KR20190020710A - Peptides for diagnosis of mosquito-borne viruses and uses thereof - Google Patents

Abstract

The present invention relates to isolated peptides for mosquito-borne virus-specific diagnosis, characterized in that no cross-reaction between mosquito-borne viruses is observed in the diagnosis of the mosquito-borne viruses, and to a rapid diagnostic kit for the mosquito-borne virus-specific diagnosis comprising the isolated peptides.

Description

Peptides for diagnosis of mosquito-borne viruses and uses thereof

The present invention is characterized in that the cross-reaction between mosquito-borne viruses does not appear when diagnosing a mosquito-borne virus, an isolated peptide for specific diagnosis of a mosquito-mediated virus, and an isolated peptide for a mosquito-mediated virus-specific diagnosis It is about a rapid diagnosis kit.

Dengue virus is a major global disease that suffers from 50 million to 100 million people per year, and is known to kill 25,000 people per year. Dengue (fever) is a mosquito-borne disease, and after showing symptoms of a cold, it progresses to dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Therefore, in order to block dengue virus infection, the development of a vaccine is very important, but in the case of this disease, it is difficult to develop a vaccine because multiple infections (second infection by another type of dengue virus) show more severe symptoms of DHF/DSS. Therefore, it is still known as the best treatment to relieve patient symptoms through fast and accurate diagnosis.

In addition, Zika virus belongs to the Flaviviridae family, such as yellow fever and dengue virus, and when infected with Zika virus, mild colds such as fever, skin rash, conjunctivitis, muscle and joint pain, headache, etc. Symptoms appear for 2 to 7 days. The symptoms are mild, so some people pass without knowing the infection, and the mortality rate is low. However, the possibility of causing microcephaly or neurological complications in newborns has been raised, and in Brazil, more than 200 microcephaly have been confirmed by January 2016 after the first case of Zika virus was reported in May 2015. However, there is currently no adequate treatment or vaccine, which cannot be prevented.

In addition, when infected with the chikunkunia virus, after an incubation period of 1 to 12 days, a high fever of about 40 degrees occurs, and severe muscle pain, headache, and joint pain appear. At the same time, a rash similar to a heat rash occurs around the arms, legs, and neck, and symptoms such as fatigue, nausea, and vomiting occur. It is difficult to discriminate because the clinical symptoms are similar to dengue. Currently, the situation cannot be prevented because there is no adequate treatment or vaccine.

Japanese Patent Publication No. 2012-504955 discloses a kit for diagnosing dengue virus comprising a recombinant antibody capable of binding to a surface antigen of dengue virus, and Korean Patent No. 10-169236 discloses a primer set for diagnosing Zika virus, A kit for diagnosing Zika virus including the same, and a method for diagnosing Zika virus using the same are disclosed.

However, the dengue, Zika, or chikunkunia virus is prevalent in the same area by the same type of mosquito, and because the infection symptoms are similar, it is difficult to accurately distinguish. In addition, it is difficult to accurately distinguish and diagnose infections of these viruses due to the similarity of the sequence. Currently, most antibody diagnostic kits use recombinant proteins to diagnose antibodies generated after infection, but in this case, there is a problem that cross-reactions between similar viruses appear.

Under this background, the present inventors have made diligent efforts to develop a method that does not show cross-reaction when diagnosing a mosquito-mediated virus, and as a result of sequencing, specific sequences that bind to the hydrophilic portion of the epitope in each virus of Dengue, Zika or Chikunkunia When a peptide for was selected and applied to an antibody diagnosis kit, it was confirmed that the virus can be diagnosed without cross-reaction with high sensitivity, and the present invention was completed.

The main object of the present invention is to provide an isolated peptide for mosquito-mediated virus-specific diagnosis, which is characterized in that no cross-reaction between mosquito-mediated viruses occurs when diagnosing a mosquito-borne virus.

Another object of the present invention is to provide a rapid diagnostic kit for specific diagnosis of a mosquito-mediated virus containing the isolated peptide.

Each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

In order to achieve the above object, one aspect of the present invention provides an isolated peptide for specific diagnosis of a mosquito-mediated virus, characterized in that no cross-reaction between mosquito-mediated viruses occurs when diagnosing a mosquito-mediated virus.

While the inventors of the present invention are conducting various studies to develop a method for diagnosing antibodies of mosquito-borne viruses with higher sensitivity, dengue, Zika, or chikunkunia viruses are diagnosed by accurately distinguishing infections of these viruses due to their sequence similarity. It was confirmed that it was not easy to do, and it was recognized that it is important to solve the problem of cross-reaction during diagnosis.

The term "mosquito-borne viruses" of the present invention refers to viruses that are transmitted through mosquitoes and cause disease in humans. The types include Japanese encephalitis virus of Flaviviridae, Dengue virus, West Nile virus, Yellow fever virus and Togaviridae. Chikungunya virus and the like. Specifically, dengue, zika, or chikunkunia virus is a mosquito-borne virus, and it is difficult to distinguish when diagnosed due to similarity in sequence. Therefore, for the purposes of the present invention, efforts were made to select a peptide that does not exhibit cross-reaction when diagnosing a mosquito-borne virus.

As an example of a virus belonging to the mosquito-borne virus, the "Flaviviridae" is a family of viruses, and uses mammals, including humans, as a natural host, and is mediated by insects such as ticks and mosquitoes. It has a propagating feature. Diseases caused by viruses belonging to the flavivirus family include hepatitis, hemorrhagic syndrome, abortion, mucosal disease, hemorrhagic fever, and encephalitis. Zika virus and the like, but are not limited thereto.

The term "cross-reaction" of the present invention means that an antibody can respond to the same epitope in different antigenic substances, and the term may be used interchangeably with cross-immune or cross-protective immunity. In most cases, multiple epitopes exist in one antigenic substance, which stimulates multiple immune responses.

In medical tests, including diagnostic tests, cross-reaction is often confused or helpful. For example, when a reaction occurs with an antigen other than the antigen of interest, it may cause disturbances that yield an error in a positive response, and antibodies for other antigens may be used to help detect other viruses.

For the purposes of the present invention, cross-reaction when diagnosing a mosquito-borne virus means that it is impossible to determine whether a virus has been infected among dengue, zika, or chikunkunia viruses by reaction with an antigen other than the antigen of interest. That is, dengue, zika, or chikunkunia viruses belong to mosquito-borne viruses, and their sequence is similar, so that cross-reaction may occur during diagnosis and may cause an error in positive reaction. Therefore, the present inventors first identified a peptide specific for dengue, zika, or chikunkunia viruses without causing a cross-reaction at the time of diagnosis.

In addition, for the purposes of the present invention, when diagnosing dengue virus, no cross-reaction with Zika or Chikunkunia virus does not appear, and when diagnosing Zika virus, no cross-reaction with dengue or Chikunkunia virus does not appear, and chikunkunia virus diagnosis Poetry may mean that there is no cross-reaction with Zika or Dengue bacteria.

The isolated peptide is characterized by having a sequence specific to a mosquito-mediated virus among the hydrophilic portions of the epitope.

The term "epitope" of the present invention refers to a site that determines antigen specificity present in an antigen molecule, and may be used interchangeably with an epitope or an epitope. For the purposes of the present invention, since the epitope directly binds to a water-soluble antibody, it is highly likely to be present in a portion with a high proportion of hydrophilic residues, so by analyzing the hydrophilicity of the epitope in the mosquito-mediated virus, dengue, zika or An epitope specific to chikunkunia virus was predicted. Specifically, a sequence portion specific to each virus among the epitopes of the virus was selected as a peptide through sequencing analysis, and as long as no cross-reaction occurs during diagnosis of Dengue, Zika, or Chikunkunia viruses, it is not limited thereto.

In a specific embodiment of the present invention, a sequence specific to dengue, zika, or chikunkunia was confirmed through multiple sequencing, and the epitope was analyzed to predict an antigenic determinant site specific to each virus (Fig. 1). ).

For the purposes of the present invention, the isolated peptide may be a peptide containing SEQ ID NO: 1 or SEQ ID NO: 2 when specific for dengue virus, and more specifically, may be a peptide of SEQ ID NO: 2, but Zika or Chikunkunia virus It is not limited thereto as long as it is a dengue virus-specific peptide that does not cause a cross-reaction upon diagnosis in a mosquito-borne virus such as.

In addition, when specific for Zika virus, it may be a peptide comprising SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, specifically, a peptide consisting of SEQ ID NO: 3, SEQ ID NO: 4 and/or SEQ ID NO: 5, sequence It may be a peptide consisting of No. 4 or SEQ ID No. 5. More specifically, it may be a peptide consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, but if it is a Zika virus-specific peptide that does not cause a cross-reaction during diagnosis in a mosquito-borne virus such as dengue or chikunkunia virus, it is limited thereto. It doesn't work.

In addition, if specific for chikunkunia virus, it may be a peptide comprising one of SEQ ID NO: 6 to SEQ ID NO: 11, and more specifically, it may be a peptide of SEQ ID NO: 9, but in a mosquito-mediated virus such as Zika or Dengue virus. It is not limited thereto as long as it is a Zika virus-specific peptide that does not cause a cross-reaction during diagnosis.

In a specific embodiment of the present invention, two dengue specific sequences selected in Example 1-1 (DenV-P1, DenV-P2), three Zika specific sequences (ZIKA-P1, ZIKA-P2, IKA-P3) Six chikungunia specific sequences (CKIK-P1, CKIK-P2, CKIK-P3, CKIK-P4, CKIK-P5, CKIK-P6) peptides were analyzed by ELISA using serum from 10 normal and infected samples. . As a result, when Dengue 2 (DenV-P2) peptide was used, Zika when 1 (ZIKA-P1), 2 (ZIKA-P2) and 3 (ZIKA-P3) peptide were used together, Chikungunia was confirmed to be able to effectively diagnose infection when using the No. 4 (CKIK-P4) peptide (Fig. 2).

From the above results, it was found that even if a peptide specific for dengue, Zika, or chikunkunia virus selected through multi-sequencing analysis, not all can be effectively used for infection diagnosis, only some specific sequences can be effectively used for infection diagnosis. I can.

In another aspect of the present invention, there is provided a rapid diagnostic kit for specific diagnosis of a mosquito-mediated virus, comprising the isolated peptide.

As a specific embodiment, the rapid diagnosis kit includes a buffer pad containing a buffer solution; Condensation pad; Specimen pad; A membrane on which the inspection line and the control line are immobilized; And an absorbent pad.

For example, the rapid diagnostic kit includes (i) an anti-human IgG-monoclonal antibody or a membrane immobilized with an anti-human IgM-monoclonal antibody; (Ii) a condensation pad comprising a gold condensate to which the isolated peptide is bound; (Iii) specimen pad; (Iv) a buffer pad containing a buffer solution; And (v) an absorbent pad. The rapid diagnosis kit as described above may be for diagnosing dengue virus.

Next, the membrane may have an anti-human IgG or anti-human IgM monoclonal antibody immobilized thereon. The anti-human IgG monoclonal antibody and anti-human IgM monoclonal antibody may play a role of binding by using an anti-viral antibody that may be contained in a specimen as an antigen at the time of diagnosis using a kit, depending on the type of virus. Either anti-human IgG monoclonal antibody (test line 1) or anti-human IgM monoclonal antibody (test line 2) may be included, or both anti-human IgG monoclonal antibody and anti-human IgM monoclonal antibody may be included. .

Specifically, in the case of Zika or Chikunkunia virus, one antibody may be immobilized on the membrane, and more specifically, an anti-human IgM monoclonal antibody may be immobilized. In addition, in the case of dengue virus, two antibodies may be immobilized on the membrane, and more specifically, both anti-human IgG monoclonal antibody and anti-human IgM monoclonal antibody may be immobilized, but Zika, Chikun It is not limited thereto as long as it can more sensitively diagnose the Kunia or Dengue virus.

As an example, each of the monoclonal antibodies can be used to determine whether the antibody is in the form of IgM or in the form of IgG when an antibody against the dengue virus is present in the patient's sample, so whether it is an antibody in the form of IgM against the dengue virus or an antibody in the form of IgG It can be used to confirm recognition, and by immobilizing each of the monoclonal antibodies to the membrane, it is possible to more effectively confirm whether an antibody against dengue virus is present in a sample of a patient. The content of the anti-human IgG-monoclonal antibody (test line 1) or anti-human IgM-monoclonal antibody (test line 2) included in the diagnostic kit is not particularly limited thereto, but preferably 0.1 to 5.0 mg/ml It can be immobilized by dispensing on the membrane at a concentration.

In addition, the membrane is used to bind each of the monoclonal antibodies, and the membrane is not particularly limited thereto, but preferably may be a nitrocellulose membrane to which the antibody can be bound, a polyvinylidene fluoride (PVDF) membrane, or the like.

Next, the condensation pad included in the kit may be configured to include a gold condensate in a form in which an isolated peptide specific for dengue, zika, or chikunkunia virus is bound to gold particles.

In addition, the isolated peptide of the present invention may be included in the condensation pad among the constitution of the rapid diagnosis kit of the present invention. That is, the condensation pad may have the isolated peptide of the present invention immobilized thereon. In addition, the isolated peptide may be included in the condensation pad in a condensed form with a gold condensate.

The gold particles bound to the isolated peptide specific for the dengue, Zika or chikunkunia virus are used as a labeling material for indicating the position of the isolated peptide specific for the dengue, Zika or chikunkunia virus. In the kit, it is finally used as a means to confirm the presence or absence of the antibody contained in the sample. The isolated peptide according to the present invention is not limited thereto as long as it is a peptide specific to the dengue virus, but specifically may be a peptide of SEQ ID NO: 2. In addition, the isolated peptide is not limited thereto as long as it is a Zika or chikunkunia virus-specific peptide, but specifically, the Zika virus-specific peptide may be SEQ ID NOs: 4 and 5, and more specifically, SEQ ID NOs: 3 and 4 And 5 may be reacted by bonding at the same time. In addition, the chikunkunia virus-specific peptide may be SEQ ID NO: 9, but is not limited thereto.

Next, the specimen pad according to the present invention may be made of a porous material, and may be configured to be positioned between the membrane and the antigen pad. The sample pad not only serves as an inlet through which samples (blood, whole blood, plasma, serum, etc.) obtained from individuals suspected of being infected with dengue virus are injected directly into the kit of the present invention, but also because of the porous material, the sample The blood cells included in the sample may be filtered through the sample pad, and may serve to transfer only liquid components included in the sample to the membrane. In this case, the porous material is not particularly limited thereto, but may be a porous alumina, porous silicate, porous synthetic resin, or the like including pores that do not allow blood cells to pass therethrough. In addition, since the sample pad serves to separate blood cells, it may be made of a material capable of adsorbing blood cells in addition to a porous material. The material capable of adsorbing such blood cells is not particularly limited thereto, but a nitrocellulose membrane, It may be a PVDF (polyvinylidene fluoride) membrane or the like.

Next, when using the kit of the present invention, the buffer pad according to the present invention may include a sample development solution containing a buffer solution for assisting the delivery of a sample in the kit and assisting the antigen-antibody reaction. It is made of a porous material, and may be configured to be connected to the condensation pad to be located at the end of the kit. The porous material is the same as defined in the sample pad.

The sample development solution may be present in the state contained in the buffer pad, or the sample development solution may be added to the buffer pad after adding the sample to the kit of the present invention, preferably after adding the sample to the kit of the present invention. The developing solution can be added to the buffer pad. The buffer solution included in the sample development solution is not particularly limited as long as it shows an effect of supporting sample delivery and antigen-antibody reaction, but a borate buffer solution, a Tris buffer solution, a phosphate buffer solution, and the like may be used. In addition, the sample development solution may contain Triton X-100, Tween 20, casein, etc., which can inhibit non-specific reactions, and sodium azide as a preservative.

Finally, the absorbent pad according to the present invention is made of a material capable of absorbing an excess sample or buffer, is connected to the membrane in the direction opposite to the sample pad, and is configured to be located at the end of the kit in the direction opposite to the buffer pad. . The absorbent pad absorbs and removes an excess sample or sample development solution, thereby preventing disturbance of the test result of the kit provided in the present invention, and the material of the absorbent pad is not particularly limited thereto, but preferably Cotton, liquid absorbent gel, or the like can be used.

On the other hand, the assay system for use in the diagnostic kit of the present invention is not limited thereto, but an ELISA plate, a deep-stick device, an immunochromatography method and a radiation split immunoassay device and a flow-through device, etc. Includes. Preferably, a diagnostic kit in the form of a strip or device using immunochromatography may be used. Diagnosis using immunochromatographic assay (ICA) is called rapid test because of its speed and simplicity, which is a monoclonal antibody against epidermal protein domain 1 of dengue virus condensed in colloidal gold particles. As the condensate moves, it binds to the dengue virus in the antigen pad. This complex reacts with the dengue antibody present in the sample (blood) and moves through the micropore of the nitrocellulose membrane by capillary action. In the meantime, it binds with anti-human IgG or anti-human IgM-monoclonal antibody that is immobilized on the inner surface of the micropores of the membrane to form a chromogenic band. By checking the degree of color development of this band, the presence or absence of the antibody, the type of the antibody, and the amount of the antibody can be visually determined.

In the diagnostic kit of the present invention, the antigen-antibody complex is detected by a color particle binding method, and the color particles include, for example, colloidal gold particles or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads ( bead). In the present invention, gold colloid is preferably used.

When a blood sample such as plasma, serum, whole blood, etc. derived from an individual suspected of having dengue, Zika, or chikunkunia virus infection is added to the sample pad included in the kit of the present invention, and the sample development solution is added to the buffer pad, the sample pad Anti-mosquito-mediated virus (dengue, zika, or chikunkunia) antibody in the sample is bound from the liquid component from which blood cells are separated, and the gold condensate separated from the condensation pad by the sample development solution is further bound, and gold-isolated peptide (Condensation pad)-An anti-mosquito-mediated virus (dengue, Zika, or chikunkunia) antibody complex is formed in the specimen, and then the antibody derived from the sample contained in the complex binds to test line 1 or 2, and the bound site The color development reaction occurs due to the gold particles.

If there is no antibody against dengue virus in the sample, a color reaction due to gold particles does not appear anywhere in test lines 1 and 2; If there is an IgG type antibody against dengue virus in the sample, a color reaction due to gold particles appears in test line 1; If there is an IgM type antibody against dengue virus in the sample, a color reaction due to gold particles appears in test line 2.

In addition, if there is no antibody against Zika or Chikunkunia in the sample, a color reaction due to gold particles does not appear anywhere in the test line; When an IgM type antibody against Zika or Chikunkunia virus is present in the sample, a color reaction due to gold particles appears in the test line.

Using the dengue virus rapid diagnostic kit of the present invention, it is possible to check whether an antibody against dengue virus is present in the sample, as well as if an antibody against dengue virus is present in the sample, the form of the antibody is IgM or IgG. You can check whether it is. In the diagnosis of dengue, it is important to determine the timing of infection. In other words, it is to judge the positive/negative as well as whether any kind of antibody (IgG or IgM) is present. The positive IgM and IgG antibodies to dengue in the initially collected blood means that you have been infected with the dengue virus within the last few weeks. If IgG is positive but IgM is very low or negative, it indicates a past infection. If the titer of the IgG antibody in the sample collected at the beginning and the sample collected after 2-4 weeks has risen 4 times or more, it is possible that the infection is recent. Negative IgM and IgG antibodies are not dengue infections and are likely symptoms of other causes, or if the antibodies are too low to measure. Thus, the possibility of dengue infection remains, as it has failed to produce measurable levels of antibodies measured early in viral exposure.

In addition, by using the Zika or Chikunkunia virus rapid diagnostic kit of the present invention, it can be confirmed whether an antibody against Zika or Chikunkunia virus is present in the sample. It is important to diagnose symptoms of Zika and Chikungunia. Therefore, Zikawa Chikungunia can confirm the presence of IgM antibodies against each virus.

In a specific embodiment of the present invention, as a result of performing a test using the kit of the present invention using the provided blood, the sensitivity was 96.7% for a dengue IgG positive sample, 96.5% for a dengue IgM positive sample, and the specificity was dengue. It was confirmed that it was 100% for the IgG-positive sample and 100% for the dengue IgM-positive sample. In addition, for the Zika IgM-positive sample, the sensitivity was 96.7% and the specificity was 98.7%, and for the Chikungunia IgM-positive sample, the sensitivity was 97.1% and the specificity was 98%.

In addition, as a result of conducting a comparative experiment with other companies' products, it was confirmed that it exhibited superior sensitivity (FIG. 6).

The above results show that the isolated peptide of the present invention is a peptide specific for dengue, Zika, or chikunkunia virus, without cross-reaction at the time of diagnosis, that is, without detecting other mosquito-borne viruses, and only the desired virus with specific high sensitivity. It suggests that it can be done.

As another aspect of the present invention, a method for diagnosing a mosquito-borne virus using the rapid diagnosis kit is provided.

The present invention is a technology for measuring antibodies in blood by binding some peptides of a mosquito-mediated virus with an antibody in a sample. It is much more sensitive than using conventional recombinant proteins or virus-like particles (VLPs). Make it measurable. In addition, when the isolated peptide is used, cross-reaction does not occur when diagnosing infection with dengue, zika, or chikunkunia virus, and thus the sensitivity and accuracy of the antibody diagnostic kit are significantly improved.

Figure 1 shows the selection of a specific sequence through multiple sequencing analysis of dengue virus and Zika virus
Figure 2 shows the selection of a peptide sequence through hydrophilic analysis of the sequence of dengue, Zika, and chikunkunia virus proteins.
3 shows the results of serological diagnosis using a peptide sequence.
4 is a schematic diagram of a kit for diagnosing a mosquito-borne virus.
Figure 5 is a diagram confirming the cross-reaction using a positive sample of dengue, zika or chikunkunia.
6 is a diagram confirming the sensitivity with other companies' products.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Dengue, Zika, Chikungunia Virus Sequence Analysis and Infection Diagnosis Peptide Selection

Example 1-1. Sequencing of Dengue, Zika and Chikungunia Viruses

Dengue, Zika and Chikungunia protein sequences were obtained from the National Center for Biological Information (www.ncbi.nlm.nih.gov). Dengue and Zika-specific sequences were identified through multiple sequencing analysis. Specifically, a part of the antigen recognized by an antibody is called an epitope, and there are numerous epitopes in one antigen. Since the epitope is directly bound to the water-soluble antibody, there is a possibility that a hydrophilic moiety occupies a high portion. Therefore, the present inventors analyzed the hydrophilicity portion to predict an epitope specific to each virus.

Among the hydrophilic portions of protein amino acids, a sequence portion specific to each virus was used as a peptide antigen, and specifically, two dengue specific sequences (DenV-P1: KGIMQAGKRSLRPQPTELKY (SEQ ID NO: 1), DenV-P2: LIDGPETAECPNTNRAWNSLEVEDY (SEQ ID NO: 2)) ) And three Zika specific sequences (ZIKA-P1: SVKNPMWRGPQRLPVPVNELPHGW (SEQ ID NO: 3), ZIKA-P2: AKTNNSFVVDGDTLKECPLEHR (SEQ ID NO: 4), ZIKA-P3: YSLECDPAVIGTAVKGREAAHSDLGYWIESEKNDTWR (SEQ ID NO: 5). Chikungunia is 6 (CKIK-P1: PMFEVEPRQVTSNDHANAR (SEQ ID NO: 6), CKIK-P2: QEIDPDSTILDIGSAPARRMMS (SEQ ID NO: 7)), CKIK-P3: MRSAEDPERLAN (SEQ ID NO: 8), CKIK-P4: RKLASAAGKVLDRNISGKIGDL (SEQ ID NO: 9), CKIK-P5: EVMAVPDTETPTFC (SEQ ID NO: 10), CKIK-P6: MAGAYPSYSTNWADEQVLK (SEQ ID NO: 11)).

Example 1-2. Serum diagnosis using peptide antigen (ELISA)

2 dengue specific sequences prepared in Example 1-1 (DenV-P1, DenV-P2), 3 Zika specific sequences (ZIKA-P1, ZIKA-P2, IKA-P3) 6 chikungunia specific sequences (CKIK- P1, CKIK-P2, CKIK-P3, CKIK-P4, CKIK-P5, CKIK-P6) peptides (peptron, Korea) were analyzed by ELISA. Specifically, the peptide antigen was diluted to 5 μg/ml using PBS, and each well of a 96 well plate (Nunc, Denmark) was coated with each type of peptide at a concentration of 100 μl/well.

The well plate was stored at 4° C. for one day, washed 3 times with 300 μl of PBS-T (1X PBS + 0.1% Tween-20), and blocked at room temperature for one hour with PBS to which 5% BSA was added, and PBS Washed again 3 times with -T.

The dengue samples for diagnosis were serum from 10 normal and 10 infected samples. The serum was diluted 1:50 with PBS, reacted for 1 hour at room temperature at a concentration of 100 μl/well, and washed three times with PBS-T. HRP-conjugated goat anti-human IgG (Sigma, USA) diluted 1:1000 was used as a secondary antibody to react. The secondary antibody was also reacted at room temperature for 1 hour, and then washed 3 times with PBS-T. For detection of the bound secondary antibody, after reacting at room temperature for 10 minutes with a TMB substrate (BioFX, SurModics, USA), 50 μl/well of 1N HCl was administered to terminate the reaction, and the reaction was measured at 450 nm to observe the reactivity.

As a result, when Dengue 2 (DenV-P2) peptide was used, Zika when 1 (ZIKA-P1), 2 (ZIKA-P2) and 3 (ZIKA-P3) peptide were used together, It can be seen that chikungunia can effectively diagnose infection when using the No. 4 (CKIK-P4) peptide (FIG. 3).

Example 2: Preparation of a rapid immunochromatographic diagnostic kit for diagnosing dengue, Zika, and chikungunia virus antibodies

Example 2-1. Composition of strip manufacturing and diagnostic kit

A buffer pad treated with a buffer solution from the bottom of the strip as shown in FIG. 4; A condensation pad in which a dengue, zika, or chikungunia recombinant antigen and gold condensate are condensed; A sample pad for injecting a sample; And the membranes to which the test line and the control line were attached/dispensed in order were sequentially attached, and an absorbent pad was attached to the top.

The strip prepared above was mounted on a plastic cassette and assembled to complete a diagnostic kit.

Example 2-2: Dispensing of antibodies to test lines and control lines for mosquito-borne virus diagnosis

Prepare anti-human IgG-monoclonal antibody and anti-human IgM-monoclonal antibody, and test line 1 (anti-human IgG) and test line 2 (anti-human IgM) sites at a concentration of 0.5 to 4.0 mg/ml on a nitrocellulose membrane. Was dispensed on. In addition, a goat anti-mouse IgG polyclonal antibody was purchased from Arista Biologicals Inc. (USA) at the control line position of the membrane, diluted with PBS at a concentration of 1.0 mg/ml, and dispensed.

Example 2-3: Preparation of gold condensate and condensation pad for diagnosis of mosquito-borne virus

When the 0.02% HAuCl ₄ solution began to boil while stirring and heating, a 0.2% sodium citrate solution was added to reduce the solution to prepare gold particles. To the gold particles thus prepared, the isolated peptides specific to dengue, zika, or chikunkunia selected in Example 1 were condensed by adding 1 mg per 100 ml of the gold particle solution. The condensed peptide-gold condensate was precipitated by centrifugation at 10,000 g and dissolved in physiological saline (PBS) containing 0.1% BSA to obtain an OD450 value of 10. The prepared solution was immediately treated on a polyester or glass pad pretreated with 0.05% Tween 20 and dried to complete a condensation pad.

Example 2-4: Buffer pad and absorbent pad

After treatment with 0.1 M Tris (pH 8.0) on a pad made of glass fiber, cotton or cellulose, it was dried and used as a buffer pad. As for the absorbent pad, a cotton pad was cut and used without any separate treatment.

Example 3: Test method of mosquito-mediated virus rapid immunochromatography diagnostic kit

Put 5 μl of human serum/plasma with viral antibody or 10 μl of typical type into the sample inlet, and add 3 drops (about 100 μl) of the reaction solution to the buffer pad. Then, the sample solution separates blood cells and moves to the membrane to react.

The diagnostic kit is a reaction solution that moves to a condensation pad to dissolve the peptide-gold condensate, and then moves to the membrane and "peptide-gold condensate-mosquito-mediated virus (dengue, Zika, or chikunkunia) in the sample) antibody-anti-human It reacts in the form of "IgG (or anti-human IgM)" and forms a colored band. On the other hand, the unreacted peptide-gold condensate reacts with the anti-mouse immunoglobulin G attached to the control line site, resulting in a colored band.

Example 4: Diagnostic efficacy test

For the diagnostic efficacy test (sensitivity and specificity) of the present invention, 660 blood (dengue IgG positive 320, dengue IgM positive 190 and dengue negative 150) from General Hospital Kuala Lumpur (GHKL, Malaysia), Hospital Gaffree e 250 blood samples (Zika IgM positive 150, Chikungunia IgM positive 100) and 300 negative samples were provided by Dankook University Hospital (Cheonan, Korea) from Guinle (Rio de Janeiro, Brazil). All of these specimens were confirmed as positive and negative by ELISA.

Using the blood thus provided, a test using the kit of the present invention was performed, and as shown in Table 1, the sensitivity was 96.7% for the Dengue IgG-positive sample, 96.5% for the Dengue IgM-positive sample, and the specificity was for the Dengue IgG-positive sample. It was confirmed that it was 100% and 100% for the dengue IgM positive sample. In addition, for the Zika IgM-positive sample, the sensitivity was 96.7% and the specificity was 98.7%, and for the Chikungunia IgM-positive sample, the sensitivity was 97.1% and the specificity was 98%.

Dengue IgG/IgM Zika IgM Chikungunya IgM responsiveness IgG: 96.7% IgM: 96.5% responsiveness 96.7% responsiveness 97.1% Specificity IgG: 100% IgM: 100% Specificity 98.7% Specificity 98.0% Detection limit IgG: 1.1s/CO IgM: 1.2s/CO Detection limit 3.41s/CO Detection limit 1.25s/CO

These results showed no cross-reaction between viral infections, and are the best results among the internationally reported Zika rapid diagnostic kits.

Example 5: Confirmation of sensitivity with other companies' products

In order to check the sensitivity of the kit developed through this study, a comparative experiment with other companies' products was conducted. Specifically, when comparing the Dengue IgG/IgM kit and other company's product (SD), the sensitivity was equal to or higher than that. In particular, it was possible to confirm the superior analysis ability in the detection of IgM antibodies. Chikungunia IgM kit also showed a sensitivity equal to or higher than that of other companies' products (CTK, J. Mitra) (FIG. 6).

The results show that the isolated peptide of the present invention is a peptide specific for dengue, Zika, or chikunkunia virus, without cross-reaction at the time of diagnosis, that is, without detecting other mosquito-mediated viruses, and only the desired virus with specific high sensitivity. It suggests that it can be done.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

<110> GenBody Inc. <120> Peptides for diagnosis of mosquito-borne viruses and uses thereof <130> KPA170715-KR-D1 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> DenV-P1 <400> 1 Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln Pro Thr 1 5 10 15 Glu Leu Lys Tyr 20 <210> 2 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> DenV-P2 <400> 2 Leu Ile Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala 1 5 10 15 Trp Asn Ser Leu Glu Val Glu Asp Tyr 20 25 <210> 3 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> ZIKA-P1 <400> 3 Ser Val Lys Asn Pro Met Trp Arg Gly Pro Gln Arg Leu Pro Val Pro 1 5 10 15 Val Asn Glu Leu Pro His Gly Trp 20 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> ZIKA-P2 <400> 4 Ala Lys Thr Asn Asn Ser Phe Val Val Asp Gly Asp Thr Leu Lys Glu 1 5 10 15 Cys Pro Leu Glu His Arg 20 <210> 5 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> ZIKA-P3 <400> 5 Tyr Ser Leu Glu Cys Asp Pro Ala Val Ile Gly Thr Ala Val Lys Gly 1 5 10 15 Arg Glu Ala Ala His Ser Asp Leu Gly Tyr Trp Ile Glu Ser Glu Lys 20 25 30 Asn Asp Thr Trp Arg 35 <210> 6 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CKIK-P1 <400> 6 Pro Met Phe Glu Val Glu Pro Arg Gln Val Thr Ser Asn Asp His Ala 1 5 10 15 Asn Ala Arg <210> 7 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CKIK-P2 <400> 7 Gln Glu Ile Asp Pro Asp Ser Thr Ile Leu Asp Ile Gly Ser Ala Pro 1 5 10 15 Ala Arg Arg Met Met Ser 20 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CKIK-P3 <400> 8 Met Arg Ser Ala Glu Asp Pro Glu Arg Leu Ala Asn 1 5 10 <210> 9 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> CKIK-P4 <400> 9 Arg Lys Leu Ala Ser Ala Ala Gly Lys Val Leu Asp Arg Asn Ile Ser 1 5 10 15 Gly Lys Ile Gly Asp Leu 20 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CKIK-P5 <400> 10 Glu Val Met Ala Val Pro Asp Thr Glu Thr Pro Thr Phe Cys 1 5 10 <210> 11 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> CKIK-P6 <400> 11 Met Ala Gly Ala Tyr Pro Ser Tyr Ser Thr Asn Trp Ala Asp Glu Gln 1 5 10 15 Val Leu Lys

Claims (9)

SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
A composition for diagnosing a chica virus infection disease comprising the peptide of claim 1.
A rapid diagnostic kit for diagnosing a chikavirus infectious disease, comprising the peptide of claim 1 or the composition of claim 2.
The method of claim 3,
Wherein the rapid diagnostic kit comprises: a buffer pad comprising a buffer solution; Condensation pad; Sample pad; A membrane on which an inspection line and a reference line are immobilized; And an absorbent pad.
The method of claim 3,
Wherein the condensation pad of the rapid diagnostic kit comprises an isolated peptide.
6. The method of claim 5,
Wherein the isolated peptide is condensed with a gold co-polymer.
5. The method of claim 4,
Wherein the membrane is immobilized with an anti-human IgG or anti-human IgM monoclonal antibody on the test line.
The method of claim 3,
Wherein the rapid diagnostic kit is in the form of a strip using immunochromatography.
A method for providing information necessary for diagnosis of chicarvirus infection disease using the rapid diagnosis kit of claim 3.

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