Treg cell detection antibody combination, kit and application

Abstract

The invention relates to a Treg cell detection antibody combination, a kit and application, and belongs to the technical field of Treg cell detection. The Treg cell detection antibody combination comprises a first antibody combination and a second antibody combination, and the first antibody combination is composed of the following antibodies: an anti-CD4 antibody, an anti-CD25 antibody, an anti-CD304 antibody, an anti-CD45RO antibody, an anti-CD20 antibody and an anti-CD127 antibody; the first antibody combination is used in one flow type tube during detection. Through flow cytometry detection, multiple marker combination gates are used, and clinical analysis is combined to preliminarily distinguish whether a sample has tumor cells or tumor types, autoimmune diseases, inflammations and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of Treg cell detection, and in particular relates to a Treg cell detection antibody combination, a kit and an application thereof. Background Art

[0002] Regulatory T cells (Treg) are a type of T cells with immunosuppressive effects, accounting for about 2% to 5% of peripheral blood mononuclear cells (PBMC) and about 5% to 10% of peripheral CD4+T cells. This type of cell participates in the regulation of immune response / immune tolerance in an "active" way. It not only participates in the regulation of autoimmune tolerance, but also plays an important role in tumor immunity and transplantation immunity. Treg cell imbalance, that is, too high or too low, will have an impact on the human body.

[0003] When using flow cytometry to detect Treg cells, the commonly used molecular markers are CD4, CD25 and CD127 or CD4, CD25 and FoxP3 to detect whether Treg cells are imbalanced. FoxP3 is an intranuclear substance, and it needs to be fixed and broken during specific detection, which is complicated to operate and has poor stability. The expression of CD127 is opposite to that of FoxP3, so CD4, CD25 and CD127 are currently used for detection. The expression level of CD127 is weak and the stability needs to be improved. Summary of the invention

[0004] The first objective of the present invention is to provide a Treg cell detection antibody combination for preliminary clinical screening of whether cancer cells are present and the type of cancer.

[0005] The second object of the present invention is to provide a Treg cell detection kit.

[0006] The third object of the present invention is to provide an application of a Treg cell detection antibody combination.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] The Treg cell detection antibody combination includes a first antibody combination and a second antibody combination, wherein the first antibody combination is composed of the following antibodies: anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody; the first antibody combination is used in one flow tube during detection.

[0009] Furthermore, the fluorescein labeling order of the anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody in the first antibody combination is EV450, FITC, PE-Cy7, PerCP, APC, PE, respectively.

[0010] Furthermore, the antibody combination also includes a second antibody combination, which consists of the following antibodies: anti-CD4 antibody, anti-CD25 antibody and anti-FoxP3 antibody, and the second antibody combination is used in one flow tube during detection.

[0011] Furthermore, the fluorescent labeling order of the anti-CD4 antibody, anti-CD25 antibody and anti-FoxP3 antibody in the second antibody combination is: EV450, FITC, PE, respectively.

[0012] Furthermore, the antibodies in the antibody combination are monoclonal antibodies.

[0013] A Treg cell detection kit comprises the above-mentioned antibody combination; when the antibody combination comprises a first antibody combination and a second antibody combination, the first antibody combination and the second antibody combination are packaged independently.

[0014] Furthermore, the kit also includes: red blood cell lysis solution and buffer PBS.

[0015] The application of Treg cell detection antibody combination is used to distinguish tumor symptoms, inflammation or autoimmune diseases associated with Treg cells.

[0016] Furthermore, it comprises a detection part and an analysis part; the detection part comprises the above-mentioned antibody combination or the above-mentioned kit, and is used to detect the antigen expression level of the individual to be tested by flow cytometry; and the analysis part is used to analyze the detection results of the detection part.

[0017] Beneficial effects of the present invention:

[0018] The Treg cell detection antibody combination of the present invention is detected by flow cytometry, uses a combination of multiple markers to set gates, and combines clinical analysis to preliminarily distinguish whether the sample has tumor cells or tumor types, whether it has autoimmune diseases, inflammation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a graph showing the results of flow cytometry screening of CD4+ in Example 1;

[0020] Figure 2 This is a graph showing the results of flow cytometry screening of CD4+ / CD25+ / CD127- in Example 1;

[0021] Figure 3This is a graph showing the results of flow cytometry screening for CD304+ in Example 1;

[0022] Figure 4 This is a graph showing the results of flow cytometry screening of CD45RO+ in Example 1;

[0023] Figure 5 This is a graph showing the results of flow cytometry screening for CD20+ in Example 1;

[0024] Figure 6 This is a graph showing the results of flow cytometry screening of CD4+ / CD25+ / FoxP3+ in Example 1. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0026] The present invention provides a Treg cell detection antibody combination, including a first antibody combination and a second antibody combination, wherein the first antibody combination is composed of the following antibodies: including a first antibody combination and a second antibody combination, wherein the first antibody combination is composed of the following antibodies: anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody; the first antibody combination is used in a flow tube during detection. In the antibody composition of this scheme, each antibody component can be commercially available. Each antibody should meet the requirements of relevant industry standards.

[0027] The fluorescein labeling order of anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody in the first antibody combination is PE, FITC, PE-Cy7, PerCP, APC, V450, respectively. In the antibody composition of the present invention, anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody are mixed in a volume ratio of 1:1:2:1:1:2 (when the titers are substantially equivalent).

[0028] The antibody combination further includes a second antibody combination, which consists of the following antibodies: anti-CD4 antibody, anti-CD25 antibody and anti-FoxP3 antibody. The second antibody combination is used in one flow tube during detection.

[0029] The fluorescein labeling order of the anti-CD4 antibody, anti-CD25 antibody and anti-FoxP3 antibody in the second antibody combination is EV450, FITC, and PE, respectively.

[0030] The antibodies in the antibody panel are monoclonal antibodies.

[0031] A Treg cell detection kit, comprising the above-mentioned antibody combination; when the antibody combination comprises a first antibody combination and a second antibody combination, the first antibody combination and the second antibody combination are packaged independently. The kit also includes: red blood cell lysate and buffer PBS. The antibody composition of this scheme can be applied to a kit, which includes: red blood cell lysate, buffer, and one or more of the flow tubes used in conjunction with a flow cytometer. These reagents and consumables are commercially available. Each reagent material can be placed in different containers respectively.

[0032] The application of the Treg cell detection antibody combination is used to distinguish cancer, inflammation or autoimmunity related to Treg cells. It includes a detection part and an analysis part; the detection part includes the antibody combination or the kit, which is used to detect the antigen expression level of the individual to be tested by flow cytometry; the analysis part is used to analyze the detection results of the detection part.

[0033] Example 1

[0034] 1. Preparation of reagents

[0035] The antibody composition of this embodiment is: CD4-PE, CD25-FITC, CD304-PE-Cy7, CD45RO-PerCP, CD20-APC and anti-CD127-RV450. The above five monoclonal antibody reagents are mixed in a volume ratio of 1:1:2:1:1:2 and loaded into a first container, and optionally a red blood cell lysate is added and loaded into a second container, and a PBS buffer is loaded into a third container. The red blood cell lysate and PBS buffer are commercially available, and the cell lysate and PBS buffer are products of Beckman Coulter.

[0036] 2. Specimen processing

[0037] According to the cell counting results, add the EDTA anticoagulated peripheral blood sample into the flow tube to ensure that the amount of cells added is about 2×10 6 Then, according to Table 1, add 15 μl of six kinds of cell membrane monoclonal antibody reagents labeled with different fluorescent substances into the flow tube, mix well with the cell suspension and incubate at room temperature and away from light for 15 minutes, add 0.5 ml of 1× hemolysin and incubate in the dark for 10 minutes to lyse red blood cells, centrifuge at 1500 rpm for 5 minutes, and finally add 3 ml of PBS buffer for washing. After centrifugation, remove the supernatant and resuspend the cells with 0.5 ml of PBS buffer. The processed specimen can be used for detection on the machine.

[0038] 3. Sample testing

[0039] The specimens were processed according to the method of "Specimen Processing" above, and tested on Beckman Coulter's 2-laser 8-color DxFlex flow cytometer. It is preferred to obtain 100,000 cells per tube (at least 10,000 is recommended), and then the data were analyzed using Kaluza software. The gates were set in the following manner when testing on the flow cytometer:

[0040] Use FSC-A / FSC-H to set gate A for doublet cell removal, SSC-A / SSC-B to continue setting gate B for adhesion removal, FSC-A / SSC-A to remove debris and dead cells, and set gate R1 for live single cells. Use CD4 / CD25 / CD127 / SSC to set gates to analyze the proportion of Treg cells in T cells. Use CD304 / SSC to set gates to analyze inflammation. Use CD45RO / CD20 to set gates to analyze the type of tumor cells.

[0041] 4. Results Analysis

[0042] 4.1 Patient Composition

[0043] A total of 62 patient samples were tested using the antibody combination of the present invention, including 41 males and 21 females, with a median age of 30 (1-69 years old). The proportion of Treg cells in peripheral blood CD4+T cells is 5%-10%, which is in the normal range. After testing, 15 patients had Treg cells in peripheral blood CD4+T cells less than 5%, 9 patients had Treg cells in peripheral blood CD4+T cells between 5%-10%, and 38 patients had Treg cells in peripheral blood CD4+T cells greater than 10%. Among the 15 patients whose Treg cells accounted for less than 5% of peripheral blood CD4+T cells, combined with clinical tests, 15 patients suffered from autoimmune syndrome. Among the 38 patients whose Treg cells accounted for more than 10% of peripheral blood CD4+T cells, there were 6 CD304+ patients. Combined with clinical tests, these 6 patients all had varying degrees of inflammatory response. Among the 38 patients whose Treg cells accounted for more than 10% of peripheral blood CD4+T cells, 23 were CD45RO+. Combined with clinical tests, all of these 23 patients suffered from T-cell lymphoma. Among the 38 patients whose Treg cells accounted for more than 10% of peripheral blood CD4+T cells, 9 were CD45RO+. Combined with clinical tests, all of these 9 patients suffered from B-cell lymphoma.

[0044] 4.2. Typical result graphs are as follows:

[0045] CD4 / CD25 / CD127 / SSC combined gating was used to circle out Treg cells, such as Figure 1 and Figure 2As shown in the figure, determine the proportion of Treg cells to T cells. When the proportion is reduced, it indicates the risk of autoimmunity. When the proportion is increased, there may be a risk of tumor or inflammation. At this time, use CD304 / SSC combined gating or CD45RO / CD20 combined gating. CD304 / SSC combined gating is used to analyze the inflammation situation, such as Figure 3 As shown in Figure 2, upregulated CD304 expression indicates an inflammatory risk, which is consistent with clinical test results. CD45RO / CD20 combined gating can be used to analyze tumor types, such as Figure 4 As shown, CD45RO expression is upregulated, indicating the risk of T cell tumors, such as Figure 5 As shown in Figure 2, CD20 expression is upregulated, indicating the risk of B cell tumors, which is consistent with the clinical test results. The proportion of Treg cells to T cells was verified using a secondary antibody combination, such as Figure 6 The antibody combination of the present invention can provide clinical efficacy evaluation.

Claims

1. A Treg cell detection antibody combination, characterized in that: It comprises a first antibody combination and a second antibody combination, wherein the first antibody combination consists of the following antibodies: anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody; the first antibody combination is used in one flow tube during detection.

2. The Treg cell detection antibody combination according to claim 1, characterized in that: The fluorescein labeling order of the anti-CD4 antibody, anti-CD25 antibody, anti-CD304 antibody, anti-CD45RO antibody, anti-CD20 antibody and anti-CD127 antibody in the first antibody combination is PE, FITC, PE-Cy7, PerCP, APC and V450 respectively.

3. The Treg cell detection antibody combination according to claim 2, characterized in that: The antibody combination also includes a second antibody combination, which consists of the following antibodies: anti-CD4 antibody, anti-CD25 antibody and anti-FoxP3 antibody. The second antibody combination is used in one flow tube during detection.

4. The Treg cell detection antibody combination according to claim 3, characterized in that: The fluorescent labeling order of the anti-CD4 antibody, anti-CD25 antibody and anti-FoxP3 antibody in the second antibody combination is: EV450, FITC, PE.

5. The Treg cell detection antibody combination according to claim 1, characterized in that: The antibodies in the antibody combination are monoclonal antibodies.

6. The Treg cell detection kit according to claim 1, characterized in that: The invention comprises the antibody combination according to any one of claims 1 to 5; when the antibody combination comprises a first antibody combination and a second antibody combination, the first antibody combination and the second antibody combination are packaged independently.

7. The Treg cell detection kit according to claim 6, characterized in that: The kit also includes: red blood cell lysis solution and buffer PBS.

8. The use of the Treg cell detection antibody combination according to claim 1, characterized in that: Used to distinguish tumor symptoms, inflammation or autoimmune diseases associated with Treg cells.

9. The use of the Treg cell detection antibody combination according to claim 8, characterized in that: It comprises a detection part and an analysis part; the detection part comprises the antibody combination according to any one of claims 1 to 5 or the kit according to claim 6 or 7, and is used to detect the antigen expression level of the individual to be tested by flow cytometry; the analysis part is used to analyze the detection results of the detection part.

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