KR101588582B1 - Biomarker composition for diagnosing sepsis comprising Ly6C cell surface antigen and method for diagnosing sepsis using the same marker - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing septicemia, comprising Ly6C cell surface antigen, and to a septicemia diagnostic method using the same. More specifically, the present invention relates to a biomarker composition for diagnosing septicemia using Ly6C cell surface antigen expressed in blood cells in an animal model with cecal ligation and puncture (CLP)-induced septicemia. The present invention further relates to a septicemia diagnostic method using the same. According to the present invention, the expression of Ly6C on a surface of white blood cell existing in blood after induction of CLP increases in accordance with the progression of septicemia, and it has been found that 46.3% out of the total blood cells expressed Ly6C in 48 hours. Therefore, Ly6C cell surface antigen of the present invention can be used as a useful biomarker for diagnosing septicemia.

Description

Biomarker composition for the diagnosis of sepsis including Ly6C cell surface antigens and method for diagnosing sepsis using the same

The present invention relates to a biomarker composition for diagnosing sepsis including Ly6C cell surface antigen expressed on hemocyte cells of an animal model of cecal ligation and puncture (CLP) induced sepsis, and a method for diagnosing sepsis using the same.

Sepsis is a disease in which a germ enters the blood and shows signs of poisoning by the toxin produced by it, or causes an infection in the whole body. Although the statistics of domestic sepsis mortality are not known precisely, in the United States, more than 750,000 deaths have been reported annually, and the annual death toll is 210,000. This accounted for 9.3% of all deaths and was close to mortality due to acute myocardial infarction and was the most common cause of death among noncardiovascular patients. This result has a great effect on the increase of the medical expenses. In the case of the US in 1995, the per capita cost of the patients with sepsis was 22,100 US dollars. This represents a total medical expenditure of $ 16.7 billion annually, which is expected to increase by more than 1.5 percent annually until 2020, and the enormous cost of medical care is expected. Clinically, sepsis is known to cause metabolic disturbance, deterioration of tissue function, perfusion disorder, resulting in systemic tissue perfusion disturbance and ultimately resulting in multiple organ failure. Despite the fact that many studies have been carried out on these symptoms, the mortality rate of sepsis patients has remained at 30% per year for the past decade, and the existing studies are not helping to improve the disease. Therefore, the identification of early diagnosis markers of sepsis is very important to understand and cope with the progress of the disease. Various methods have been used in sepsis studies since the 1960s. Since the animal model of infection induced by double cecal ligation and puncture (CLP) shows very similar results to human sepsis, this method has been used by many researchers for many years as an animal model of sepsis studies come. Therefore, the present invention aims to develop a disease marker that exists in the blood after inducing CLP induced sepsis in a mouse sepsis model.

Japanese Patent Application Laid-Open No. 1998-179163 (July 7, 1998)

It is an object of the present invention to provide a biomarker composition for diagnosing sepsis including Ly6C cell surface antigen and a method for diagnosing sepsis using the same.

The present invention provides a biomarker composition for diagnosing sepsis including Ly6C cell surface antigen.

The present invention also provides a sepsis diagnosis kit comprising an antibody or an aptamer that specifically binds to a Ly6C cell surface antigen.

In addition, the present invention provides a method for providing information necessary for diagnosis of sepsis, comprising the step of measuring the expression level of Ly6C cell surface antigen from a sepsis patient sample.

The present invention relates to a biomarker composition for diagnosing septicemia comprising Ly6C cell surface antigen and a method for diagnosing sepsis using the same, and more particularly, to a method for diagnosing septicemia using Ly6C cell surface antigens, Biomarker composition for the diagnosis of sepsis using Ly6C cell surface antigen and a method for diagnosing sepsis using the same. According to the present invention, the expression of Ly6C on the surface of leukocyte cells present in the blood after CLP induction increased with the progression of sepsis, and 46.3% of the whole blood cells were expressed at 48 hours. Thus, Ly6C cell surface antigens can be used as useful biomarkers for the diagnosis of sepsis.

Figure 1 shows the results of CLP induced mice and survival analysis. Anesthetized by intraperitoneal injection of ketamine (80 mg / kg) and xylazine (12 mg / kg) in C57BL / 6 mice (23 g / In the control group, the cecum was removed after 1 cm incision in the abdominal wall, and then inserted into the abdominal cavity again without any treatment. In the CLP-guided group, 50% of the cecum was ligated in the immediate lower chamber of the ileocecal valve after the cecum was drained, and once through the 21-gauge needle, Then, it was inserted into the abdominal cavity again. The abdominal wall muscles and skin layers were sutured and subcutaneously administered with physiological saline (1 ml / mouse). After the surgery, analgesic (diclofenac sodium) was injected into the buttocks, put in a breeding facility, and water and feed were given and the survival rate was observed at the time described.
FIG. 2A shows the blood levels of CLP-induced mice according to the time of day described, and the degree of Ly6C expressed on the surface of white blood cells was analyzed using a flow cytometer. FIG. 2B is a schematic diagram of the results obtained three times.
FIG. 3A shows the blood of CLP-induced mice according to the time zone described, and the degree of Ly6-G expressed on the surface of leukocyte cells was analyzed using a flow cytometer. FIG. 3B shows the results obtained by performing the triplet operation.
Figure 4A shows the spleen of CLP-induced mice according to the time periods described and analyzed morphological changes. FIG. 4B shows the results of analysis of the degree of Ly6C and Ly6-G expressed on the surface of splenocyte cells separated according to the time zone described using a flow cytometer.

The present invention provides a biomarker composition for diagnosing sepsis including Ly6C cell surface antigen. Specifically, the sepsis may be cecal ligation and puncture (CLP) induced, but not limited thereto.

The "Ly6C" of the present invention is also known as Ly6c, Ly-6C and Ly-6C1, and NCBI accession no. XM_006520521 (AA682074, AA959465), but is not limited thereto.

The term " diagnosing " herein is used to determine the susceptibility of an object to a particular disease or disorder, to determine whether an object currently has a particular disease or disorder, Determining the prognosis of the object, or therametrics (e.g., monitoring the status of the object to provide information about the therapeutic efficacy).

Also provided is a kit for diagnosing sepsis comprising an antibody or an aptamer that specifically binds to the Ly6C cell surface antigen of the present invention.

The term " antibody " as used herein refers to a specific immunoglobulin as indicated in the art and directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to Ly6C cell surface antigen as a biomarker for sepsis diagnosis of the present invention, and an antibody can be produced according to a conventional method in the art. The forms of the antibodies include polyclonal or monoclonal antibodies, including all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes a special antibody such as a humanized antibody.

Alternatively, an aptamer that specifically binds to the biomarker of the present invention may be used in place of the antibody of the present invention, and the aptamer may be an oligonucleotide or a peptide molecule.

In addition, the kit of the present invention comprises an antibody specifically binding to a marker component, a secondary antibody conjugate conjugated with a label that develops upon reaction with the substrate, a coloring substrate solution to be colored with the label, A reaction stop solution, and the like, and may be manufactured from a number of separate packaging or compartments including the reagent components used.

The marker of the secondary antibody conjugate is preferably a conventional coloring agent that undergoes a chromogenic reaction and is preferably selected from the group consisting of HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein fluorescein such as isothiocyanate and rhodamine-B-isothiocyanate, and dye may be used.

Also, the present invention provides a method for detecting the expression level of Ly6C cell surface antigen, comprising: measuring the expression level of Ly6C cell surface antigen from a sepsis patient sample; Comparing the expression level of the Ly6C cell surface antigen with a normal control sample; And determining that the expression level of the Ly6C cell surface antigen of the patient sample is higher than that of the normal control sample.

Specifically, the expression level of the Ly6C cell surface antigen can be measured through an antigen-antibody reaction, and more particularly, the antigen-antibody reaction can be carried out according to various quantitative or qualitative immunoassay protocols conventionally developed have. The immunoassay format may be selected from the group consisting of enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining, flow cytometry but are not limited to, fluorescence activated cell sorting (FACS), enzyme substrate staining, and antigen-antibody aggregation.

As used herein, the term " patient " refers to a subject suspected of having sepsis and includes any individual whose expression of Ly6C cell surface antigen changes due to sepsis, preferably a human, or a rodent including a mouse.

As used herein, the term " patient sample " refers to a sample, such as a tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differs from a normal control in expression level of a Ly6C cell surface antigen as a biomarker for sepsis diagnosis But is not limited thereto. Preferably, the sample of the patient is a sample obtained from blood.

Hereinafter, the present invention will be described in detail with reference to examples which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

< Example  > CLP  Sepsis in an animal model of induced sepsis Diagnostic marker  excavation

1. Antibody

PE-conjugated anti-mouse Ly-6G and PE-conjugated anti-mouse Ly-6C were purchased from BD Pharamingen (San Diego, USA).

2. Experimental animals

The experimental design of 8-week-old female C57BL / 6 mice used in this invention was approved by the animal laboratory of Inje University College of Medicine (approval No. 2013-048) and purchased from Daejeon, Korea.

3. CLP Induced Sepsis Animal Model

The control group was anesthetized by injecting ketamine (80 mg / kg) and xylazine (12 mg / kg) in the abdominal cavity of C57BL / 6 mice (23 g / mouse). The abdominal wall was incised by 1 cm, and the cecum was removed and inserted into the abdominal cavity again without any treatment. The abdominal wall muscles and skin layers were sutured and subcutaneously administered with physiological saline (1 ml / mouse). The CLP induction group was anesthetized by injecting a mixture of ketamine (80 mg / kg) and xylazine (12 mg / kg) in the abdominal cavity of C57BL / 6 mice (23 g / mouse). After a 1 cm incision in the abdominal wall, the cecum was removed and a 50% cecum was ligated in the immediate lower chamber of the ileocecal valve. After a single pass through the 21-gauge needle, After treatment, it was inserted into the abdominal cavity again. The abdominal wall muscles and skin layers were sutured and physiological saline (1 ml / mouse) was subcutaneously administered. After the operation, analgesic (diclofenac sodium) was injected into the buttocks, put into a breeding facility, and water and feed were observed.

4. Immunocytochemistry of mouse blood using concentration gradient centrifugation

Blood is collected in heparin tubes and blood is diluted with PBS. 4 ml of diluted blood is slowly added to 2 ml of Histopaque-1077 (Sigma-Aldrich) to make a concentration gradient. Centrifuge the sample tube by setting the centrifuge to 400 g, 20 min, 20 ° C, No brake. Peripheral blood mononuclear cells, which are centrifuged in a new tube, are transferred using a pasteur pipette equipped with a syringe rubber. After 10% RPMI medium is filled, set it to 1700rpm, 5min, 20 ℃, brake and centrifuge. After centrifugation, the supernatant is removed and the remaining pellet is obtained. Suspend with FACS buffer and count the number of cells.

5. Flow cytometry

Blood was collected under various experimental conditions and the cells were washed with FACS buffer (PBS containing 1% FCS and 0.1% NaN ₃ ) and blocked with Fc blocker (mIgG, Sigma-Aldrich) for 15 minutes at 4 ° C to prevent nonspecific binding Respectively. After incubation for 20 min at 4 ° C with PE, FITC-conjugated antibody was analyzed by FACSort and CellQuestPro software (BD science, USA).

6. Results

In this experiment, we observed the mortality of C57BL / 6 mice in inducing CLP. The survival rate of 10 animals in the control group was 100% for 4 days and survived after that. In contrast, the survival rate was 100% for 12 hours, 75% for 24 hours, 43% for 48 hours and 37% for 72 hours among 30 CLP-induced mice (FIG. 1). Based on these data, the changes of cytokines in the serum after the blood of the mice were obtained, and the changes of cell surface antigens were analyzed by flow cytometry.

Expression of Ly6C on the hemocyte surface showed various expression with time in the CLP-induced group (Fig. 2A). Expression of Ly6C in the control group was increased to 13% at 12 hours, 27% at 24 hours, 46% at 48 hours, and 33% at 72 hours when CLP was induced 2B). On the other hand, the expression of Ly6G on the surface of the blood cells was remarkably low (FIG. 3A). Expression of Ly6G was about 1.6% in the control group, and slightly increased to 2% at 12 hours, 7.7% at 24 hours, and 17.9% at 48 hours when CLP was induced, and decreased to 13% at 72 hours (Fig. 3B). Analysis of the spleen of CLP-induced mice showed no difference in size and number of cells between the control and CLP-induced mice (Fig. 4A). In addition, splenocytes were isolated and analyzed for the expression of Ly6C and Ly6G, but there was no difference in the expression of these cell surface factors between the control and CLP-induced groups (FIG. 4B). In conclusion, the expression of Ly6C on the surface of leukocyte cells in the blood after CLP induction increased with the progression of sepsis, and 46.3% of the whole blood cells were expressed at 48 hours.

7. Conclusion

The mortality rate of the CLP induced sepsis animal model increased with disease progression and the survival rate was 37% for 4 days (96 hours). The cytokines in the serum of CLP induced animals showed very similar changes to the known results with progress of the disease. Expression of Ly6C increased 1.38 times at 12 hours and 2.88 times at 24 hours and increased to 4.98 times at 48 hours, which corresponds to 57% of deaths. Therefore, the expression of Ly6C was found to increase in blood cells in the blood with progress of sepsis.

Claims (7)

A biomarker composition for the diagnosis of sepsis comprising Ly6C cell surface antigens expressed in hemocyte cells. The biomarker composition for diagnosing sepsis according to claim 1, wherein the sepsis is induced by cecal ligation and puncture (CLP). A kit for diagnosing sepsis comprising an antibody or an aptamer that specifically binds to a surface antigen of Ly6C cells expressed in a hemocyte. 4. The kit for diagnosing septicemia according to claim 3, wherein the antibody is a monoclonal antibody or a polyclonal antibody. Measuring the expression level of Ly6C cell surface antigen expressed in hemocyte from a sepsis patient sample;
Comparing the expression level of the Ly6C cell surface antigen with a normal control sample; And
And determining that the expression level of the Ly6C cell surface antigen of the patient sample is higher than that of the normal control sample. 6. The method according to claim 5, wherein the expression level of the Ly6C cell surface antigen is measured through an antigen-antibody reaction. The method according to claim 6, wherein the antigen-antibody reaction is selected from the group consisting of an enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, Western blotting, immunoprecipitation, immunohistochemical staining ), A flow cytometry method, a fluorescence activated cell sorting method (FACS), an enzyme substrate staining method, and an antigen-antibody aggregation method. .

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