METHOD FOR EVALUATING SEVERITY OF DENGUE VIRUS INFECTION IN INDIVIDUAL, DETECTION DEVICE AND DETECTION

Abstract

Provided is a method for evaluating the severity of the dengue virus infection in an individual, executed by means of detecting a biological sample of an individual in vitro. The method comprises: detecting whether a non-structural protein 1 is present in a biological sample in vitro to obtain a first detection result; detecting whether a complex is present in the biological sample in vitro to obtain a second detection result, wherein the complex comprises the non-structural protein 1 and a thrombin, or comprises the non-structural protein 1 and a prothrombin; and evaluating the severity of the dengue virus infection in the individual via the first detection result and the second detection result. Also provided are a detection device and a detection kit for evaluating the severity of the dengue virus infection in an individual.

Claims

1. A method for evaluating the severity of the dengue virus infection in an individual, which is executed by means of detecting an in vitro biological sample of an individual, and comprises: detecting whether a non-structural protein 1 is present in an in vitro biological sample to obtain a first detection result; detecting whether a complex is present in the in vitro biological sample to obtain a second detection result, wherein the complex contains the non-structural protein 1 and thrombin, or contains the non-structural protein 1 and prothrombin; and evaluating the severity of the dengue virus infection in the individual via the first detection result and the second detection result.

2. The method of claim 1, wherein the individual belongs to a group B patient with dengue virus infection or a group C patient with dengue virus infection if one of the complex containing the non-structural protein 1 and thrombin and the complex containing the non-structural protein 1 and prothrombin as well as the non-structural protein 1 are simultaneously present in the biological sample.

3. The method of claim 1, wherein the step of detecting whether the non-structural protein 1 is present in the in vitro biological sample is carried out by using an antibody specifically recognizing the non-structural protein 1.

4. The method of claim 1, wherein the step of detecting whether the complex is present in the in vitro biological sample is carried out by using an antibody specifically recognizing the complex.

5. The method of claim 4, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

6. The method of claim 1, wherein the biological sample includes one of followings: blood, urine, saliva and lymph.

7. The method of claim 1, wherein the individual is a mammal.

8. The method of claim 7, wherein the mammal is a human.

9. The method of claim 1, wherein the non-structural protein 1 is a secreted protein or a membrane-associated protein.

10. The method of claim 1, wherein the detection is carried out by using any one of following methodologies: an enzyme linked immunosorbent assay (ELISA), dot blotting, lateral flow assay (LFA), multiplex immunoassay, radioimmunoassay (RIA), immunoradiometric assay (IRMA), fluorescent immunoassay (FIA), chemiluminescent immunoassay and immunonephelometry.

11. A detection device for evaluating the severity of the dengue virus infection in an individual, comprising: a first detection unit for detecting whether a non-structural protein 1 is present in an in vitro biological sample to obtain a first detection result; and a second detection unit for detecting whether a complex is present in the in vitro biological sample to obtain a second detection result, wherein the complex contains the non-structural protein 1 and thrombin, or contains the non-structural protein 1 and prothrombin, wherein the severity of the dengue virus infection in the individual is evaluated via the first detection result and the second detection result.

12. The detection device of claim 11, wherein the biological sample includes one of followings: blood, urine, saliva and lymph.

13. The detection device of claim 11, wherein the individual is a mammal.

14. The detection device of claim 13, wherein the mammal is a human.

15. The detection device of claim 11, wherein the individual belongs to a group B patient with dengue virus infection or a group C patient with dengue virus infection if one of the complex containing the non-structural protein 1 and thrombin and the complex containing the non-structural protein 1 and prothrombin as well as the non-structural protein 1 are simultaneously present in the biological sample.

16. The detection device of claim 11, wherein the non-structural protein 1 is a secreted protein or a membrane-associated protein.

17. A detection kit comprising the detection device of claim 12.

18. The method of claim 3, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

19. The detection kit of claim 17, wherein the individual belongs to a group B patient with dengue virus infection or a group C patient with dengue virus infection if one of the complex containing the non-structural protein 1 and thrombin and the complex containing the non-structural protein 1 and prothrombin as well as the non-structural protein 1 are simultaneously present in the biological sample.

20. The detection kit of claim 17, wherein the non-structural protein 1 is a secreted protein or a membrane-associated protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows a flowchart of a method for evaluating the severity of the dengue virus infection in an individual according to the present invention; and

[0038] FIG. 2 shows a schematic diagram of a preferred embodiment according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention is further illustrated by the following embodiments and examples. However, it should be understood that the embodiments and examples are only for illustrative purposes and in no way a limitation of the present invention.

Embodiments

[0040] An embodiment of the present invention will be described below with reference to the related drawings. The embodiment describes a method for evaluating the severity of the dengue virus infection in an individual with high sensitivity and high accuracy. Said method executed by means of detecting an in vitro biological sample of an individual. The flowchart of said method is shown in FIG. 1, and the steps comprise: detecting whether a non-structural protein 1 (NS1) is present in an in vitro biological sample to obtain a first detection result (Step S01); detecting whether a complex is present in the in vitro biological sample to obtain a second detection result, wherein the complex contains NS1 and thrombin or NS1 and prothrombin (Step S02); and evaluating the severity of the dengue virus infection in the individual via the first detection result and the second detection result (Step S03).

[0041] In the Step S01 of this embodiment, the in vitro biological sample is obtained from the individual with or suspected of having dengue virus infection. In the examples of the present invention, the individual is a human. In the examples of the present invention, the biological sample is a serum.

[0042] In the Step S02 of this embodiment, NS1 and thrombin or NS1 and prothrombin conjugated mutually. That is, both NS1 and thrombin or both NS1 and prothrombin have positions capable of interaction or covalent bonding. In the embodiment of the present invention, NS1 and thrombin or NS1 and prothrombin can also indirectly conjugate via other molecules. In the embodiment of the present invention, the serum has multiple complexes, and the partial complexes contain NS1 and thrombin and the partial complexes contain NS1 and prothrombin. In the embodiment of the present invention, the complexes can simultaneously contain NS1 and thrombin as well as NS1 and prothrombin, and the numbers of NS1, thrombin and prothrombin are not limited. The mutual conjugating relation may also be the mutual conjugation of one NS1 and two thrombin or prothrombin, or the mutual chimerism of two or more NS1 and one thrombin or one prothrombin. The invention is not limited herein. In an example of the present invention, the form of NS1 include, but are not limited to, a secreted protein secreted to out of cell, and a membrane-associated protein binding on the surface of host cell. In another example of the present invention, NS1 can be in the form without post-translational modification, or in the form of post-translational modification, such as glycosyation, phosphorylation and the like. In an example of the present invention, the thrombin or prothrombin can be isolated or purified from an organism, or be a functional protein made by artificial synthesis. These separation, purification and artificial synthesis techniques can be understood by a person ordinarily skilled in the art.

[0043] In the Step S03 of this embodiment, if the first detection result is that NS1 is present in the in vitro biological sample as well as the second detection result is that the complex is present in the in vitro biological sample, that is, if one of the complex containing NS1 and thrombin and the complex containing NS1 and prothrombin as well as NS1 are simultaneously present in the biological sample, it indicates that the individual belongs to a group B patient with dengue virus infection or a group C patient with dengue virus infection. In an example of the present invention, the first detection result is that NS1 is present in the in vitro biological sample, and the second detection result is that the complex containing NS1 and thrombin is present in the in vitro biological sample.

[0044] The present invention also provides another embodiment, which relates to a detection device. The detection device is used for evaluating the severity of the dengue virus infection in an individual. The detection device comprises a first detection unit and a second detection unit. The first detection unit is used for detecting whether NS1 is present in an in vitro biological sample to obtain a first detection result, and the second detection unit is used for detecting whether a complex is present in the in vitro biological sample to obtain a second detection result, wherein the complex contains NS1 and thrombin, or contains NS1 and prothrombin. Then, the severity of the dengue virus infection in the individual is evaluated via the first detection result and the second detection result.

[0045] In this embodiment, the source of the in vitro biological sample is as mentioned in the Step S01 of the above embodiment.

[0046] In this embodiment, the relation of NS1 and thrombin or NS1 and prothrombin is as mentioned in the Step S02 of the above embodiment.

[0047] In this embodiment, if the first detection result is that NS1 is present in the in vitro biological sample as well as the second detection result is that the complex is present in the in vitro biological sample, the indicated meaning is as mentioned in the Step S03 of the above embodiment.

[0048] The present invention also provides a further embodiment, which relates to a detection kit. Said detection kit comprises the above-mentioned detection device. In addition, the constitution of the detailed components of the detection device, variation form and connection relation with other components are the same as mentioned in the above embodiment, and are not elaborated any further here.

[0049] Hereinafter, the present invention will provide a representative example by using serum from patients with dengue virus infection, and illustrate a method for evaluating the severity of the dengue virus infection in an individual according to the present invention in order to supplement to the above description and illustrate that the method of the present invention has high sensitivity and high accuracy at the same time. However, it should be noted that the following descriptions are used to describe the present invention in detail in order to be practiced by a person ordinarily skilled in the art, but not to limit the scope of the present invention.

Example the Evaluating Method of the Present InventionSensitivity Test for Evaluating the Severity of the Dengue Virus Infection in an Individual

Experimental Materials: Preparation of the Individual Serum Sample

[0050] All serum samples donated by the experimental individuals participating in this study are obtained after getting their informed consents. A total of 26 human individuals participated in this study, and all of them were confirmed diagnosed as patients with dengue virus infection by the Centers for Disease Control (CDC, Taiwan). The serum samples are obtained from these patients by collecting blood from the blood vessel of each patient via needles and then centrifuging at 2500 rpm for 25 minutes at room temperature.

Experimental Methods

[0051] This experiment was performed using the SD BIOLINE Dengue Duokit (Standard diagnostic Inc.) and following the manufacturer's operating instructions. First, the rapid tests used to perform this experiment were divided into six groups including one experimental group and five comparison groups, that is, Comparison groups 1 to 5. Each rapid test group includes a control line marked as C. The rapid tests of Comparison groups 1 and 5 includes test lines marked as G and M, wherein the test line marked as G contained anti-human IgG antibodies as capture antibodies, and the test line marked as M contained anti-human IgM antibodies as capture antibodies, and the colloidal gold pad contained membrane protein of dengue virus-colloidal gold. Comparison groups 2, 3, 4 and 5 and Experimental group contained test lines marked as T, which contained 1 L of mouse anti-NS1 monoclonal antibodies (Cat. 12100/12110, Leadgene Biomedical Inc., Taiwan) as capture antibodies, and the colloidal gold pad contained mouse anti-NS1 monoclonal antibody-colloidal gold. Moreover, the colloidal gold pads of Comparison groups 3, 5 and Experimental group further contained 1 L of sheep anti-thrombin polyclonal antibodies as detecting antibodies in addition to the colloidal gold-labeled mouse anti-NS1 monoclonal antibodies. Subsequently, 80 L of serum sample was added to a sample pad set in each group of rapid tests and reacted. At the 15th minute of the reaction, the results were interpreted with the naked eyes.

Experimental Results

[0052] FIG. 2 is a schematic diagram of each group of rapid tests, wherein the meanings of the symbols of the rapid tests have been described in the Experimental Methods section of this example, and are not elaborated any further here. Table 1 is a statistical table of the data created after performing the experiment according to the above-mentioned Experimental Method and interpreting the results according to the schematic diagram of FIG. 2 (see below).

TABLE-US-00001 TABLE 1 Group No. Detected Ag or Ab Sensitivity Comparison Anti-dengue IgG antibody and/or 9/26 (34.61%) group 1 anti-dengue IgM antibody Comparison NS1 19/26 (73.07%) group 2 Comparison Complex containing NS1 and 22/26 (84.61%) group 3 thrombin Comparison NS1 and anti-dengue IgG antibody 23/26 (88.46%) group 4 and/or anti-dengue IgM antibody Comparison Complex containing NS1 and 23/26 (88.46%) group 5 thrombin and anti-dengue IgG antibody and/or anti-dengue IgM antibody Experimental NS1 and complex containing NS1 25/26 (96.15%) group and thrombin

[0053] As shown in FIG. 2 and Table 1, for Comparison group 1, the anti-dengue IgG antibody and/or anti-dengue IgM antibody were detected in the serum samples from 9 of 26 patients with dengue virus infection, and the sensitivity was 34.61%. For Comparison group 2, NS1 was detected in the serum samples from 19 of 26 patients with dengue virus infection, and the sensitivity is 73.07%. For Comparison group 3, the complex containing NS1 and thrombin was detected in the serum samples from 22 of 26 patients with dengue virus infection, and the sensitivity was 84.61%. For Comparison group 4, NS1 and anti-dengue IgG antibody and/or anti-dengue IgM antibody were detected in the serum samples from 23 of 26 patients with dengue virus infection, and the sensitivity was 88.46%. For Comparison group 5, the complex containing NS1 and thrombin and anti-dengue IgG antibody and/or anti-dengue IgM antibody were detected in the serum samples from 23 of 26 patients with dengue virus infection, and the sensitivity was 88.46%. For Experimental group, NS1 and complex containing NS1 and thrombin were detected in the serum samples from 25 of 26 patients with dengue virus infection, and the sensitivity was 96.15%. The results of this example indicate: the method according to the present invention may accurately detect the dengue virus infection in an individual, and thus have high sensitivity and high accuracy. In particular, the applicant found that the individual belongs to a group B patient with dengue virus infection or a group C patient with dengue virus infection if NS1 and the complex containing NS1 and thrombin are simultaneously present in the serum sample of patient with dengue virus infection (not shown in data). Therefore, the applicant believes that the method according to the present invention can be used to evaluate the severity of dengue virus infection in an individual, and has high sensitivity and high accuracy.

[0054] The above description is only exemplary rather than restrictive. Any equivalent modification or change without departing from the spirit and scope of the present invention shall be included in the scope of the appended claims.