CN114133449A

CN114133449A - Antibody composition for detecting lymphocyte surface PD-1 receptor and application thereof

Info

Publication number: CN114133449A
Application number: CN202111530256.XA
Authority: CN
Inventors: 徐兵; 张黎; 王彩燕; 李志峰; 屈浩; 刘红芹
Original assignee: Tianjin Kuangbo Tongsheng Biotechnology Co ltd; First Affiliated Hospital of Xiamen University
Current assignee: Tianjin Kuangbo Tongsheng Biotechnology Co ltd; First Affiliated Hospital of Xiamen University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-04

Abstract

The present invention provides an antibody composition for detecting PD-1 receptor on lymphocyte surface and application thereof, and the antibody composition for detecting PD-1 receptor on lymphocyte surface includes anti-PD-1 antibody, anti-CD45 antibody, Any one or a combination of at least two of an anti-CD3 antibody, an anti-CD4 antibody, or an anti-CD8 antibody. The invention also provides a kit for detecting the PD-1 receptor on the lymphocyte surface and a method for detecting the expression rate of the PD-1 receptor on the lymphocyte surface. With flow cytometry, the antibody composition for detecting the PD-1 receptor on the lymphocyte surface of the present invention can quickly and accurately detect the PD-1 expression of various types of lymphocytes, with easy operation and high detection efficiency. High, and realize the continuous detection and dynamic observation of related samples, which provides the basis for related drug treatment.

Description

Antibody composition for detecting lymphocyte surface PD-1 receptor and application thereof

Technical Field

The invention belongs to the technical field of medical detection, and particularly relates to an antibody composition for detecting a lymphocyte surface PD-1 receptor and application thereof.

Background

Programmed cell death protein 1(PD-1 for short) is an immunoglobulin superfamily I type transmembrane glycoprotein of 50-55 KD, belongs to one of CD28 superfamily members, is mainly expressed in activated lymphocytes and monocytes, interacts with ligand PD-L1/PD-L2, and participates in the inhibition of immune response in peripheral tissues. Normally, the immune system will respond to foreign antigens accumulated in the lymph nodes or spleen to promote the proliferation of T cells with antigen specificity, and programmed cell death protein-1 (PD-1) binds to ligand PD-L1/PD-L2 and can conduct inhibitory signals, thereby inhibiting the proliferation of T cells. The PD-1 molecule regulates the strength and the breadth of immune response through the high and low expression level thereof.

When a point inhibitor medicament is screened through PD-1/PD-L1 axis immunity in clinic, the detection of PD-L1 expression on tumor tissues through immunohistochemical staining is mainly taken as a preferred scheme of accompanying diagnosis according to the consensus of relevant experts at home and abroad, but the accuracy, standardized judgment and the like of the point inhibitor medicament still have problems. Meanwhile, the biopsy tissue specimen is difficult to source, and the aims of multiple detection and dynamic observation cannot be fulfilled.

The detection of the expression level of PD-1 in peripheral blood is a useful mark for understanding the overall condition of the PD-1/PD-L1 axis, can realize multiple detections and dynamic observations, helps clinicians to understand the immune state of organisms, and is helpful for judging the progress of the disease course, making a treatment scheme, and analyzing the curative effect and prognosis of drugs, thereby making more accurate assessment.

Therefore, it is a problem to be solved that how to provide a product and a method for rapidly and accurately detecting the lymphocyte surface PD-1 receptor in human peripheral blood or bone marrow fluid.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the antibody composition for detecting the lymphocyte surface PD-1 receptor and the application thereof, the PD-1 expression level on the lymphocyte surface can be detected by matching with a flow cytometer, the operation is simple, the sensitivity is high, the specificity is good, the detection can be completed in 1 hour, and the application value is high.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an antibody composition for detecting a lymphocyte surface PD-1 receptor, which comprises an anti-PD-1 antibody, and any one or a combination of at least two of an anti-CD 45 antibody, an anti-CD 3 antibody, an anti-CD 4 antibody, or an anti-CD 8 antibody.

In the invention, the anti-CD 45 antibody, the anti-CD 3 antibody, the anti-CD 4 antibody and the anti-CD 8 antibody can mark cell membrane surface antigens CD45, CD3, CD4 and CD8, qualitatively distinguish lymphocytes, detect the lymphocytes by using the PD-1 antibody, and rapidly and accurately analyze the expression condition of the lymphocyte PD-1.

In the invention, the expression condition of the lymphocyte PD-1 receptor is detected by adopting flow cytometry. Flow Cytometry (FCM) is a means of quantitatively analyzing and sorting single cells or other biological particles at the functional level, and can analyze tens of thousands of cells at high speed, measure multiple parameters from one cell at the same time, and, in cooperation with the above antibody composition, can rapidly and accurately analyze the expression of the receptor PD-1 on the surface of the lymphocyte.

Preferably, the anti-PD-1 antibody comprises a fluorescent monoclonal antibody for PD-1-APC.

Preferably, the anti-CD 45 antibody comprises a CD45-QB500 fluorescent monoclonal antibody.

Preferably, the anti-CD 3 antibody comprises a CD3-percp fluorescent monoclonal antibody.

Preferably, the anti-CD 4 antibody comprises a CD4-FITC fluorescent monoclonal antibody.

Preferably, the anti-CD 8 antibody comprises a CD8-PE fluorescent monoclonal antibody.

Preferably, the weight portion of the PD-1-APC fluorescent monoclonal antibody in the antibody composition for detecting a lymphocyte surface PD-1 receptor is 8-13 parts, for example, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, or 13 parts, and other specific points within the range of the values can be selected, and are not repeated herein.

Preferably, the weight part of the CD45-QB500 fluorescent monoclonal antibody in the antibody composition for detecting a lymphocyte surface PD-1 receptor is 4-6 parts, for example, 4 parts, 4.5 parts, 5 parts, 5.5 parts, or 6 parts, and other specific points in the numerical range can be selected, and are not repeated herein.

Preferably, the weight portion of the CD3-percp fluorescent monoclonal antibody in the antibody composition for detecting a lymphocyte surface PD-1 receptor is 5-12 parts, for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, or 12 parts, and other specific points within the range of the values can be selected, and are not repeated herein.

Preferably, the weight part of the CD4-FITC fluorescent monoclonal antibody in the antibody composition for detecting a lymphocyte surface PD-1 receptor is 5-12 parts, for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, or 12 parts, and other specific points within the range of the values can be selected, and are not repeated herein.

Preferably, the weight portion of the CD8-PE fluorescent monoclonal antibody in the antibody composition for detecting a lymphocyte surface PD-1 receptor is 5-12 parts, for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, or 12 parts, and other specific points within the range of the values can be selected, and are not repeated herein.

Preferably, the antibody composition for detecting the lymphocyte surface PD-1 receptor comprises 8-13 parts by weight of PD-1-APC fluorescent monoclonal antibody, 4-6 parts by weight of CD45-QB500 fluorescent monoclonal antibody, 5-12 parts by weight of CD3-percp fluorescent monoclonal antibody, 5-12 parts by weight of CD4-FITC fluorescent monoclonal antibody and 5-12 parts by weight of CD8-PE fluorescent monoclonal antibody.

In a second aspect, the present invention provides a kit for detecting a lymphocyte surface PD-1 receptor, which comprises the antibody composition for detecting a lymphocyte surface PD-1 receptor according to the first aspect.

Preferably, the kit for detecting the lymphocyte surface PD-1 receptor further comprises erythrocyte lysate and auxiliary reagents.

Preferably, the auxiliary reagent comprises a phosphate buffer.

In a third aspect, the present invention provides a method for detecting the expression rate of a PD-1 receptor on the surface of a lymphocyte, which comprises:

the antibody composition for detecting a lymphocyte surface PD-1 receptor according to the first aspect and/or the kit for detecting a lymphocyte surface PD-1 receptor according to the second aspect is used for detecting a sample.

Preferably, the method comprises:

mixing the sample with the antibody composition for detecting the lymphocyte surface PD-1 receptor, incubating in a dark place, cracking red blood cells, detecting on a machine, and analyzing.

Preferably, the analysis comprises:

setting a lymphocyte gate to obtain lymphocytes;

the expression rate of PD-1 receptor of each lymphocyte group is obtained by circling the graph in the lymphocyte.

As a preferred technical scheme, the method for detecting the expression rate of the PD-1 receptor on the surface of the lymphocyte comprises the following steps:

mixing the sample with the antibody composition for detecting the lymphocyte surface PD-1 receptor, and incubating in a dark place;

cracking erythrocytes by using erythrocyte lysate, and cleaning;

collecting cells, detecting on a machine, and analyzing;

the analysis comprises the following steps:

setting a lymphocyte gate to obtain lymphocytes;

As a preferred technical scheme, the method for detecting the expression rate of the PD-1 receptor on the surface of the lymphocyte specifically comprises the following steps:

(1) 4-6 parts of CD45-QB500 fluorescent monoclonal antibody, 5-12 parts of CD3-percp fluorescent monoclonal antibody, 5-12 parts of CD4-FITC fluorescent monoclonal antibody, 5-12 parts of CD8-PE fluorescent monoclonal antibody and 8-13 parts of PD-1-APC fluorescent monoclonal antibody are added to the bottom of the flow type sample loading tube.

(2) And (3) sucking the uniformly mixed bone marrow blood or peripheral blood sample, and adding the sample into the bottom of the tube to prevent the sample from touching the wall of the flow-type sample loading tube.

(3) Vortexing and mixing, and incubating for 15-20 minutes at room temperature in a dark place.

(4) And adding 0.5-3 mL of erythrocyte lysate into the tube, whirling, uniformly mixing, and incubating for 8-12 minutes at room temperature in a dark place.

(5) Centrifuging for 4-7 minutes at 300g, and discarding the supernatant.

(6) Adding 2-5 mL of phosphate buffer solution into the tube, whirling and uniformly mixing, centrifuging for 4-7 minutes at 300g, and discarding the supernatant.

(7) 0.5-1 mL of phosphate buffer was added to the tube and mixed thoroughly.

(8) Filtering the suspended matters in the sample by a 200-mesh filter screen and detecting on a machine.

(9) And (3) analysis:

setting a lymphocyte gate to obtain lymphocytes;

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

(1) the antibody composition for detecting the lymphocyte surface PD-1 receptor can be matched with the flow cytometry, so that the expression level of an immune checkpoint (PD-1) marker can be evaluated quickly and accurately;

(2) the kit for detecting the lymphocyte surface PD-1 receptor can detect various samples such as peripheral blood, marrow fluid and the like, has simple and convenient operation and high detection efficiency, can complete detection only within 1 hour, can realize multiple detection and dynamic observation, overcomes the defects of difficult rechecking and no standardization of result judgment of immunohistochemical staining detection expression on tumor tissues, and can meet the clinical requirements on rapid detection, medication guidance and disease prognosis judgment.

Drawings

FIG. 1 shows the flow measurement of CD3+ T lymphocytes in peripheral blood;

FIG. 2 shows the flow-based detection of CD3+ CD4+ T lymphocytes and CD3+ CD8+ T lymphocytes in peripheral blood;

FIG. 3 shows the flow-based assay of a negative control for the expression of PD-1 from CD3+ T lymphocytes in peripheral blood;

FIG. 4 shows the results of flow-based detection of negative control for PD-1 expression of T lymphocytes of CD3+ CD4+ in peripheral blood;

FIG. 5 shows the results of flow-based detection of negative control for PD-1 expression of T lymphocytes of CD3+ CD8+ in peripheral blood;

FIG. 6 shows the result of flow measurement of the PD-1 expression rate of CD3+ T lymphocytes in peripheral blood;

FIG. 7 shows the flow-based measurement of the PD-1 expression rate of T lymphocytes of CD3+ CD4+ in peripheral blood;

FIG. 8 shows the flow measurement of PD-1 expression rate of T lymphocytes of CD3+ CD8+ in peripheral blood;

FIG. 9 shows the results of flow-based assay of CD3+ T lymphocytes in bone marrow;

FIG. 10 shows the flow-through assay of CD3+ CD4+ T lymphocytes and CD3+ CD8+ T lymphocytes in bone marrow;

FIG. 11 is a flow chart of a negative control for PD-1 expression of CD3+ T lymphocytes in bone marrow;

FIG. 12 is a flow chart of a negative control for PD-1 expression of T lymphocytes CD3+ CD4+ in bone marrow;

FIG. 13 is a flow chart of a negative control for PD-1 expression of T lymphocytes CD3+ CD8+ in bone marrow;

FIG. 14 shows the result of flow-based assay of PD-1 expression rate of CD3+ T lymphocytes in bone marrow;

FIG. 15 is a flow chart showing the expression rate of PD-1 from CD3+ CD4+ T lymphocytes in bone marrow;

FIG. 16 shows the flow-based assay of the expression rate of PD-1 in CD3+ CD8+ T lymphocytes in bone marrow.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

The material and the method are as follows:

the PD-1-APC fluorescent monoclonal antibody is purchased from Tianjin Kuangbo Biotechnology Ltd, a product number A6024R 12;

the CD45-QB500 fluorescent monoclonal antibody is purchased from Tianjin Kuangbo Biotechnology Ltd, a cargo number A6015V 32;

the CD4-FITC/CD8-PE/CD3-percp fluorescent monoclonal antibody was purchased from Tianjin Kuangbo Biotechnology Co., Ltd, cat # Z6410011.

Example 1

The embodiment provides an antibody composition for detecting a lymphocyte surface PD-1 receptor, which comprises 10 parts by weight of a PD-1-APC fluorescent monoclonal antibody, 5 parts by weight of a CD45-QB500 fluorescent monoclonal antibody, 7 parts by weight of a CD3-percp fluorescent monoclonal antibody, 7 parts by weight of a CD4-FITC fluorescent monoclonal antibody and 7 parts by weight of a CD8-PE fluorescent monoclonal antibody;

wherein the CD45-QB500 fluorescent monoclonal antibody, the CD3-percp fluorescent monoclonal antibody and the CD4-FITC fluorescent monoclonal antibody are combined reagents of a CD4-FITC/CD8-PE/CD3-percp fluorescent monoclonal antibody, and 21 parts in total are added.

Example 2

The embodiment provides an antibody composition for detecting a lymphocyte surface PD-1 receptor, which comprises 8 parts by weight of a PD-1-APC fluorescent monoclonal antibody, 6 parts by weight of a CD45-QB500 fluorescent monoclonal antibody, 5 parts by weight of a CD3-percp fluorescent monoclonal antibody, 5 parts by weight of a CD4-FITC fluorescent monoclonal antibody and 5 parts by weight of a CD8-PE fluorescent monoclonal antibody;

the CD45-QB500 fluorescent monoclonal antibody, the CD3-percp fluorescent monoclonal antibody and the CD4-FITC fluorescent monoclonal antibody are combined reagents of a CD4-FITC/CD8-PE/CD3-percp fluorescent monoclonal antibody, and 15 parts in total are added.

Example 3

The embodiment provides an antibody composition for detecting a lymphocyte surface PD-1 receptor, which comprises 13 parts by weight of a PD-1-APC fluorescent monoclonal antibody, 4 parts by weight of a CD45-QB500 fluorescent monoclonal antibody, 12 parts by weight of a CD3-percp fluorescent monoclonal antibody, 12 parts by weight of a CD4-FITC fluorescent monoclonal antibody and 12 parts by weight of a CD8-PE fluorescent monoclonal antibody;

the kit comprises a CD45-QB500 fluorescent monoclonal antibody, a CD3-percp fluorescent monoclonal antibody and a CD4-FITC fluorescent monoclonal antibody, wherein the CD45-QB500 fluorescent monoclonal antibody, the CD3-percp fluorescent monoclonal antibody and the CD4-FITC fluorescent monoclonal antibody are combined reagents, namely a CD4-FITC/CD8-PE/CD3-percp fluorescent monoclonal antibody, and 36 parts in total are added.

Example 4

This example provides a kit for detecting a lymphocyte surface PD-1 receptor, which comprises the antibody composition for detecting a lymphocyte surface PD-1 receptor of example 1;

the kit for detecting the lymphocyte surface PD-1 receptor further comprises erythrocyte lysate and an auxiliary reagent, wherein the auxiliary reagent is phosphate buffer solution.

Example 5

The only difference from example 4 is that the antibody composition for detecting a lymphocyte surface PD-1 receptor in example 2 was used in place of the antibody composition for detecting a lymphocyte surface PD-1 receptor in example 1, and the remaining raw materials and components were the same as in example 4.

Example 6

The only difference from example 4 is that the antibody composition for detecting a lymphocyte surface PD-1 receptor in example 3 was used in place of the antibody composition for detecting a lymphocyte surface PD-1 receptor in example 1, and the remaining raw materials and components were the same as in example 4.

Test example 1

In this test example, the kit for detecting a lymphocyte surface PD-1 receptor in example 4 was used to detect the expression rate of the lymphocyte surface PD-1 receptor in a blood sample, and the following steps were performed:

(1) taking two branch flow type sample loading tubes, and adding 10 parts of PD-1-APC fluorescent monoclonal antibody, 5 parts of CD45-QB500 fluorescent monoclonal antibody and 21 parts of CD4-FITC/CD8-PE/CD3-percp fluorescent monoclonal antibody at the bottom of one tube; the other one was added with 5 parts of CD45-QB500 fluorescent monoclonal antibody and 21 parts of CD4-FITC/CD8-PE/CD3-percp fluorescent monoclonal antibody (as a negative control).

(2) Respectively sucking 100 mu L of uniformly mixed peripheral blood samples, and respectively adding the samples to the tube bottoms of the two flow-type sample loading tubes to avoid the samples from touching the tube walls of the flow-type sample loading tubes.

(3) Vortexed and mixed, incubated at room temperature for 20 minutes in the absence of light.

(4) 1mL of erythrocyte lysate is added into each of the two tubes, vortexed and mixed, and incubated for 10 minutes at room temperature in the dark.

(5) Centrifuge at 300g for 5 minutes and discard the supernatant.

(6) 3mL of phosphate buffer solution was added to each of the two tubes, vortexed and mixed, and the mixture was centrifuged at 300g for 5 minutes, and the supernatant was discarded.

(7) 0.5mL of phosphate buffer was added to each tube and mixed thoroughly.

(8) And filtering suspended matters in the sample by using a 200-mesh filter screen, and detecting by using an up-flow cytometer.

(9) And (3) analysis:

setting a lymphocyte gate on CD45/SSC, and stopping sample suction after 1 ten thousand lymphocytes are obtained;

using a flow cytometer circle, the PD-1 receptor expression rate of each population of lymphocytes was obtained.

The detection results are shown in fig. 1 to 8, wherein fig. 1 is a flow detection result of CD3+ T lymphocytes in peripheral blood; FIG. 2 shows the flow-based detection of CD3+ CD4+ T lymphocytes and CD3+ CD8+ T lymphocytes in peripheral blood; FIG. 3 shows the flow-based assay of a negative control for the expression of PD-1 from CD3+ T lymphocytes in peripheral blood; FIG. 4 shows the results of flow-based detection of negative control for PD-1 expression of T lymphocytes of CD3+ CD4+ in peripheral blood; FIG. 5 shows the results of flow-based detection of negative control for PD-1 expression of T lymphocytes of CD3+ CD8+ in peripheral blood; FIG. 6 shows the result of flow measurement of the PD-1 expression rate of CD3+ T lymphocytes in peripheral blood; FIG. 7 shows the flow-based measurement of the PD-1 expression rate of T lymphocytes of CD3+ CD4+ in peripheral blood; FIG. 8 shows the flow-based measurement of the expression rate of PD-1 in CD3+ CD8+ T lymphocytes in peripheral blood.

The picture data were collated, and the results are shown in table 1.

TABLE 1

Item	Results (%)	Reference value (%)	Prompting
				Total T lymphocytes	44.1	56.0～86.0	↓
Helper T lymphocyte	42.3	27.0～51.0	-
				Cytotoxic T lymphocytes	50.7	15.0～44.0	↑
Total T lymphocyte PD-1 expression Rate	34.2	5.61～29.43	↑
				PD-1 expression rate of helper T lymphocyte	36.4	6.60～34.34	↑
Cytotoxic T lymphocyte PD-1 expression Rate	42.9	4.21～44.89	-

As can be seen from the above-mentioned graphs in the table, the proportion of total T lymphocytes in the sample is low, the proportion of cytotoxic T lymphocytes of CD3+ CD8+ is high, the PD-1 expression rates of both total T lymphocytes and helper T lymphocytes are increased, and the proliferation of T lymphocytes is inhibited.

The results show that the antibody composition for detecting the lymphocyte surface PD-1 receptor can be used for quickly and accurately detecting the expression condition of the lymphocyte surface PD-1 receptor in a peripheral blood sample by matching with a flow detection technology, and has the advantages of simple method, short time, accurate result and important significance in clinical detection and medication guidance.

Test example 2

This test example used the kit for detecting a lymphocyte surface PD-1 receptor of example 5 to detect the expression rate of a lymphocyte surface PD-1 receptor in a blood sample in the same manner as in test example 1.

Test example 3

This test example used the kit for detecting a lymphocyte surface PD-1 receptor of example 6 to detect the expression rate of a lymphocyte surface PD-1 receptor in a blood sample in the same manner as in test example 1.

The results showed that the test results of test examples 2 and 3 were very close to the test result of test example 1. For the sake of brevity, no further description is provided herein.

Test example 4

In this test example, the kit for detecting a lymphocyte surface PD-1 receptor in example 4 was used to detect the expression rate of a lymphocyte surface PD-1 receptor in a bone marrow sample, and the following steps were performed:

(2) Respectively sucking 100 mu L of the uniformly mixed bone marrow fluid sample, and respectively adding the bone marrow fluid sample to the tube bottoms of the two flow-type sample loading tubes to avoid the sample from touching the tube walls of the flow-type sample loading tubes.

(5) Centrifuge at 300g for 5 minutes and discard the supernatant.

(7) 0.5mL of phosphate buffer was added to each tube and mixed thoroughly.

(9) And (3) analysis:

The detection results are shown in fig. 9 to 16, wherein fig. 9 is a flow detection result of CD3+ T lymphocytes in bone marrow; FIG. 10 shows the flow-through assay of CD3+ CD4+ T lymphocytes and CD3+ CD8+ T lymphocytes in bone marrow; FIG. 11 is a flow chart of a negative control for PD-1 expression of CD3+ T lymphocytes in bone marrow; FIG. 12 is a flow chart of a negative control for PD-1 expression of T lymphocytes CD3+ CD4+ in bone marrow; FIG. 13 is a flow chart of a negative control for PD-1 expression of T lymphocytes CD3+ CD8+ in bone marrow; FIG. 14 shows the result of flow-based assay of PD-1 expression rate of CD3+ T lymphocytes in bone marrow; FIG. 15 is a flow chart showing the expression rate of PD-1 from CD3+ CD4+ T lymphocytes in bone marrow; FIG. 16 shows the flow-based assay of the expression rate of PD-1 in CD3+ CD8+ T lymphocytes in bone marrow.

The picture data were collated, and the results are shown in table 2.

TABLE 2

As can be seen from the above pictures and tables, the bone marrow samples showed slightly higher cytotoxic T lymphocyte ratio of CD3+ CD8+, and increased PD-1 expression rates of total T lymphocytes and helper T lymphocytes.

The results show that the antibody composition for detecting the lymphocyte surface PD-1 receptor can be used for quickly and accurately detecting the expression condition of the lymphocyte surface PD-1 receptor in a bone marrow sample by matching with a flow detection technology, has the advantages of simple method, short time and accurate result, and has important significance in clinical detection and medication guidance.

In conclusion, the antibody composition for detecting the lymphocyte surface PD-1 receptor can qualitatively distinguish the lymphocytes and respectively detect the expression quantity of the lymphocyte surface PD-1 receptor of each group, and has accurate result, good sensitivity and high detection efficiency; the detection method is easy to operate, short in time consumption, realizes continuous detection and dynamic observation of related samples, is beneficial to judging the immune state and disease course progress of an organism, and provides guidance for drug treatment.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. An antibody composition for detecting PD-1 receptor on lymphocyte surface, characterized in that, the antibody composition for detecting PD-1 receptor on lymphocyte surface comprises anti-PD-1 antibody, anti-CD45 antibody, anti-CD45 antibody, Any one or a combination of at least two of CD3 antibody, anti-CD4 antibody or anti-CD8 antibody.

2. The antibody composition for detecting PD-1 receptor on the surface of lymphocytes according to claim 1, wherein the anti-PD-1 antibody comprises a PD-1-APC fluorescent monoclonal antibody;

Preferably, the anti-CD45 antibody comprises CD45-QB500 fluorescent monoclonal antibody;

Preferably, the anti-CD3 antibody comprises CD3-percp fluorescent monoclonal antibody;

Preferably, the anti-CD4 antibody comprises CD4-FITC fluorescent monoclonal antibody;

Preferably, the anti-CD8 antibody comprises a CD8-PE fluorescent monoclonal antibody.

3. The antibody composition for detecting PD-1 receptor on lymphocyte surface according to claim 2, characterized in that, in parts by weight, the antibody composition for detecting PD-1 receptor on lymphocyte surface comprises PD -1-APC fluorescent monoclonal antibody 8-13 copies, CD45-QB500 fluorescent monoclonal antibody 4-6 copies, CD3-percp fluorescent monoclonal antibody 5-12 copies, CD4-FITC fluorescent monoclonal antibody 5-12 copies and CD8 -5 to 12 copies of PE fluorescent monoclonal antibodies.

4. A kit for detecting PD-1 receptors on lymphocytes, wherein the kit for detecting PD-1 receptors on lymphocytes comprises the detection kit according to any one of claims 1 to 3. Antibody compositions for surface PD-1 receptors.

5. The kit for detecting PD-1 receptors on lymphocytes according to claim 4, wherein the kit for detecting PD-1 receptors on lymphocytes further comprises erythrocyte lysate and auxiliary reagents;

Preferably, the auxiliary reagent includes phosphate buffered saline.

6. A method for detecting the expression rate of PD-1 receptor on the surface of lymphocytes, wherein the method comprises:

Using the antibody composition for detecting the PD-1 receptor on the lymphocyte surface according to any one of claims 1 to 3 and/or the reagent for detecting the PD-1 receptor on the lymphocyte surface according to any one of claims 4 to 5 box to test the sample.

7. The method for detecting the expression rate of PD-1 receptor on the surface of lymphocytes according to claim 6, wherein the method comprises:

Mix the sample with the antibody composition for detecting PD-1 receptor on the surface of lymphocytes, incubate in the dark, lyse the red blood cells, detect and analyze on the machine.

8. The method for detecting the expression rate of PD-1 receptor on the surface of lymphocytes according to claim 7, wherein the analysis comprises:

Set the lymphocyte gate to obtain lymphocytes;

Circle the figure in the lymphocytes to obtain the PD-1 receptor expression rate of each group of lymphocytes.

9. The method for detecting the expression rate of PD-1 receptor on the surface of lymphocytes according to claim 7 or 8, wherein the method comprises:

Mix the sample with the antibody composition for detecting PD-1 receptor on the surface of lymphocytes, and incubate in the dark;

Use red blood cell lysate to lyse red blood cells and wash them;

Collect cells, test on the machine, and analyze;

The analysis includes:

Set the lymphocyte gate to obtain lymphocytes;

10. The antibody composition for detecting PD-1 receptor on the surface of lymphocytes according to any one of claims 1 to 3, and the kit for detecting PD-1 receptor on the surface of lymphocytes according to any one of claims 4 to 5 Or the application of the method for detecting the expression rate of PD-1 receptor on the surface of lymphocytes according to any one of claims 6 to 9 in detecting the expression of PD-1 receptor on the surface of lymphocytes.