 0.025 ng / mL for recombinant DBV protein. Figure 11 ).

[0190] 2.8 Specificity Validation of Immunochromatographic Test Strips

[0191] To verify the specificity of the developed immunochromatographic test strip, this invention simultaneously tested DBV-infected positive serum samples, Hantavirus-infected positive serum samples, dengue virus-infected positive serum samples, influenza A virus-infected positive samples, and normal human samples (negative control). The results showed that, except for DBV-infected samples, all other samples showed only a clear band at the control line; while DBV-positive samples showed a detection band simultaneously. Figure 12 The results showed that this immunochromatographic test strip is highly specific for DBV virus.

[0192] 2.9 Detection and Analysis of DBV in Human Serum Using Immunochromatographic Test Strips

[0193] To assess clinical applicability, this test strip was used to test 10 positive serum samples (samples 1-10) from DBV-infected patients, with serum from healthy individuals serving as a negative control. Clinical sample test results are as follows: Figure 13 As shown, this test strip can effectively detect positive serum samples from DBV-infected patients (samples 1-9). Both PCR and LFIA test strip results were consistently positive (A), while PCR and LFIA test strip results for 18 healthy serum samples (samples 11-28) were consistently negative (B). As summarized in Table 3, only one sample with a relatively low viral load (sample 10, ct=37.7) showed a negative result on the test strip but a positive result on the PCR test. A comprehensive analysis of the molecular detection method and the LFIA test strip designed in this study yielded a Kappa coefficient of 0.92, indicating excellent consistency between the test strip and the results. Furthermore, the LFIA test strip showed a sensitivity of 90%, a specificity of 100%, and a Youden index of 0.9. These conclusions demonstrate that this test strip exhibits excellent consistency with the gold standard PCR method. All tests were repeated three times with consistent results at weekly intervals (Table 4), demonstrating the excellent stability of the prepared test strips.

[0194] Table 3. Analysis of DBV in samples using paper-based lateral flow immunochromatography.

[0195]

[0196]

[0197] Note: (-) represents a negative result, (+) represents a positive result.

[0198] Table 4 Stability of LFIAs

[0199]

[0200] Note: - indicates no color development; + indicates weak color development; ++ indicates strong color development; +++ indicates clear color development.

[0201] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment of Dabie Bandar virus, characterized in that, The antibody or antigen-binding fragment of the Dabie Bandar virus contains a heavy chain variable region and a light chain variable region. The heavy chain variable region contains heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and the light chain variable region contains light chain CDR1, light chain CDR2 and light chain CDR3. Wherein: the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are as shown in SEQ ID NO.1, 2, and 3, respectively; and the amino acid sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 are as shown in SEQ ID NO.4, 5, and 6, respectively; or The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NO.7, 8 and 9, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.10, 11 and 12, respectively.

2. The antibody against Dabie Bandar virus or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or antigen-binding fragment of the Dabie Bandar virus contains the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 90% identity with SEQ ID NO. 13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 14 or an amino acid sequence having at least 90% identity with SEQ ID NO. 14; or The heavy chain variable region of the Dabie Bandar virus antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 90% identity with SEQ ID NO. 15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence having at least 90% identity with SEQ ID NO.

16.

3. A polynucleotide molecule or a carrier comprising the thereof, characterized in that, The polynucleotide molecule encodes an antibody or antigen-binding fragment of the Dabie Bandar virus as described in any one of claims 1-2.

4. The polynucleotide molecule or carrier comprising it according to claim 3, characterized in that, The vector is selected from plasmid vectors.

5. The polynucleotide molecule or carrier comprising it according to claim 4, characterized in that, The plasmid vector is selected from the PTT5 vector.

6. A modified host cell or a population of host cells comprising the same, characterized in that, The modified host cell contains an antibody or antigen-binding fragment of the Dabie Bandar virus as described in any one of claims 1-2, or a polynucleotide molecule or a vector containing the same as described in any one of claims 3-5.

7. The modified host cell or host cell population containing the modified host cell according to claim 6, characterized in that, The modified host cell is selected from prokaryotic cells.

8. The modified host cell or host cell population containing the modified host cell according to claim 7, characterized in that, The prokaryotic cells were selected from Escherichia coli.

9. An antibody against Dabie Bandar virus or an antibody derivative thereof containing an antigen-binding fragment, characterized in that, The antibody derivative comprises a complex formed by directly or indirectly conjugating an antibody or antigen-binding fragment of the Dabie Bandar virus as described in any one of claims 1-2 to a detectable marker.

10. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 9, characterized in that, The detectable markers include radionuclides, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, photosensitizing diagnostic agents, and plasma nanomaterials.

11. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The radionuclides include 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 86 Y、 90 Y、 89 Zr、 120 I, 123 I, 124 I, 125 I, 131 I, 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 55 Co、 72 As.

12. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The chemiluminescent agents include luminol, isoluminol, aromatic acridine esters, imidazole, acridine salts, and oxalate esters.

13. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The bioluminescent agents include luciferin, luciferase, and jellyfish luminescent protein.

14. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III).

15. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

16. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The photosensitive diagnostic agents include dihydroxysilyl phthalocyanine, methylene blue, protoporphyrin, hematoporphyrin, and photoporphyrin.

17. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 10, characterized in that, The detectable markers are selected from plasma nanomaterials.

18. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 17, characterized in that, The plasma nanomaterials include colloidal gold nanoparticles and colloidal silver nanoparticles.

19. The antibody against Dabie Bandar virus or an antibody derivative thereof of its antigen-binding fragment according to claim 18, characterized in that, The plasma nanomaterial is selected from colloidal gold nanoparticles.

20. A product for detecting Dabie Bandar virus, characterized in that, The product for detecting Dabie Bandar virus includes an antibody or antigen-binding fragment of Dabie Bandar virus as described in any one of claims 1-2, or an antibody derivative of an antigen-binding fragment of Dabie Bandar virus as described in any one of claims 9-19.

21. The product for detecting Dabie Bandar virus according to claim 20, characterized in that, The products include test strips, reagent kits, reagents, and chips.

22. The product for detecting Dabie Bandar virus according to claim 21, characterized in that, The test strip includes a test strip, a test strip sheet containing the test strip, a test strip card containing the test strip, and a test pen containing the test strip.

23. The product for detecting Dabie Bandar virus according to claim 22, characterized in that, The test strips are selected from test strips.

24. The product for detecting Dabie Bandar virus according to claim 23, characterized in that, The test strips are selected from immunochromatographic test strips.

25. The product for detecting Dabie Bandar virus according to claim 24, characterized in that, The immunochromatographic test strip includes a sample pad for adding the sample, a binding pad for immobilizing the detection antibody-detectable marker, a reaction pad with a detection line T and a control line C, and an absorption pad.

26. The product for detecting Dabie Bandar virus according to claim 25, characterized in that, The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the detection antibody are shown in SEQ ID NO.7, 8 and 9, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.10, 11 and 12, respectively.

27. The product for detecting Dabie Bandar virus according to claim 25, characterized in that, The detection line T is coated with a capture antibody. The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 of the capture antibody are shown in SEQ ID NO.1, 2 and 3, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 of the capture antibody are shown in SEQ ID NO.4, 5 and 6, respectively.

28. The product for detecting Dabie Bandar virus according to claim 25, characterized in that, The quality control line C is coated with anti-IgG antibodies.

29. The product for detecting Dabie Bandar virus according to claim 26, characterized in that, The heavy chain variable region of the detection antibody contains the amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence having at least 90% identity with SEQ ID NO.15, and the light chain variable region of the detection antibody contains the amino acid sequence shown in SEQ ID NO.16 or an amino acid sequence having at least 90% identity with SEQ ID NO.

16.

30. The product for detecting Dabie Bandar virus according to claim 27, characterized in that, The heavy chain variable region of the capture antibody contains the amino acid sequence shown in SEQ ID NO.13 or an amino acid sequence having at least 90% identity with SEQ ID NO.13, and the light chain variable region of the capture antibody contains the amino acid sequence shown in SEQ ID NO.14 or an amino acid sequence having at least 90% identity with SEQ ID NO.

14.

31. The product for detecting Dabie Bandar virus according to claim 25, characterized in that, The test strip also includes a backing plate, and the sample pad, conjugate pad, reaction pad, and absorbent pad are adhered to the backing plate.

32. A method for preparing the product for detecting Dabie Bandar virus according to any one of claims 20-31, characterized in that, The method includes: combining a sample pad for adding a sample, a binding pad for immobilizing a detection antibody-detectable marker, a reaction pad with a detection line T and a control line C, and an absorbent pad on a backing plate.

33. The method according to claim 32, characterized in that, The concentration of the capture antibody used is 1.0-3.0 mg / mL.

34. The method according to claim 33, characterized in that, The concentration of the capture antibody used is 1.5 mg / mL.

35. The method according to claim 32, characterized in that, The concentration of the anti-IgG antibody used is 1.0-3.0 mg / mL.

36. The method according to claim 35, characterized in that, The concentration of the anti-IgG antibody used is 2.0 mg / mL.

37. The method according to claim 32, characterized in that, The method further includes a method for preparing a detection antibody-detectable marker, comprising: adjusting the pH of a colloidal gold nanoparticle solution to 7.4-8.0, mixing the colloidal gold nanoparticle solution with the detection antibody to make the mass concentration of the detection antibody 19.5 μg / mL-50.0 μg / mL, and blocking the detection antibody with a blocking agent.

38. The method according to claim 37, characterized in that, The mass concentration of the detection antibody was set to 23.4 μg / mL.

39. The method according to claim 37, characterized in that, The sealing agent includes serum albumin, skim milk, and skim milk powder.

40. The method according to claim 37, characterized in that, The method also includes a method for preparing colloidal gold solution, comprising: diluting HAuCl4·3H2O suspension, heating, adding sodium citrate solution, and continuing to heat for 5-15 minutes after the solution turns bright wine red, then removing the heating device and allowing it to cool.

41. The method according to claim 40, characterized in that, Continue heating for 10 minutes after the solution turns a bright wine red color.

42. A method for preparing an antibody or antigen-binding fragment of the Dabie Bandar virus according to any one of claims 1-2, characterized in that, The method includes the following steps: culturing the modified host cells or host cell populations containing the modified host cells as described in any one of claims 6-8, and isolating the antibody or antigen-binding fragment of the Dabie Bandar virus as described in any one of claims 1-2 from the culture.

43. A method for preparing the modified host cell or host cell population containing the thereof as described in any one of claims 6-8, characterized in that, The method includes the following steps: introducing the polynucleotide molecule of any one of claims 3-4 or a vector containing the polynucleotide molecule into a host cell.

44. A method for detecting Dabie Bandar virus in a test sample for non-diagnostic purposes, characterized in that, The method includes: contacting the sample to be tested with an antibody or antigen-binding fragment of the Dabie Banda virus as described in any one of claims 1-2, an antibody derivative of the Dabie Banda virus or antigen-binding fragment as described in any one of claims 9-19, or a product for detecting Dabie Banda virus as described in any one of claims 20-31, and detecting the formation of an immune complex between the Dabie Banda virus and the antibody or antigen-binding fragment.

45. The use of the antibody or antigen-binding fragment of the Dabie Bandar virus according to any one of claims 1-2, the antibody or antibody derivative of the antigen-binding fragment of the Dabie Bandar virus according to any one of claims 9-19, and the product for detecting the Dabie Bandar virus according to any one of claims 20-31 in the preparation of a product for detecting the Dabie Bandar virus.

46. ​​The use of the antibody or antigen-binding fragment of the Dabie Banda virus according to any one of claims 1-2, the antibody or antibody derivative of the antigen-binding fragment of the Dabie Banda virus according to any one of claims 9-19, and the product for detecting Dabie Banda virus according to any one of claims 20-31 in the preparation of a product for diagnosing whether a subject has a disease or condition caused by Dabie Banda virus.

47. The application according to any one of claims 45-46, characterized in that, The products include test strips, reagent kits, reagents, and chips.

48. The application according to claim 47, characterized in that, The test strip includes a test strip, a test strip sheet containing the test strip, a test strip card containing the test strip, and a test pen containing the test strip.

49. The application according to claim 48, characterized in that, The test strips are selected from test strips.

50. The application according to claim 49, characterized in that, The test strips are selected from immunochromatographic test strips.

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