WO2016167573A1

WO2016167573A1 - Biomarker composition for diagnosing still's disease, diagnostic kit, and diagnostic method

Info

Publication number: WO2016167573A1
Application number: PCT/KR2016/003898
Authority: WO
Inventors: 손성향; 김현아
Original assignee: 아주대학교산학협력단
Priority date: 2015-04-15
Filing date: 2016-04-14
Publication date: 2016-10-20

Abstract

The present invention relates to a biomarker composition for diagnosing Still's disease, a diagnostic kit, and a diagnostic method and, more specifically, the present invention can accurately diagnose Still's disease in young children and adults using the expression level of CD11b or CD32 and the frequency ratio of granulocytes to lymphocytes as indexes.

Description

Biomarker Compositions, Diagnostic Kits, and Diagnostic Methods for Still's Disease Diagnosis

The present invention relates to a composition for diagnosing Still's disease, a diagnostic kit, and a method for providing information necessary for diagnosis.

Still's disease is a systemic form of juvenile rheumatoid arthritis that has not yet been identified since it was first described and develops in both children and adults. Adult Still's disease, which occurs in adults, has been reported all over the world since it was reported by Bywaters in 1971, and is known as a rare disease or a cause of unknown fever.

In general, adult STI is a benign disease that is reported to have a good prognosis. However, some patients have reported fatal deaths. Patients may die if accompanied by hemophilia, liver failure, or infection. It may be in a state where it is. Domestic research has also reported the prognosis of death in about 10% of affected patients.

The diagnosis of Still's disease is mainly based on clinical findings, including high fever, arthralgia or arthritis, characteristic skin lesions, lymphadenopathy, hepatomegaly, splenomegaly, meningitis and sore throat, leukocytosis, increased blood infiltration rate, Diagnosis is based on laboratory findings such as antibody negative and rheumatoid factor negative.

Much of the work being done in adult Still's disease has been carried out in patients with serum inflammatory cytokines, simple enzymatic immunoassays (ELISA), or genetic studies on acute reactive substances. There was no report. In addition, due to the lack of understanding of the pathogenesis mechanism, treatment is based on experience, and there is no consensus on this.

Genetic studies of adult Still's disease have also been shown to correlate with human leucocyte antigens (HLAs), and have been associated with HLA-DR4 and HLA-DR7, HLA-Bw35, and HLA-Cw4. However, foreign researchers have reported adult Stilde disease in only one of the identical twins, focusing more on environmental factors than on genetics.

Human parvo virus B19, cytomegalovirus, rubella virus, and echovirus7 have been reported. (Toxoplasma gondii) and Yersinia enterocolitica have also been linked.

These associations with bacterial and viral infections also reflect the importance of innate immunity in the pathogenesis of adult Still's disease. Studies on the pathogenesis of adult Stilde disease have been very limited in the study of cytokine changes and inflammatory cells in the inflammatory state of the acute phase, and related cytokines include interleukin-1 (IL-1) and tumors. Necrosis factor-α (tumor necrosis factor-α, TNF-α), IL-6, IL-18 and the like are known.

Although viral and bacterial infections are thought to be the causative mechanism of the pathogenesis of adult STI, there is still little research on how such infections cause STI.

In the early stages of innate immunity, damage-associated molecular patterns (DAMPs) and pattern-recognition receptors that play an important role in recognizing microbe-associated molecular patterns (MAMPs) are important. Although gaining attention as a key, there have been no reports of changes in pattern recognition receptors in adult STI patients.

Accordingly, an object of the present invention is to provide a biomarker composition capable of diagnosing Still's disease.

Another object of the present invention is to provide a kit for diagnosing Still's disease.

Another object of the present invention is to provide a method for providing information necessary for diagnosing Still's disease.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing Stillse disease comprising at least one protein selected from CD11b or CD32.

In order to achieve the above another object, the present invention provides a stye disease diagnostic kit comprising an antibody that specifically binds to CD11b or CD32 cell surface antigen.

In order to achieve the above another object, the present invention comprises the steps of measuring the expression level of one or more proteins selected from CD11b or CD32 from the sample; Comparing the expression level with the expression level of a normal control sample; And diagnosing Still's disease when at least one expression level selected from CD11b or CD32 of the sample is higher than that of the normal control sample.

In another aspect, the present invention comprises the steps of measuring the frequency ratio of granulocytes to lymphocytes from the sample; Comparing the frequency ratio with the frequency ratio of a normal control sample; And it provides a method for providing information necessary for diagnosing Still's disease, if the frequency ratio of granulocytes to lymphocytes of the sample is higher than the normal control sample.

According to the present invention, when compared to peripheral blood mononuclear cells (PBMCs) of patients with Still's disease, the frequency of CD11b-expressing cells and CD32-expressing cells is higher, and lymphocytes The frequency ratio of the granulocytes to the other also shows a higher difference, which is why still's disease comprising the antibody specifically binding to the CD11b or CD32 cell surface antigen, the diagnostic biomarker composition comprising one or more proteins selected from CD11b or CD32 It can be useful as a diagnostic kit and a method for providing information necessary for diagnosing Still's disease, which can improve the therapeutic effect by accurately diagnosing Still's disease in children and adults.

1 is a table showing the frequency of living cells (P1%), the frequency of cells expressing CD11b, and the frequency of cells expressing CD32 in total peripheral blood mononuclear cells (PBMC),

2 is an image showing the frequency of living cells (P1%), the frequency of cells expressing CD11b and the frequency of cells expressing CD32 in PBMC,

3 is a table showing the ratio of granulocytes, lymphocytes and lymphocytes to lymphocytes in PBMC,

Figure 4 is an image showing the ratio of granulocytes, lymphocytes in PBMC.

The inventor of the present invention, while studying a biomarker for diagnosing Still's disease, the frequency of CD11b-expressing cells, the frequency of CD32-expressing cells and the frequency of granulocytes for lymphocytes in normal people and patients with Still's disease. The difference in the ratio was found to complete the present invention.

The frequency means the number of cells expressing the CD11b, the number of cells expressing CD32, and the number of frequencies occupied by granulocytes or lymphocytes.

Accordingly, the present invention provides a biomarker composition for diagnosing Still's disease, which comprises at least one protein selected from CD11b or CD32.

According to one embodiment of the present invention, the frequency of CD11b-expressing cells and CD32-expressing cells in the whole peripheral blood mononuclear cells (PBMC) of the Still's disease patients, respectively, of the PBMCs of normal or rheumatic patients Expression was higher than the frequency of cells expressing CD11b or CD32.

In addition, according to another embodiment of the present invention, the proportion of granulocytes in PBMCs of patients with Stillse disease was higher than that of granulocytes in PBMCs of normal or rheumatic patients, and the proportion of lymphocytes was lower.

In addition, the present invention provides a kit for diagnosing Still's disease, which comprises an antibody that specifically binds to CD11b or CD32 cell surface antigen.

The antibody is a polyclonal antibody or a monoclonal antibody.

The polyclonal antibody can be prepared by injecting a CD11b or CD32 protein or fragment thereof into an external host with an immunogen according to methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep or rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection and are usually administered with an adjuvant to increase antigenicity. Serum is periodically collected from external hosts to collect serum that shows improved titers and specificity for antigen or to separate and purify antibodies therefrom.

The monoclonal antibodies can be prepared by the technology of generating immortalized cell lines by fusion known to those skilled in the art. For example, the protein expressed by the gene is immunized in mice by immunizing mice or by synthesizing peptides and binding to bovine serum albumin. Antigen-producing B lymphocytes isolated from mice are fused with myeloma of human or mouse to produce immortalized hybridomas, indirect enzyme-linked immunosorbent assays with the hybridoma cells. Monoclonal antibodies can be prepared by confirming the production of monoclonal antibodies using linked immunoabsorbent assays (ELISA), selecting positive clones, incubating them, and then separating or purifying the antibodies or injecting them into the abdominal cavity of rats. have.

The monoclonal antibody is generally quantified by color reaction using a secondary antibody to which an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) is bound, and a substrate thereof. Alternatively, the antibody may be directly quantitated using a combination of an AP or HRP enzyme or the like with a monoclonal antibody directed against the protein. In addition, antibodies with various fluorescent labels, including fluorescein isothiocyanat (FITC), phycoerythrin (PE), and the like can also be used.

The reaction between the protein and the antibody is Western blot, immunoprecipitation (IP), enzyme-linked immunoabsorbent assays (ELISA), immunohistochemistry (IHC) and flow cytometry (Flow) Protein identification experiments, such as cytometry analysis (FACS), can be confirmed, but are not limited thereto.

Kits comprising such antibodies typically include lyophilized antibodies, buffers, stabilizers, inactive proteins, and the like, wherein the antibodies are prepared by radionuclides, fluorescors, enzymes, and the like. Can be labeled.

In another aspect, the present invention comprises the steps of measuring the expression level of at least one protein selected from CD11b or CD32; Comparing the expression level with the expression level of a normal control sample; And diagnosing Still's disease when at least one expression level selected from CD11b or CD32 of the sample is higher than that of the normal control sample.

In addition, the present invention comprises the steps of measuring the frequency ratio of granulocytes to lymphocytes from the sample; Comparing the frequency ratio with the frequency ratio of a normal control sample; And it provides a method for providing information necessary for diagnosing Still's disease, if the frequency ratio of granulocytes to lymphocytes of the sample is higher than the normal control sample.

The frequency ratio of granulocytes to lymphocytes can be diagnosed as Still's disease when 3.0 to 50.0.

The sample uses blood, and more preferably, it is preferable to use peripheral blood mononuclear cells (PBMC) isolated from blood, but is not limited thereto.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<< 실시예Example 1> 말초혈액 1> Peripheral Blood 단핵세포Monocytes (peripheral blood mononuclear cell, (peripheral blood mononuclear cell, PBMCPBMC ) 제조) Produce

Human PBMCs were isolated from human blood from healthy volunteer donors using Ammonium-Chloride-Potassium (ACK) Lysis Buffer. Ethylenediaminetetraacetic acid (EDTA) was added to 2 mL of blood in a tube treated with 15 inversions. It was then placed in 40 mL ACK lysis buffer and left at room temperature for 5 minutes. White blood cells (WBC) were collected by centrifugation at 300 xg for 5 minutes, the supernatant was discarded, and the precipitated cells were mixed in 5 ml PBS. PBMC was prepared by repeating the centrifugation twice for 5 minutes at a speed of 300 xg at 4 ° C.

<< 실시예Example 2> 말초혈액(peripheral blood mononuclear cell, 2> peripheral blood mononuclear cells, PBMCPBMC )에서 )in CD11bCD11b 또는 CD32를 발현하는 세포의 빈도 측정 Or measuring the frequency of cells expressing CD32

The frequency of CD11b expressing cells and CD32 expressing cells in PBMCs obtained according to Example 1 from normal (19 patients), rheumatoid patients (RA) (19) and Still's disease patients (18) were measured, respectively. .

To this end, red blood cells are removed from each blood, lymphocytes are collected and reacted with anti-CD11b and anti-CD32 antibodies with different fluorescence, and then the number of cells with antibodies is counted using flow cytometry (FACS). And the frequency which these cells occupy in the whole cell was analyzed.

As a result, as shown in Figures 1 and 2, it was confirmed that the frequency of each of the cells expressing CD11b and CD32 is significantly higher compared to the normal control group and the rheumatoid patient experimental group.

At this time, the frequency (P1%) of living cells in the total cells did not have a big difference as a whole.

<< 실시예Example 3> 말초혈액(peripheral blood mononuclear cell, 3> peripheral blood mononuclear cells, PBMCPBMC )에서 과립구 및 림프구의 빈도 측정Frequency of granulocytes and lymphocytes

In the same manner as in Example 2, the frequency of granulocytes and lymphocytes was measured in PBMCs obtained from normal (19 patients), rheumatoid patients (RA) (19), and Still's disease patients (18), and the ratio of granulocytes to lymphocytes was measured. Confirmed.

As a result, as shown in Figures 3 and 4, the frequency of granulocytes was higher than the normal control group and rheumatic patients experimental group, it was confirmed that the frequency of lymphocytes is significantly lower than the normal control group and rheumatic patients experimental group.

In particular, as a result of confirming the ratio of granulocytes to lymphocytes, as shown in FIG. .

Taken together, when the PBMC of patients with Stilde disease was compared with the normal control group, the frequency of CD11b expressing cells and CD32 expressing cells was higher, and the ratio of granulocytes to lymphocytes was also higher. It can be used to diagnose Still's disease.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Biomarker composition for diagnosing Still's disease comprising at least one protein selected from CD11b or CD32.
Still's disease diagnostic kit comprising an antibody that specifically binds to CD11b or CD32 cell surface antigen.
The method of claim 2,

The antibody is Stills disease diagnosis kit, characterized in that the monoclonal antibody or polyclonal antibody.
Measuring at least one protein expression level selected from CD11b or CD32 from the sample;

Comparing the expression level with the expression level of a normal control sample; And

Diagnosing Still's disease when the expression level of at least one selected from CD11b or CD32 of the sample is higher than that of the normal control sample.
Measuring the frequency ratio of granulocytes to lymphocytes from the sample;

Comparing the frequency ratio with the frequency ratio of a normal control sample; And

And diagnosing Still's disease when the frequency ratio of granulocytes to lymphocytes of the sample is higher than a normal control sample.
The method of claim 5,

The frequency ratio of granulocytes to lymphocytes is 3.0 to 50.0 providing the information necessary for diagnosing Still's disease.
The method according to claim 4 or 5,

And said sample is blood.
The method according to claim 4 or 5,

And said sample is a peripheral blood mononuclear cell (PBMC).