KR102314073B1 - Composition for detecting target material comprising ficolin-1 and method for detecting using the same - Google Patents

Abstract

The present invention relates to a composition for detecting a target substance containing picoline-1 and a detection method using the same. According to the present invention, by using picoline-1 as a capture agent, it is possible to perform multiple diagnosis with high sensitivity in a short time with different target substances. By using the present invention, it is possible to quickly and accurately diagnose and detect infectious diseases, and through this, it can be widely used in the field of molecular diagnostic technology development for various new infectious bacteria and infectious viruses, as well as efficient treatment of infectious diseases through rapid treatment. It is expected to contribute greatly to treatment.

Description

Composition for detecting target material comprising ficolin-1 and method for detecting using the same

The present invention relates to a composition for detecting a target substance containing picoline-1 and a detection method using the same.

Arboviruses (or arthropod-borne viruses) are viral infectious diseases transmitted by the bite of hematophagous arthropods, such as mosquitoes, ticks, and sand flies. Among them, mosquito-borne viruses such as Zika virus (ZIKV), Dengue fever (DENV), and Chikungunya (CHIKV) have become an important public health problem worldwide due to global spread, and in 2016 The World Health Organization (WHO) has declared a global health emergency in anticipation of a global pandemic of the Zika virus. Recently, 390 million cases of arbovirus infection have been reported each year, including asymptomatic infections. As the world becomes a daily life zone and the habitats of vectors expand due to global warming, such infectious diseases are expected to occur continuously around the world. Therefore, there is an urgent need for research in the diagnostic field to prevent these diseases from becoming indigenous and to prevent their spread in advance through rapid diagnosis, quarantine, and vector management.

Currently, for diagnosis of such virus infection, PCR is performed by isolating the virus from blood or detecting antigen or gene from a sample for patients who show clinical symptoms such as fever, rash, joint pain, and headache, but viral infection Because of these similar symptoms, it takes a lot of time and money for rapid clinical identification and detection, as well as difficulty in confirming the diagnosis. In the case of serology, it is possible to diagnose infection and measure immunity by detecting virus-specific IgM in serum collected during the acute stage of the disease, but it is less sensitive than antigen detection and is not appropriate for the first 5-6 days immediately after infection.

Ficolin is an oligomeric protein composed of a complex of monomers composed of a collagen-like domain and a fibrinogen-like domain. The N-terminal domain specifically recognizes carbohydrates to distinguish foreign microorganisms, and the human innate immune system It is known as a protein that initiates the activation of the lectin complement pathway in There are three types of ficolin-1 (M-ficolin), ficolin-2 (L-ficolin), and ficolin-3 (H-ficolin) in humans, and among them, ficolin-1 passes through the fibrinogen domain. It is known to activate the lectin complement pathway by recognizing N-acetylated residues in sialic acid of microbial carbohydrates.

Accordingly, as a result of the present inventors' efforts to develop a composition for detecting a target substance based on ficolin-1, when ficolin-1 is used as a capture agent, ficolin-1 is a target-antibody complex (target- Antibodies complex) and non-specific binding, it was confirmed that two or more different target substances can be detected simultaneously with high sensitivity in a short time, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for detecting a target material comprising picoline-1 and a method for detecting a target material using the same.

In order to achieve the above object, the present invention provides a composition for detecting a target material comprising ficolin-1.

In addition, the present invention provides a kit for detecting a target substance based on ficolin-1.

In addition, the present invention provides a method for detecting a target substance using a kit for detection based on ficolin-1.

According to the present invention, by using picoline-1 as a capture agent, it is possible to perform multiple diagnosis with high sensitivity in a short time with different target substances. By using the present invention, it is possible to quickly and accurately diagnose and detect infectious diseases, and through this, it can be widely used in the field of molecular diagnostic technology development for various new infectious bacteria and infectious viruses, as well as efficient treatment of infectious diseases through rapid treatment. It is expected to contribute greatly to treatment.

Figure 1 shows the interaction characteristics of ficolin-1 with the envelope protein (EP) of DENV and CHIKV (a) ELISA, (b) co-immunoprecipitation (Co-immunoprecipitation), (c) dot blot It is a diagram showing the results of analysis through the assay.
Figure 2 shows a schematic diagram of a ficolin-1 based assay strip according to the present invention.
3 is a diagram showing the detection results of DENV and CHIKV using a ficolin-1 based assay strip according to the present invention.

The present invention provides a composition for detecting a target material comprising ficolin-1.

Hereinafter, the present invention will be described in detail.

The present inventors have found that when ficolin-1 is used as a capture agent, as ficolin-1 binds non-specifically with a target-antibodies complex, two or more different It was confirmed that the target material can be detected at the same time, and it was confirmed that the composition for detecting a target material based on picolin-1 has a very high value for the purpose of detecting a target material from a sample with high sensitivity.

In the present invention, the target material is not limited thereto, but may be any one selected from the group consisting of proteins, peptides, amino acids, nucleic acids, hormones, steroids, vitamins, drugs, bacteria and viruses.

In the present invention, the virus may be preferably an arbovirus, more preferably a tick or mosquito-borne virus, and even more preferably a mosquito-borne virus. .

The tick-borne virus may be Kyasanur forest disease virus or tick-borne encephalitis virus, but is not limited thereto.

The mosquito-borne virus is Dengue virus (DENV), Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), St. Louis encephalitis virus (SLE) ), West Nile virus (WNV), Spondweni virus (SPOV), Zika virus (ZIKV), yellow fever and Chikungunya virus (CHIKV) It may be any one selected from the group consisting of, but is not limited thereto.

In addition, in the present invention, the bacteria may be one or two or more types of exogenous bacteria (including bacteria, Gram-negative bacteria, or Gram-positive bacteria), but is not limited thereto.

The composition for detection of the present invention may be provided in an immobilized state using various known methods on a suitable carrier or support in order to increase the speed and convenience of detection. Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polyesterin, gabbro, filter paper, ion exchange resin, plastic film, plastic tubing, glass, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, cups, flat packs. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface) or planar (sheet, test strip).

In addition, the present invention provides a kit for detection based on ficolin-1 comprising the composition for detection.

In the present invention, the detection kit is not limited thereto, but may be an RT-PCR kit, a microarray chip kit, or an immunoassay kit, and the immunoassay kit is a Luminex assay kit, a protein microarray. kit or ELISA kit.

The Luminex assay kit, protein microarray kit, and ELISA kit include a polyclonal antibody and a monoclonal antibody against the protein, and a secondary antibody against the polyclonal antibody and monoclonal antibody to which a label is bound.

Examples of the type of the kit in the present invention include an immunochromatographic strip kit, a Luminex assay kit, a protein microarray kit, an ELISA kit, or an immunological dot kit. is not limited.

The kit may additionally include essential elements necessary for performing the ELISA. The ELISA kit contains an antibody specific for a marker protein. Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit may also include an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibody, such as labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and substrates thereof or capable of binding the antibody. other materials and the like.

In addition, the kit may additionally include essential elements necessary for performing a protein microarray. The microarray kit contains antibodies specific for a marker protein bound to a solid phase. Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The protein microarray kit may also include an antibody specific for a control protein. Other protein microarray kits include reagents capable of detecting bound antibodies, for example, labeled secondary antibodies, chromophores, enzymes (eg, fused to antibodies) and their substrates or other substances capable of binding antibodies. and the like.

The detection kit is most preferably provided in the form of a lateral flow assay based on immunochromatographic analysis (lateral flow assay, or lateral flow immunoassay) kit. Accordingly, the diagnostic kit may be provided as an immunochromatographic assay strip, and more preferably as a side flow immunoassay strip.

Lateral flow immunoassay (LFA) is a method for diagnosing and detecting target substances (antigens, antibodies) in a sample using a membrane to which gold particles are bound or an antibody is fixed, and capillary movement of a liquid sample. As a result, it can be stored for a long time even at room temperature and the results can be obtained in a short time without special equipment, so it is widely used for field analysis. However, the lateral flow immunoassay has problems with low assay sensitivity and lack of quantitation compared to ELISA as it relies on visual identification evaluation.

In one embodiment of the present invention, ficolin-1, an oligomeric protein composed of a complex of monomers consisting of a collagen-like domain and a fibrinogen-like domain, is used as a capture agent. Initiating a -1 based assay strip (strip for lateral flow immunoassay), when ficolin-1 is used as a capture agent, the target (target material) to be detected is specifically bound to the envelope protein of a virus or bacteria. It is confirmed that it is fast and accurate and can detect multiple objects at the same time. Through this, the present inventors have found that using the ficolin-1 based assay strip according to the present invention, while following the basic measurement principle of the conventional side flow immunoassay strip, select ficolin-1 as a capture agent to quickly and with high sensitivity It was confirmed that a large number of pathogens can be detected simultaneously.

More specifically, in the ficolin-1 based assay strip according to the present invention, a sample pad (sample pad) to which a sample (or sample) is applied, a target (antigen)-antibody complex to move, a membrane for development, and a sample are continuously and an absorption pad for moving. A test line and a control line are formed on the membrane in a direction from the sample pad to the absorption pad, a capturing agent that binds to a target (antigen) is fixed to the test line, and a detection antibody is bound to the control line. The secondary antibody may be immobilized.

In the present invention, the "sample pad" means a pad capable of diffusion flow by receiving a sample to be analyzed, and is composed of a material having sufficient porosity to receive and contain a sample to be analyzed. Such porous materials include fibrous papers, microporous membranes made of cellulose materials, cellulose derivatives such as cellulose and cellulose acetate, nitrocellulose, glass fibers, naturally occurring cotton, fabrics such as nylon, or porous gels. , but not limited thereto.

In the ficolin-1 based assay strip according to the present invention, a sample to be detected and an antibody solution prepared in advance by targeting the sample instead of a bonding pad or a releasing pad coated with a detection antibody in the strip (antibody solution) is mixed to form a target-antibodies complex, and the mixture is applied to a sample pad of a strip and developed.

In the present invention, the antibody solution includes a detection antibody bound to nanoparticles.

In the present invention, the nanoparticles refer to nanoparticles acting as a detectable label. The nanoparticles include noble metals of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), and ruthenium (Ru); Titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum (Mo), tungsten (W), ruthenium (Ru), a transition metal of osmium (Os); metals such as iron (Fe), nickel (Ni), and cobalt (Co); Metal nanoparticles, such as metal oxides of magnesium oxide (MgO), titanium dioxide (TiO ₂ ), vanadium pentoxide (V ₂ O ₅ ), zinc oxide (ZnO), latex beads, magnetic beads, It may be any one selected from the group consisting of quantum dots, upconversion nanoparticles (UCNP), graphene-nanoparticle complexes, and color dyed particles, preferably Silver may be gold (Au) nanoparticles or latex beads, most preferably latex beads, but is not limited thereto.

In one embodiment of the present invention, the antibody was labeled using colored latex beads.

In the present invention, the detection antibody refers to an antibody that specifically binds to a target substance to be detected or analyzed, and includes antibody fragments as long as it has binding specificity.

The detection antibody may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

The binding of the nanoparticles to the detection antibody includes, for example, an ionic bond, a covalent bond, a metal bond, a coordination bond, a hydrogen bond, and a van der Waals bond, but is not limited thereto.

In the present invention, the “membrane” may be made of any variety of materials through which a sample material can pass. For example, naturally occurring, synthetic, or synthetically modified materials such as polysaccharides (eg, cellulosic materials, paper, cellulose derivatives such as cellulose acetate and nitrocellulose); polyether sulfone; polyethylene; nylon; polyvinylidene fluoride (PVDF); Polyester; polypropylene; silica; An inorganic material uniformly dispersed in a porous polymer matrix with a polymer such as vinyl chloride, vinyl chloride-propylene copolymer and vinyl chloride-vinyl acetate copolymer, for example deactivated alumina, diatomaceous earth, MgSO ₄ , or other inorganic fines matter; naturally occurring (such as cotton) and synthetic (such as nylon or rayon) fabrics; porous gels such as silica gel, agarose, dextran and gelatin; polymer films such as polyacrylamide; and the like, preferably polymeric materials such as, but not limited to, nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester and polypropylene.

In the present invention, the “absorbent pad” may be located adjacent to or near the end of the membrane. The absorbent pad generally receives a fluid sample that travels through the entire membrane and can help promote capillary action and diffuse flow of the fluid through the membrane.

The assay strip according to the present invention may be configured by sequentially connecting the sample pad, the membrane, and the absorbent pad on the same support.

The support may be formed of any material as long as it can support and transport the sample pad, membrane and absorbent pad, but it is generally preferred to be liquid impermeable so that fluid of the sample that diffuses through the membrane does not leak through the support. do. For example, glass; Polymeric materials may include, but are not limited to, polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate, and polymelamine.

In the present invention, a test line and a control line are sequentially formed on the membrane in a direction from the sample pad to the absorption pad.

In the present invention, a capture agent that binds to an antigen (target material) to be detected or analyzed is fixed to the “test line”.

In the present invention, the secondary antibody that binds to the detection antibody is immobilized on the “control line”. The secondary antibody of the control line generates a detection signal by binding to the detection antibody bound to the nanoparticles.

As used herein, the term “antigen” refers to a target substance to be analyzed for concentration or presence, and may be used interchangeably with “target substance” in the present specification, for example, proteins, peptides, amino acids, nucleic acids, hormones. , steroids, vitamins, drugs, may be any one selected from the group consisting of bacteria and viruses, preferably bacteria or viruses, but is not limited thereto.

In the ficolin-1 based assay strip, when a sample is loaded into a sample pad, the sample is developed through a test line and a control line by capillary action. At this time, the target material present in the sample passes through the membrane and binds to the ficolin-1 and antibody immobilized on the test line and the control line to detect the presence of the target material.

In the present invention, the sample may be a target-antibody complex formed by mixing a sample in which a target material (target) to be detected or analyzed is expected to exist and the antibody solution according to the present invention before being added to the strip.

In the present invention, the antibody solution may include a detection antibody bound to nanoparticles. As described above, metal nanoparticles, latex beads, etc. may be used without limitation as the type of nanoparticles, and in an embodiment of the present invention, anti-CHIKV and anti-DENV to which colored latex beads are combined. It is confirmed that chikungunya virus and dengue virus can be simultaneously detected in a sample using an antibody solution containing

The kit for detection based on ficolin-1 of the present invention may further include a reagent and a detection device required for detection of a target substance.

In addition, the present invention provides a method for detecting a target substance using the detection kit according to the present invention.

The method may preferably be to use a ficolin-1 based assay strip, and more preferably a step of injecting a sample containing a target material into the sample pad of the ficolin-1-based assay strip according to the present invention. ; and confirming color development on the membrane.

As used herein, the term “sample” refers to a biological material suspected of containing an antigen to be analyzed. Examples include blood, interstitial fluid, saliva, eyepiece fluid, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, nasal fluid, hemoptysis, joint blood, intraperitoneal fluid, vaginal fluid, menses, amniotic fluid and semen. and may be derived from any biological source, such as a physiological fluid. Meanwhile, in the present specification, the term “sample” is also used interchangeably with “sample”.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Assay of binding of ficolin-1 to viral envelope proteins

Streptavidin binding peptide (SBP)-tagged ficolin-1, HIS-tagged CHIKV E2 Δ glycoprotein and DENV E Δ glycoprotein were expressed in insect cells using recombinant baculovirus and affinity Those purified by chromatography were used.

1-1. ELISA

Binding of ficolin-1 to envelope glycoprotein was analyzed by ELISA. First, the purified envelope proteins of CHIKV and DENV diluted to a concentration of 1 μg/ml with bovine serum albumin (BSA) or carbonate buffer (50 mM Na ₂ CO ₃ , 50 mM NaHCO _{3 , pH9.0) were plated on a microtiter plate.} was added and reacted overnight at 4° C. for coating. Antigen-coated wells were treated with phosphate buffer (PBS) containing 2% BSA to block non-specific binding, and binding buffer containing 10 μM of SBP-tagged ficolin-1 (20 mM Tris-HCl (pH 7.4)) , 150mM NaCl ₂ , 0.05% Tween 20, 0.1% (w/v) BSA, 10mM MgCl ₂ and 10mM CaCl ₂ ) were added and incubated at room temperature for 1 hour. After washing, HRP-conjugated streptavidin (Thermo Fisher Scientific, USA) was added and incubated, then washed again with PBST 6 times, and TMB (Sigma, USA) substrate solution was added to each well and a stop solution (stop solution; Abcam Inc., USA) to stop the reaction. After the reaction, absorbance was measured at an OD value of 450 nm using a 96-well plate ELISA reader. Between each step, it was washed 5 times with PBS containing 0.05% Tween 20. The results are shown in a of FIG. 1 .

As shown in FIG. 1 a, it was confirmed that ficolin-1 had high binding affinity to DENV2 and CHIKV envelope proteins.

1-2. zero- Immunoprecipitation (co- immunoprecipitation )

Binding of ficolin-1 to envelope glycoprotein was analyzed by co-immunoprecipitation. First, 10 μM of SBP-tagged ficolin-1 was incubated with streptavidin magnetic beads. Magnetic beads coated with ficolin-1 were blocked with 2% BSA, and immunoprecipitation analysis was performed with a binding buffer containing purified HIS-tagged EP (5 μM). After washing 5 times, the samples were separated by electrophoresis using SDS-PAGE (12%~4% gradient; Invitrogen, USA). Thereafter, western blotting was performed using 1 μg/ml of rabbit anti-HIS polyclonal antibody and anti-rabbit IgG (BIO-RAD, USA) conjugated with HRP. The results are shown in FIG. 1 b.

As shown in FIG. 1 b , it was confirmed that a significant band at 50KDa appeared for DENV2 and CHIKV envelope proteins compared to the control group BSA. From the above results, it was confirmed that ficolin-1 has high binding affinity to DENV2 and CHIKV envelope proteins.

1-3. dot blot test (dot blot analysis)

The interaction between ficolin-1 and virus was analyzed through protein dot blot analysis (dot blot analysis). 2 μl of CHIKV (1 × 10 ⁷ TCIC ₅₀ /mL) and DENV (1 × 10 ⁷ TCIC ₅₀ /mL) were spotted on Hybond C-extra nitrocellulose membrane (BIO-RAD, USA). Thereafter, the membrane was blocked with TBS (Tris-buffered saline) containing 0.05% Tween 20, 10 mM CaCl ₂ and 5% skim milk for 1 hour at room temperature, and 4 with SBP-tagged ficolin-1 10 μM. Incubated overnight at °C. After washing three times for 20 minutes, the membrane was further incubated with HRP-conjugated streptavidin at room temperature for 1 hour. After washing, interactions were detected with an enhanced chemiluminescence (ECL) kit. The results are shown in FIG. 1 c .

As shown in FIG. 1 c , it was confirmed that ficolin-1 had high affinity with both DENV-2 and CHIKV viruses.

Example 2. Preparation of ficolin-1 based assay strips

The ficolin-1 based assay strip according to the present invention includes a sample pad, a bonding pad or a releasing pad, a nitrocellulose membrane (NC) and an absorption pad for sample injection. consists of Each antibody was fixed by spraying onto the nitrocellulose membrane using a dispenser (DCI100; Zeta Corporation, Korea). The test line and the control line were prepared using ficolin-1 (10 μM) as a capturing agent and anti-mouse IgG (0.03 mg/ml), respectively. The nitrocellulose membrane sprayed with the antibody was dried at room temperature. Anti-CHIKV mAb bound to red latex beads and anti-DENV mAb bound to blue latex beads were used as detection antibodies and blocked with ficolin-1 (1 μM). The prepared ficolin-1 based assay strip according to the present invention is shown in FIG. 2 .

Example 3. Verification of the effectiveness of the test strip

In order to verify the effect of the test strip prepared in Example 2, the following experiment was performed. Colored latex beads in activation solution of 25 mM tris-HCl (pH 7.4), 100 mM NaCl, 10 mM CaCl ₂ , 10 mM MgCl ₂ and 0.1% v/v Tween-20 (Thermo Fisher Scientific, USA) Antibody solution was prepared by adding anti-CHIKV and anti-DENV bound (latex bead). Each virus sample was mixed with 50 μl detection antibody solution and incubated for 5 minutes to form target-antibody complexes. Each mixture was then pipetted onto the sample pad of the test strip. It was confirmed that the target-antibody complex interacts with the capture agent, ficolin-1, while the mixture moves along the strip by capillary action. Each virus was tested at 1 × 10 ⁷ , 1 × 10 ⁶ , 1 × 10 ⁵ , 1 × 10 ⁴ , 1 × 10 ³ TCID ₅₀ /ml, and in a negative control group using PBST+HAS (human serum albumin) buffer. The same test was performed for All chromogenic signals were generated by red or blue latex beads. The results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that both anti-DENV mAb (blue) and anti-CHIKV mAb (red) were detected up ^{to 1×10 5} TCID _{50 /ml in both DENV-2 and CHIKV.} Through the above results, it was confirmed that the assay strip according to the present invention can rapidly detect a plurality of pathogens simultaneously with high sensitivity by using ficolin-1 as a capture agent.

As mentioned above, although preferred embodiments of the present invention have been described in detail, the technical scope of the present invention is not limited to the above-described embodiments and should be interpreted according to the claims. At this time, those skilled in the art should consider that many modifications and variations are possible without departing from the scope of the present invention.

Claims (12)

One type of detection antibody labeled with nanoparticles that specifically binds to Chikungunya virus (CHIKV), a target, and generates a detection signal, and a target, Dengue virus, DENV) and one type of detection antibody labeled with nanoparticles that generate a detection signal;
ficolin-1, which is a capture agent that binds to the target material; and
a support to which the ficolin-1 is immobilized;
As a picoline-1-detecting antibody complex for simultaneous detection of Chikungunya virus (CHIKV) and Dengue virus (DENV), comprising:
each specific detection antibody binds to the target material to form a target-antibodies complex;
The nanoparticles labeled with the detection antibody generate different detection signals;
Characterized in that, as the target-antibodies complex binds to ficolin-1 immobilized on the support, the target material is simultaneously detected,
A picoline-1-detecting antibody complex for simultaneous detection of Chikungunya virus (CHIKV) and Dengue virus (DENV). delete delete delete One type of detection antibody labeled with nanoparticles that specifically binds to Chikungunya virus (CHIKV), a target, and generates a detection signal, and a target, Dengue virus, DENV) and one type of detection antibody labeled with nanoparticles that generate a detection signal;
ficolin-1, which is a capture agent that binds to the target material;
a support to which the ficolin-1 is immobilized;
Chikungunya virus (CHIKV) and dengue virus (Chikungunya virus, CHIKV) and dengue virus ( Dengue virus, DENV) as a kit for simultaneous detection,
each specific detection antibody binds to the target to form a target-antibodies complex;
The nanoparticles labeled with the detection antibody generate different detection signals;
Characterized in that, as the target-antibodies complex binds to ficolin-1 immobilized on the support, the target material is simultaneously detected,
A kit for simultaneous detection of Chikungunya virus (CHIKV) and Dengue virus (DENV). The kit according to claim 5, wherein the support is an assay strip. 7. The method of claim 6, wherein the analysis strip comprises: a sample pad into which a sample containing a target material is input; a membrane comprising a test line to which ficolin-1 capable of binding to the target material is immobilized; and an absorbent pad, the kit. The kit according to claim 7, wherein the membrane further comprises a control line on which a secondary antibody binding to the detection antibody is immobilized. The kit according to claim 6, wherein the strip is characterized in that a sample pad, a membrane, and an absorbent pad are sequentially connected on a support. delete According to claim 5, wherein the nanoparticles are gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iridium (Ir), rhodium (Rh), ruthenium (Ru) noble metals; Titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum (Mo), tungsten (W), ruthenium (Ru), a transition metal of osmium (Os); metals such as iron (Fe), nickel (Ni), and cobalt (Co); Metal nanoparticles such as magnesium oxide (MgO), titanium dioxide (TiO2), vanadium pentoxide (V2O5), zinc oxide (ZnO), latex beads, magnetic beads, quantum dots ), upconversion nanoparticles (UCNP), graphene (graphene) - characterized in that any one selected from the group consisting of nanoparticles and color dyed nanoparticles (color dyed particles), the kit. A method of simultaneously detecting Chikungunya virus (CHIKV) and Dengue virus (DENV) using the kit of claim 5 .

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