阅读说明：本技术 双阴性b细胞与调节性b10细胞在制备自身免疫性疾病诊断试剂中的应用 (Application of double negative B cell and regulatory B10 cell in preparation of autoimmune disease diagnostic reagent ) 是由 胡凡磊 栗占国 郑茜 方翔宇 贾园 于 2019-11-11 设计创作，主要内容包括：本发明公开了双阴性B细胞与调节性B10细胞在制备自身免疫性疾病诊断试剂中的应用,该应用中,所述诊断试剂能分别检测病患的双阴性B细胞含量和调节性B10细胞含量。双阴性B细胞：调节性B10细胞比值在正常人和自身免疫病患者中存在明显差别,这种差别能够用于对自身免疫性疾病进行诊断。这两种细胞的检测,只需要通过外周血取样,用流式细胞术即可,判断其比例以及计算比值也十分简单方便；此外,通过设定cut-off值(ROC曲线分析推荐值),可保证较高的准确率,无需结合其他标志物进行综合判断即可确定自身免疫病,对于自身免疫病的诊断和药物治疗效果都能有很好的指示,更有利于临床应用。(The invention discloses application of double negative B cells and regulatory B10 cells in preparation of an autoimmune disease diagnostic reagent, wherein in the application, the diagnostic reagent can be used for respectively detecting the content of double negative B cells and the content of regulatory B10 cells of a patient. Double negative B cells: the regulatory B10 cell ratio is clearly different in normal humans and autoimmune patients, and this difference can be used to diagnose autoimmune diseases. The detection of the two cells only needs to sample peripheral blood and use flow cytometry, and the proportion is judged and the ratio is calculated very simply and conveniently; in addition, by setting a cut-off value (ROC curve analysis recommended value), higher accuracy can be ensured, the autoimmune disease can be determined without being comprehensively judged by combining other markers, good indication can be provided for diagnosis and drug treatment effect of the autoimmune disease, and clinical application is facilitated.)

1. The application of double negative B cell and regulatory B10 cell in preparing autoimmune disease diagnostic reagent can detect the content of double negative B cell and regulatory B10 cell in patient separately to further calculate the ratio.

2. The use of claim 1, wherein the autoimmune disease is at least one of rheumatoid arthritis, systemic lupus erythematosus, and sjogren's syndrome.

3. The use of claim 1, wherein the diagnostic reagent is an antibody capable of detecting double negative B cells and an antibody capable of detecting regulatory B10 cells.

4. The use according to claim 1, wherein the test object of the diagnostic reagent is human peripheral blood.

5. An autoimmune disease diagnostic reagent comprising an antibody for detecting double negative B cells and an antibody for detecting regulatory B cells.

6. The diagnostic reagent of claim 5, wherein the antibody is fluorescently labeled for detecting double negative B cells: anti-human CD19, anti-human CD3, anti-human CD27 and anti-human IgD, and a fluorescent label for detecting regulatory B10 cells: anti-human CD19, anti-human CD3, anti-human CD24, and anti-human CD 27; the fluorescent marker of the double-negative B cell and the fluorescent marker of the regulatory B10 cell are matched to distinguish the double-negative B cell from the regulatory B10 cell.

7. The diagnostic reagent of claim 6, wherein the antibody is CD19-APC/Cy7, CD3-PerCP/Cy5.5, CD27-APC and IgD-Pacific Blue for detecting double negative B cells, and CD19-APC/Cy7, CD3-PerCP/Cy5.5, CD24-FITC and CD27-APC for detecting regulatory B10 cells.

8. The diagnostic reagent of claim 5, further comprising a reference range of the ratio of normal human double negative B cell to regulatory B cell content.

Technical Field

The invention relates to the technical field of disease diagnosis, in particular to application of double negative B cells and regulatory B10 cells in preparation of an autoimmune disease diagnostic reagent.

Background

Autoimmune diseases (AID) are diseases caused by the immune dysfunction of the body caused by various factors, which leads to the immune reaction of the body to self antigens and the damage of self tissues. AID can be subdivided into more than 100 diseases, and no effective cure means exists at present, but timely and regular treatment can obviously improve symptoms and delay the progress of diseases, so the AID is very important for diagnosing the diseases.

Among the AID diseases, the most common are Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Sjogren Syndrome (SS), scleroderma, Idiopathic platelet purpura (ITP), Autoimmune hemolytic anemia (AIHA), etc., in which the patients of rheumatoid arthritis, systemic lupus erythematosus and Sjogren's syndrome are in the front.

The diagnosis of the above three diseases still has the problems of low sensitivity and poor specificity, and usually the detection result of one index needs to be combined with the detection results of other indexes to jointly judge and draw a conclusion.

AID disease is caused by the generation of autoantibodies which attack self tissues due to the imbalance of immune functions of the body, in other words, is a pathological state in which the homeostasis of autoimmunity is broken, and the biggest characteristic is the imbalance of immunity. For a long time, the relevant studies of immune imbalance in AID diseases have focused mainly on T cells, such as helper Th17 cells, follicular helper T cells (Tfh), regulatory T cells (Treg), etc. The role of B cells in the pathogenic process of AID has been mainly studied in the pathological processes such as autoantibody production, and there is no systematic study on immune imbalance.

Disclosure of Invention

At present, the relation between B cells and immune diseases is mostly focused on a single pathological process, and no combined diagnosis content exists.

In the research process, the applicant finds that double-negative B cells are obviously amplified in autoimmune diseases such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Sjogren Syndrome (SS) and the like. More importantly, in these diseases, double negative B cells: the regulatory B10 cell ratio was significantly increased (compared to the ratio of normal human and osteoarthritic double negative B cells: regulatory B10 cells). After treatment, double negative B cells: the regulatory B10 cell ratio returned to normal. It was shown that the detection of double negative B cells and regulatory B10 cells and the calculation of the ratio thereof can be an effective method for diagnosing autoimmune diseases.

Based on the above research conclusion, the invention provides the following technical scheme:

the invention provides application of double negative B cells and regulatory B10 cells in preparation of an autoimmune disease diagnostic reagent, wherein the diagnostic reagent can be used for respectively detecting the content of double negative B cells and the content of regulatory B10 cells of a patient so as to further calculate the ratio of the double negative B cells to the regulatory B10 cells.

By detecting the content thereof, and calculating double negative B cells: the ratio of B10 cells can be regulated to determine whether autoimmune diseases exist, which is very convenient.

Among these, double negative B cells are a group of cells lacking both IgD and the memory cell marker CD27 among the B cells undergoing immunoglobulin-type switching, and having the potential to produce autoantibodies. The regulatory B10 cells are a population of CD19⁺CD24^hiCD27⁺Is a marked cell and has the function of immunoregulation. The imbalance of the ratio of the two is directly related to the onset of AID. The unbalanced ratio refers to the ratio of the double negative B cells to the regulatory B10 cells of a normal human, for example, the optional cutoff value is 0.8068 (more than this value, the diagnosis is positive).

Optionally or preferably, in the above application, the autoimmune disease is at least one of rheumatoid arthritis, systemic lupus erythematosus and sjogren's syndrome. The disease can be accurately judged as the result of the imbalance of the proportion of the two, but the specific types of the diseases cannot be further distinguished.

Alternatively or preferably, in the above application, the diagnostic reagent is an antibody capable of detecting double negative B cells and an antibody capable of detecting regulatory B10 cells. The antibody detection result can accurately calculate and compare double negative B cells: ratio of regulatory B10 cells.

Alternatively or preferably, in the above application, the detection target of the diagnostic reagent is human peripheral blood. The damage to the patient to be detected is small.

The invention also provides an autoimmune disease diagnostic reagent, which comprises an antibody for detecting double negative B cells and an antibody for detecting regulatory B cells.

Alternatively or preferably, in the above diagnostic reagent, the antibody is a fluorescent-labeled antibody for detecting double negative B cells: anti-human CD19, anti-human CD3, anti-human CD27 and anti-human IgD, and a fluorescent label for detecting regulatory B10 cells: anti-human CD19, anti-human CD3, anti-human CD24, and anti-human CD 27; the fluorescent marker of the double-negative B cell and the fluorescent marker of the regulatory B10 cell are matched to distinguish the double-negative B cell from the regulatory B10 cell.

Alternatively or preferably, in the diagnostic reagent, the antibody is CD19-APC/Cy7, CD3-PerCP/Cy5.5, CD27-APC and IgD-Pacific Blue for detecting double negative B cells, and CD19-APC/Cy7, CD3-PerCP/Cy5.5, CD24-FITC and CD27-APC for detecting regulatory B10 cells.

Alternatively or preferably, the diagnostic reagent further comprises a reference range of the content ratio of normal human double negative B cells to regulatory B cells.

Compared with the prior art, the invention has the following beneficial effects:

double negative B cells: the ratio of the regulatory B10 cells is obviously different between normal people and autoimmune patients, and the ratio can be judged by flow cytometry only by sampling peripheral blood, and the ratio is calculated, so that the detection is very simple and convenient; in addition, by setting a cut-off value (ROC curve analysis recommended value), higher accuracy can be ensured, the autoimmune disease can be determined without being comprehensively judged by combining other markers, good indication can be provided for diagnosis and drug treatment effect of the autoimmune disease, and clinical application is facilitated.

Drawings

FIG. 1 is a circle gate strategy for double negative B cells and regulatory B10 cells in the examples;

FIG. 2 is a representative graph of the flow results of double negative B cells and regulatory B10 cells in peripheral blood of normal Humans (HC) and autoimmune patients (RA, SLE, SS) and disease control Osteoarthritis (OA) patients in the examples;

FIG. 3 is a scatter plot of the statistics of the ratios of double negative B cells and regulatory B10 cells in peripheral blood of normal Humans (HC) to patients with autoimmune diseases (RA, SLE, SS) and disease control Osteoarthritis (OA) in the examples;

FIG. 4 is a schematic diagram showing the flow results of double negative B cells and regulatory B10 cells and their ratios in peripheral blood before and after treatment of RA patients in the example and a scattergram of statistical results;

FIG. 5 is a graph of ROC with RA, SLE, SS classified as disease group, HC and OA classified as control group;

in the above figures, DNB cells represent double negative B cells, and B10 cells represent regulatory B10 cells.

Detailed Description

The technical solutions of the present invention are explained and illustrated in detail below with reference to specific embodiments so that those skilled in the art can better understand the technical solutions of the present invention.

In the following examples, unless otherwise specified, all of the biomaterials and reagents used are commercially available or may be prepared according to conventional techniques, and the assay methods are also conventional in the art.