阅读说明：本技术 一种检测pd-l1和cd8抗原的免疫荧光试剂盒及应用方法 (Immunofluorescence kit for detecting PD-L1 and CD8 antigens and application method ) 是由 郭志敏 樊晓婷 郭素杰 于 2019-06-12 设计创作，主要内容包括：本发明涉及一种检测PD-L1和CD8抗原的免疫荧光试剂盒及应用其的检测方法；所述的试剂盒包括以下试剂：缓冲液CYP1、CYP2、CYPP、封闭液、检测PD-L1的特异性抗体以及相应的荧光二抗、荧光标记的CD45抗体、荧光标记的CD8抗体、抗体稀释液、细胞核染色液。本发明还提出应用所述的试剂盒进行抗原检测的方法。本发明的试剂盒检测结果可以对实际用药进行指导,不仅能够反映整体肿瘤,包括原位癌和可能存在的不可见的微小转移灶负荷,同样可以反映CTC中PD-L1的表达情况；根据PBMC中PD-L1和CD8阳性细胞比例,可以为治疗药物的精准选择提供参考；在取材上比基于肿瘤组织的检测技术更加方便。(The invention relates to an immunofluorescence kit for detecting PD-L1 and CD8 antigens and a detection method using the immunofluorescence kit; the kit comprises the following reagents: buffer solutions CYP1, CYP2, CYPP, a confining liquid, a specific antibody for detecting PD-L1, a corresponding fluorescent secondary antibody, a fluorescent-labeled CD45 antibody, a fluorescent-labeled CD8 antibody, an antibody diluent and a cell nucleus staining solution. The invention also provides a method for detecting antigen by using the kit. The detection result of the kit can guide the actual medication, not only can reflect the whole tumor including in-situ cancer and the possible invisible micrometastasis load, but also can reflect the expression condition of PD-L1 in CTC; according to the proportion of PD-L1 and CD8 positive cells in PBMCs, a reference can be provided for accurate selection of therapeutic drugs; the method is more convenient in material drawing than the detection technology based on the tumor tissue.)

1. An immunofluorescence kit for detecting PD-L1 and CD8 antigens comprises the following reagent components:

name (R) Number of Unit of Total volume/mass CYP1 buffer solution 1 Bottle (Ref. TM. bottle) 80ml CYP2 buffer solution 1 Bottle (Ref. TM. bottle) 15ml CYPP buffer solution 1 Pipe 4.5ml Sealing liquid 1 Pipe 3ml PD-L1 antibody 1 Pipe 200μl CD8 antibody 1 Pipe 100μl CD45 antibody 1 Pipe 200μl Fluorescent secondary antibody 1 Pipe 200μl Antibody diluent 1 Pipe 3ml Dyeing liquid 1 Pipe 300μl

Wherein, the CYP1 buffer solution is 0.01M PBS, purified water is added, the mixture is fully stirred, dissolved and adjusted to pH value of 7.20-7.40, and then split charging is carried out;

the CYP2 buffer solution is prepared by adding 0.1 percent Triton X-100 and 0.5 percent Trehalose into 0.01M PBS solution, fully stirring and dissolving, and subpackaging;

the CYPP buffer solution is prepared by adding 0.2 percent Triton X-100 into 0.01M PBS solution, fully stirring and dissolving, and subpackaging;

the confining liquid contains PBS and BSA or selects animal serum and 0.5-10% BSA or Fc receptor antibody;

the PD-L1 antibody is 10 times concentrated solution of the PD-L1 antibody, and is subpackaged in 1.5ml of brown light-avoiding tube;

the CD8 antibody is Alexa

the CD45 antibody is Alexa

the fluorescent secondary antibody is Alexa

the antibody diluent contains Tris-HCl buffer solution and bovine serum albumin;

the staining solution is prepared by taking DAPI powder, diluting with ultrapure water, adding an anti-quenching agent ThermoFisher,subpackaging; or Hoechst or propidium iodide.

2. The immunofluorescence kit for detecting PD-L1 and CD8 antigens as claimed in claim 1, wherein the buffer solution CYP1, CYP2, CYPP contains surfactant and protein protective agent, and the surfactant can adopt 0-10% of Triton X-100, saponin, Tween, NP-40, SDS or polyethylene glycol; the protein protective agent can adopt 0-10% of BSA, Trehalose (Trehalose) or sucrose.

3. A method for detecting PD-L1 and CD8 antigens using the kit of claim 1 or 2, wherein the method comprises the steps of:

step one, CTC enrichment and PBMC smear, which comprises the following specific operation steps:

s1, taking blood and other body fluid samples, and enriching CTC in the blood and other body fluid samples;

s2, taking peripheral blood, and separating and smearing PBMCs in the peripheral blood;

step two, carrying out immunofluorescence detection on the enriched CTC and PBMC cell smear, wherein the specific detection method comprises the following steps:

s1 and CYP1 washing the glass slide for 3min multiplied by 3 times, each time is 100-150 mu L, and the whole specimen area is covered completely;

s2, absorbing the redundant liquid on the glass slide, adding CYPP to act for 5min, and washing CYP1 for 3min multiplied by 3 times; sucking off redundant liquid, adding 100-150 mul of closed liquid, and sealing at a high temperature for 20-30 min;

s3, sucking out redundant sealing liquid, adding 100 mu L of diluted PD-L1 antibody and CD45 antibody or CD8 antibody, and incubating for 1-2 h in a room-temperature wet box in a dark place; wherein CTC is incubated with PD-L1+ CD45 antibody and PBMC cell smears are incubated with CD8 and PD-L1+ CD45 antibody, respectively;

s4, keeping out of the sun, taking CYP 2100-150 mu L/film for 3min multiplied by 3 times, and absorbing excessive water;

s5, adding 100 mu l of diluted fluorescent secondary antibody, and incubating in a wet box in a dark place; 25-30 min at 37 ℃ or 30-60 min at room temperature; CYP2 washing 3min x 4 times, absorbing excessive water;

s6, counterdyeing: adding 10 mu L of DAPI staining solution into the specimen area;

s7, covering a cover glass, sucking peripheral liquid, and storing in the dark at 2-8 ℃ or-20 ℃ for the next step;

step three, microscopic observation is carried out on the sample: placing the CTC sample under a fluorescence microscope, scanning the whole sample area, and switching to a high power microscope for further identification if a sheet-shaped or ring-shaped signal is found; recording the coordinates of each positive cell, and taking pictures of 40 × objective lens under different channels respectively; the observation can be carried out by using an oil lens when necessary; scanning the whole sample area by placing the PBMC sample under a fluorescence microscope, and recording the number of PD-L1 or CD8 positive cells and the total number of cells;

step four, judging results, specifically:

CTC positive cells: marking the target protein nucleated cell with positive fluorescence signal and no fluorescence signal of the surface antigen of the blood-borne leucocyte as a positive cell;

CTC negative cells: the cells have blood-derived leukocyte surface antigen fluorescence signals, and whether the target protein has fluorescence or not is marked as negative;

PBMC positive cells: the target protein is positive in fluorescence signal and has complete cell nucleus, and the target protein is counted as a positive cell;

PBMC negative cells: the fluorescence signals of the target protein are negative and are all marked as negative.

4. The method for detecting the PD-L1 and CD8 antigens as claimed in claim 3, wherein the CTC detection in the detection method is combined with a fluorescence in situ hybridization technique, and the specific steps are any one of the following steps: (i) performing immunofluorescence detection and then performing fluorescence in situ hybridization; (ii) firstly, carrying out fluorescence in situ hybridization and then carrying out immunofluorescence detection; (iii) and performing immunofluorescence detection and fluorescence in situ hybridization at the same time.

5. The method for detecting the antigen PD-L1 and CD8 according to claim 3 or 4, characterized in that the method further comprises the step of processing the sample using a repair method comprising enzymatic digestion repair, EDTA repair, autoclaving or microwave repair.

6. The method for detecting the antigens PD-L1 and CD8 as claimed in claim 5, wherein said method further comprises the step of immunofluorescence detection of the enriched cells against the antibody PD-L1 and the leukocyte common antigen CD 45.

7. The method for detecting PD-L1 and CD8 antigen as claimed in claim 3 or 4, wherein in the step S1 in the first step, various subtypes of CTC are obtained by a negative enrichment method of removing plasma, red blood cells and white blood cells; then, the slide is naturally dried under the condition of room temperature or constant humidity of 20 percent of room temperature, and cells and antigens are fixed by using a fixing agent.

8. The method for detecting the PD-L1 and CD8 antigens as claimed in claim 3 or 4, wherein the step S2 in the first step is performed by the following steps:

(1) taking 0.5ml of peripheral blood, adding 0.5ml of 1 XPBS, and blowing, beating and uniformly mixing;

(2) separating 0.5ml of lymphocytes, and slightly superposing the uniformly mixed peripheral blood on the upper layer of the lymphocyte separation layer;

(3) centrifuging at 1500rpm for 5 min;

(4) carefully aspirating the centrifuged buffy coat to recover as many cells as possible without mixing with the red blood cell pellet;

(5) adding 10ml of 1 XPBS, reversing, uniformly mixing, centrifuging at 1500rpm for 5 min;

(6) discarding the supernatant, resuspending with 0.5ml of 1 × PBS, blowing, mixing uniformly, counting by a blood counting plate, and smearing after proper dilution;

(7) naturally drying the glass slide by using the room temperature or the constant humidity condition of 20 percent of the room temperature;

(8) the cells and antigens are fixed using a fixative.

9. The method of claim 8, wherein the fixative is selected from the group consisting of ethanol, methanol, formalin, acetone, glacial acetic acid, formaldehyde, paraformaldehyde, glutaraldehyde, acrolein, picric acid, PLP solution, and ECD-G solution.

10. The method for detecting the antigen PD-L1 and CD8 of claim 3 or 4, wherein the exclusion criteria in the judgment of the result in the fourth step is that if the fragmentation of the cell nucleus can not be determined to be a single cell, the aggregation of multiple cell nuclei can not be marked as a positive cell.

Technical Field

The invention relates to a medical detection method, in particular to detection of cancer cells in body fluid and detection of immune-related cells in PBMC, and particularly relates to a kit for performing programmed death-ligand (PD-L1) antigen immunofluorescence detection in circulating tumor cells and detection of the proportion of PD-L1 and CD8 in PBMC, which is suitable for detection of PD-L1 and CD8 of different types of samples, and also relates to an application method of the kit in detection of PD-L1 and CD 8.

Background

At present, the incidence of cancer is high, and the health of human beings is seriously threatened. Some cancers are difficult to find in early stage, and are often diagnosed in late stage, such as lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer and the like. Accordingly, the 5-year survival rate for advanced cancer is significantly lower than for early stage patients, and early detection of accurate treatment for early diagnosis can significantly prolong the survival time of patients.

Costimulatory molecules are a class of accessory molecules involved in immune responses, including both positive molecules that promote T cell proliferation and negative molecules that induce and maintain T cell immune tolerance to apoptosis. A costimulation path formed by programmed death factors (PD-1 and PD-L1(programmed death-ligand 1, also called B7-H1) has an important regulation function on the activation of lymphocytes, inhibits the proliferation and over-activation of T cells by combining with receptor PD-1, plays a role in negative regulation in the process of cellular immune response, and plays a role in regulating humoral immunity by influencing the secretion of cytokines. The PD-L1/PD1 path plays an important role in the immune monitoring process of the body in the autoimmune tolerance and the escape of tumor cells. The molecular B7-H1 (also called PD-L1) with similar structure to immunoglobulin on the surface of tumor cells is expressed in a large amount in the tumor microenvironment to inhibit the killing of the lymphocytes to the tumor. The blocking of PD-1/PD-L1 binding by specific antibodies can enhance immune response, antibody therapy (anti-PD therapy for short) designed based on this principle to block the binding of PD-1 and PD-L1 has been used on thousands of cancer patients, and has proven significant efficacy in more than ten advanced cancers, including lung cancer, kidney cancer, melanoma, head and neck cancer, bladder cancer, breast cancer, liver cancer, stomach cancer, esophageal cancer, brain glioma, colon cancer, Hodgkin's lymphoma, etc., and PD therapy is the most effective immunotherapy at present.

Depending on the presence or absence or the amount of infiltrating immune cells in the tumor tissue microenvironment, these can be classified into immunogenic tumors (i.e., hot tumors) and non-immunogenic tumors (i.e., cold tumors), which are thought to exist in hot tumors, but the killing of T cells is inhibited by the expression of tumor cells PD-L1. The treatment with the immune monitoring point inhibitor can relieve the immune suppression of immune cells, so that tumor cells can be killed. For the patients, the effectiveness of the PD-1/PD-L1 antibody used alone is higher, and the survival time is longer. For "cold" tumor patients with no or few immune cells in the tumor tissue, the immune checkpoint inhibitor has poor curative effect, even if the PD-L1 molecule on the tumor cell is blocked, the tumor tissue cannot be effectively killed due to the lack of the corresponding immune cells, and the immune cells are required to be gathered in the tumor tissue in combination with other treatment methods.

At present, tumor diagnosis mainly depends on three methods, namely pathology, imaging and serology. Pathology is the gold standard for tumor diagnosis, but tissue sampling is somewhat traumatic and limited to the site of material selection, while due to the heterogeneity of tumor tissue, single-point tissue sampling is not sufficient to reflect the overall tumor status and dynamic monitoring of repeated sampling is difficult. Imaging methods have a wide range of roles in diagnosing tumors, but imaging generally suffers from radiation damage. In addition, imaging generally reflects only information such as tumor size, and it is difficult to determine the malignancy of a tumor. More importantly, the imaging can only detect the tumor tissue with the diameter of more than 2mm, and the diagnosis sensitivity of the smaller tumor tissue is poor, so that the hysteresis exists. Serology is generally used for assisting in judging information such as cancer treatment curative effect at present, and sampling is convenient, but sensitivity and specificity are poor, and correlation with pathophysiology is poor. Similarly, there are some limitations in clinical practice at present in terms of treatment. For tumor patients, especially patients with metastasis, the treatment scheme is usually judged only according to information of primary foci at present, and detection and treatment of metastatic foci and even micrometastasis foci are omitted. In the treatment process, the treatment effect is mainly judged by imaging or serology at present, and certain limitation exists in the aspects of accuracy of judgment of the treatment effect and the like.

At present, the detection of the expression level of PD-L1 is mainly carried out by an immunohistochemical method, and the detection of PD-L1 in situ cancer tissues cannot directly reflect the level of PD-L1 in body fluid and metastasis due to the existence of tumor heterogeneity and invisible reasons of micrometastases. Detection of PD-L1 in tissues was performed on tissue sections, and individual sections did not reflect the overall PD-L1 expression level of the patient. And the detection of the expression level of the PD-L1 of a tumor patient alone cannot accurately reflect the effect of the patient receiving the PD treatment.

Therefore, a new method is urgently needed, the overall PD-L1 expression level of a tumor patient and the immune cell state of the patient are detected, the detection results of the two are combined to predict the effect of the patient on PD treatment, a reference is provided for selecting a disease treatment scheme, and clinical application is better guided. Detection of PD-L1 in Circulating Tumor Cells (CTCs) in combination with detection of PD-L1 and CD8 in PBMCs may provide some solutions to the above limitations. CTC refers to tumor cells that fall off from primary foci or metastases into the blood circulation during tumor formation or progression, and detection of CTC suggests the presence of primary foci or metastases in the body, or the presence of precancerous lesion cells, which are "seed" cells for formation of metastases and direct evidence of tumor development. In recent years there has been growing evidence to show: the tumor disseminates into the blood early in the development of the tumor, even before the formation of the imagewise visible lesion, and this dissemination may be present throughout the tumor development. Thus, detection of CTCs facilitates early tumor discovery, aids in diagnosis, or provides a reference for selection of treatment regimens. Meanwhile, the combination of dynamic detection of the expression level of PD-L1 in CTC and the ratio of PD-L1 and CD8 in PBMC can understand the sensitivity of patients to therapeutic drugs and discover drug resistance signals early.

Disclosure of Invention

The invention aims to solve the problem that an immunofluorescence method is not adopted in the prior art to simultaneously detect the expression level of PD-L1 in CTC and the proportion of PD-L1 and CD8 positive cells in PBMC, and provides an immunofluorescence kit for detecting PD-L1 and CD8 antigens and an application method thereof.

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

an immunofluorescence kit for detecting PD-L1 and CD8 antigens comprises the following reagent components:

name (R)	Number of	Unit of	Total volume/mass
				CYP1 buffer solution	1	Bottle (Ref. TM. bottle)	80ml
CYP2 buffer solution	1	Bottle (Ref. TM. bottle)	15ml
				CYPP buffer solution	1	Pipe	4.5ml
Sealing liquid	1	Pipe	3ml
				PD-L1 antibody	1	Pipe	200μl
CD8 antibody	1	Pipe	100μl
				CD45 antibody	1	Pipe	200μl
Fluorescent secondary antibody	1	Pipe	200μl
				Antibody diluent	1	Pipe	3ml
Dyeing liquid	1	Pipe	300μl

Wherein, the CYP1 buffer solution is 0.01M PBS, purified water is added, the mixture is fully stirred, dissolved and adjusted to pH value of 7.20-7.40, and then split charging is carried out;

the CYP2 buffer solution is prepared by adding 0.1 percent Triton X-100 and 0.5 percent Trehalose into 0.01M PBS solution, fully stirring and dissolving, and subpackaging;

the CYPP buffer solution is prepared by adding 0.2 percent Triton X-100 into 0.01M PBS solution, fully stirring and dissolving, and subpackaging;

the confining liquid contains PBS and BSA or selects animal serum and 0.5-10% BSA or Fc receptor antibody;

the PD-L1 antibody is 10 times concentrated solution of the PD-L1 antibody, and is subpackaged in 1.5ml of brown light-avoiding tube;

the CD8 antibody is Alexa

594 labeled CD8 antibody, dispensed in 1.5ml brown light-avoiding tube;

the CD45 antibody is Alexa647 labeled CD45 antibody, dispensed in 1.5ml brown light-avoiding tube;

the fluorescent secondary antibody is Alexa

488-labeled fluorescent secondary antibody is subpackaged in 1.5ml of brown light avoiding tube;

the antibody diluent contains Tris-HCl buffer solution and bovine serum albumin;

the staining solution is prepared by taking DAPI powder, diluting with ultrapure water, adding an anti-quenching agent ThermoFisher,

subpackaging; or Hoechst or propidium iodide.

Preferably, the buffer solutions CYP1, CYP2 and CYPP contain a surfactant and a protein protective agent, and the surfactant can adopt 0-10% of Triton X-100, saponin, Tween, NP-40, SDS or polyethylene glycol; the protein protective agent can adopt 0-10% of BSA, Trehalose (Trehalose) or sucrose.

According to another object of the present invention, the method for detecting PD-L1 and CD8 antigens by using the above kit comprises the following steps:

step one, CTC enrichment and PBMC smear, which comprises the following specific operation steps:

s1, taking blood and other body fluid samples, and enriching CTC in the blood and other body fluid samples;

s2, taking peripheral blood, and separating and smearing PBMCs in the peripheral blood;

step two, carrying out immunofluorescence detection on the enriched CTC and PBMC cell smear, wherein the specific detection method comprises the following steps:

s1 and CYP1 washing the glass slide for 3min multiplied by 3 times, each time is 100-150 mu L, and the whole specimen area is covered completely;

s2, absorbing the redundant liquid on the glass slide, adding CYPP to act for 5min, and washing CYP1 for 3min multiplied by 3 times; sucking off redundant liquid, adding 100-150 mul of closed liquid, and sealing at a high temperature for 20-30 min;

s3, sucking out redundant sealing liquid, adding 100 mu L of diluted PD-L1 antibody and CD45 antibody or CD8 antibody, and incubating for 1-2 h in a room-temperature wet box in a dark place; wherein CTC is incubated with PD-L1+ CD45 antibody and PBMC cell smears are incubated with CD8 and PD-L1+ CD45 antibody, respectively;

s4, keeping out of the sun, taking CYP 2100-150 mu L/film for 3min multiplied by 3 times, and absorbing excessive water;

s5, adding 100 mu l of diluted fluorescent secondary antibody, and incubating in a wet box in a dark place; 25-30 min at 37 ℃ or 30-60 min at room temperature; CYP2 washing 3min x 4 times, absorbing excessive water;

s6, counterdyeing: adding 10 mu L of DAPI staining solution into the specimen area;

s7, covering a cover glass, sucking peripheral liquid, and storing in the dark at 2-8 ℃ or-20 ℃ for the next step;

step three, microscopic observation is carried out on the sample: placing the CTC sample under a fluorescence microscope, scanning the whole sample area, and switching to a high power microscope for further identification if a sheet-shaped or ring-shaped signal is found; recording the coordinates of each positive cell, and taking pictures of 40 × objective lens under different channels respectively; the observation can be carried out by using an oil lens when necessary; scanning the whole sample area by placing the PBMC sample under a fluorescence microscope, and recording the number of PD-L1 or CD8 positive cells and the total number of cells;

step four, judging results, specifically:

CTC positive cells: marking the target protein nucleated cell with positive fluorescence signal and no fluorescence signal of the surface antigen of the blood-borne leucocyte as a positive cell;

CTC negative cells: the cells have blood-derived leukocyte surface antigen fluorescence signals, and whether the target protein has fluorescence or not is marked as negative;

PBMC positive cells: the target protein is positive in fluorescence signal and has complete cell nucleus, and the target protein is counted as a positive cell;

PBMC negative cells: the fluorescence signals of the target protein are negative and are all marked as negative.

Preferably, the detection method combines CTC detection and fluorescence in situ hybridization, and comprises the following specific steps: (i) performing immunofluorescence detection and then performing fluorescence in situ hybridization; (ii) firstly, carrying out fluorescence in situ hybridization and then carrying out immunofluorescence detection; (iii) and performing immunofluorescence detection and fluorescence in situ hybridization at the same time.

Still preferably, the detection method further comprises a step of processing the sample using a repair method including enzymatic digestion repair, EDTA repair, high pressure repair, or microwave repair.

Preferably, the detection method further comprises the step of performing immunofluorescence detection on the enriched cells against the PD-L1 antibody and the leukocyte common antigen CD 45.

More preferably, in step S1 of the first step, various subtypes of CTCs are obtained by a negative enrichment method of removing plasma, red blood cells and white blood cells; then, naturally drying the glass slide under the condition of room temperature or constant humidity of 20 percent of room temperature, and fixing cells and antigens by using a fixing agent;

still preferably, in the step S2 of the step one, the following method is adopted:

(1) taking 0.5ml of peripheral blood, adding 0.5ml of 1 XPBS, and blowing, beating and uniformly mixing;

(2) separating 0.5ml of lymphocytes, and slightly superposing the uniformly mixed peripheral blood on the upper layer of the lymphocyte separation layer;

(3) centrifuging at 1500rpm for 5 min;

(4) carefully aspirating the centrifuged buffy coat to recover as many cells as possible without mixing with the red blood cell pellet;

(5) adding 10ml of 1 XPBS, reversing, uniformly mixing, centrifuging at 1500rpm for 5 min;

(6) discarding the supernatant, resuspending with 0.5ml of 1 × PBS, blowing, mixing uniformly, counting by a blood counting plate, and smearing after proper dilution;

(7) naturally drying the glass slide by using the room temperature or the constant humidity condition of 20 percent of the room temperature;

(8) the cells and antigens are fixed using a fixative.

Still preferably, the fixative is selected from ethanol, methanol, formalin, acetone, glacial acetic acid, formaldehyde, paraformaldehyde, glutaraldehyde, acrolein, picric acid, PLP solution or ECD-G solution.

Preferably, the exclusion criteria in the judgment of the result in the fourth step is that if the fragmentation of the cell nucleus cannot be determined to be a single cell, the aggregation of a plurality of cell nuclei cannot be marked as a positive cell.

Drawings

FIG. 1 shows the 3 fluorescence displays of the same sample, which are PD-L1, leukocyte universal antigen CD45, nuclear dye DAPI, and FIG. respectively.

Detailed Description

In order to further understand the objects, structures, features and functions of the present invention, the following embodiments are described in detail.

The detection principle of the invention is that firstly, a known method is adopted to enrich circulating tumor cells and other rare cells, wherein the enrichment method can adopt the methods described in Chinese patents 200810097889.4, 201310057307.0 and 2015104411229; then, according to the antigen-antibody reaction principle, the expression of the target protein in the enriched cells is detected by adopting a known immunofluorescence detection method. The kit of the invention carries out fluorescence labeling on a target cell and leukocyte common antigen CD45, and screens target protein positive and CD45 negative cells, thereby judging and counting CTC positive to specific protein in blood. On the basis, the target protein positive and CD45 negative candidate non-humoral rare nucleated cells are classified according to the fluorescence intensity of the target protein and the expression of the target protein is classified according to high, medium and low expression.

However, the application of immunofluorescence detection method to the detection of PD-L1 expression in CTCs has several difficulties in practice, such as easy detachment of cells from the slide, weak fluorescence, non-specific staining, high matrix background, etc.; meanwhile, how to rapidly and accurately detect the activation state of the immune system of a patient in the kit is also a difficult point to be solved. Through multiple screening researches, a suitable method is finally obtained, the difficulties are solved, meanwhile, verification is carried out in the practical process, and the detection precision and accuracy of the detection result are greatly improved compared with those of the prior art.

Specifically, the application method of the kit for detecting PD-L1 and CD8 antigens comprises the following steps:

step one, CTC enrichment and PBMC smear, which comprises the following specific operation steps:

s1, taking blood and other body fluid samples, wherein CTCs can be enriched: wherein the enrichment method can adopt any separation and enrichment method of CTC, such as erythrocyte removal, negative enrichment, positive enrichment, density gradient separation, membrane filtration, microfluidic method or flow cytometry; or coating a blood sample on a plurality of glass slides without enrichment; wherein the CTCs are tumor cells that are shed from primary foci or metastases into the blood circulation during tumor formation and progression, and are typically enriched using flow cytometry sorting, magnetic bead positive sorting, preferably negative enrichment; in a preferred embodiment, various subtypes of CTCs are obtained using a negative enrichment process that removes plasma, red blood cells, white blood cells; naturally drying the glass slide by using the room temperature or the constant humidity condition of 20 percent of the room temperature; fixing cells and antigens by using a fixing agent;

s2, taking peripheral blood, separating and smearing PBMCs in the peripheral blood: wherein the PBMC refers to peripheral blood mononuclear cells (peripheral blood mononuclear cells) and comprises lymphocytes, monocytes and the like, and can be obtained by separating a lymphocyte separation solution; in a preferred embodiment, the following method is used:

(1) taking 0.5ml of peripheral blood, adding 0.5ml of 1 XPBS, and blowing, beating and uniformly mixing;

(2) separating 0.5ml of lymphocytes, and slightly superposing the uniformly mixed peripheral blood on the upper layer of the lymphocyte separation layer;

(3) centrifuging at 1500rpm for 5 min;

(4) carefully aspirating the centrifuged buffy coat to recover as many cells as possible without mixing with the red blood cell pellet;

(5) adding 10ml of 1 XPBS, reversing, uniformly mixing, centrifuging at 1500rpm for 5 min;

(6) discarding the supernatant, resuspending with 0.5ml of 1 × PBS, blowing, mixing uniformly, counting by a blood counting plate, and smearing after proper dilution;

(7) naturally drying the glass slide by using the room temperature or the constant humidity condition of 20 percent of the room temperature;

(8) the cells and antigens are fixed using a fixative.

And (3) fixing the enriched sample by adopting a fixing solution, wherein the fixing solution is selected from the following components: ethanol, methanol, formalin, acetone, glacial acetic acid, formaldehyde, paraformaldehyde, glutaraldehyde, acrolein, picric acid, PLP liquid (periodate-lysine-paraformaldehyde fixed liquid), ECD-G liquid (carbodiimide-glutaraldehyde), and the like.

Step two, carrying out immunofluorescence detection on the enriched CTC and PBMC cell smear, wherein the specific detection method comprises the following steps:

s1 and CYP1 washing the glass slide for 3min multiplied by 3 times, each time is 100-150 mu L, and the whole specimen area is covered completely;

s2, absorbing the redundant liquid on the glass slide, adding CYPP to act for 5min, and washing CYP1 for 3min multiplied by 3 times; sucking off redundant liquid, adding 100-150 mul of closed liquid, and sealing at a high temperature for 20-30 min;

s3, sucking out redundant sealing liquid, adding 100 mu L of diluted PD-L1 antibody and CD45 antibody or CD8 antibody, and incubating for 1-2 h in a room-temperature wet box in a dark place; wherein CTC is incubated with PD-L1+ CD45 antibody and PBMC cell smears are incubated with CD8 and PD-L1+ CD45 antibody, respectively;

s4, keeping out of the sun, taking CYP 2100-150 mu L/film for 3min multiplied by 3 times, and absorbing excessive water;

s5, adding 100 mu l of diluted fluorescent secondary antibody, and incubating in a wet box in a dark place; 25-30 min at 37 ℃ or 30-60 min at room temperature; CYP2 washing 3min x 4 times, absorbing excessive water;

s6, counterdyeing: adding 10 mu L of DAPI staining solution into the specimen area;

and S7, covering a cover glass, sucking peripheral liquid, and storing in the dark at the temperature of 2-8 ℃ or-20 ℃ for the next step.

Step three, microscopic observation is carried out on the sample: placing the CTC sample under a fluorescence microscope, scanning the whole sample area, and switching to a high power microscope for further identification if a sheet-shaped or ring-shaped signal is found; recording the coordinates of each positive cell, and taking pictures of 40 × objective lens under different channels respectively; the observation can be carried out by using an oil lens when necessary; the PBMC samples were placed under a fluorescent microscope and the entire sample area was scanned and the number of PD-L1 or CD8 positive cells and total cells recorded.

Step four, judging results, specifically:

CTC positive cells: marking the target protein nucleated cell with positive fluorescence signal and no fluorescence signal of the surface antigen of the blood-borne leucocyte as a positive cell;

CTC negative cells: the cells have blood-derived leukocyte surface antigen fluorescence signals, and whether the target protein has fluorescence or not is marked as negative;

PBMC positive cells: the target protein is positive in fluorescence signal and has complete cell nucleus, and the target protein is counted as a positive cell;

PBMC negative cells: the fluorescence signals of the target protein are negative and are all marked as negative.

Exclusion criteria: if the fragmentation of the cell nucleus cannot be confirmed to be a single cell, or if a plurality of cell nuclei are aggregated, it cannot be marked as a positive cell.

In the above steps, fluorescein is required to be used, which can be directly labeled on the antibody or detected by a fluorescently labeled secondary antibody; the fluorescein labeling the antibody has a different emission spectrum, preferably a combination of green fluorescence (e.g., Alexa Fluor 488), red fluorescence (e.g., Alexa Fluor 594), or deep red fluorescence (e.g., Alexa Fluor 647); the Fluorescein for labeling the antibody is not limited to Alexa Fluor series Fluorescein, and a series of Fluorescein-labeled antibodies such as Dylight, cyanine dye, Fluorescein Isothiocyanate (FITC), biotin (biotin), quantum dots, Texas Red, Phycoerythrin (PE), phycocyanin (APC), rhodamine (rhodomine), or a combination of different series of Fluorescein-labeled antibodies can be used.

The CD45 antibody is an antibody against a leukocyte universal antigen for use in depleting blood-derived cells, and may be selected from the following cell surface antigens: CD3, CD11b, CD14, CD16, CD19, CD34, CD35, CD36, CD38, CD41, CD58, CD61, CD66b, CD235 a; immunofluorescence detection on tissues or cell lines may omit the CD45 antibody.

In addition, the CTC detection method of the invention can be used in conjunction with Fluorescence In Situ Hybridization (FISH) techniques, with any of the following steps: (i) performing immunofluorescence detection and then performing fluorescence in situ hybridization; (ii) firstly, carrying out fluorescence in situ hybridization and then carrying out immunofluorescence detection; (iii) and performing immunofluorescence detection and fluorescence in situ hybridization at the same time.

Buffer solutions CYP1, CYP2 and CYPP used in the detection process of the invention contain surfactants and protein protective agents, and the surfactants can adopt 0-10% of Triton X-100, saponin, Tween, NP-40, SDS and polyethylene glycol; the protein protective agent can adopt 0-10% of BSA, Trehalose (Trehalose) and sucrose; in a preferred embodiment, the confining liquid used in the detection process can also be selected from: animal serum, 0.5-10% BSA, Fc receptor antibody; in a preferred embodiment, the assay process can employ a remediation method to treat the sample, including enzymatic digestion remediation, EDTA remediation, high pressure remediation, microwave remediation, and the like.

The nuclear dye used in the detection process is not limited to DAPI, and Hoechst, propidium iodide and the like can be adopted; in immunofluorescence detection, incubation processes of specific antibodies and cells can be divided into liquid staining and solid staining, wherein the liquid staining is to prepare the cells into cell suspension, carry out operations such as cell permeabilization and antibody incubation in the suspension, and then transfer the cells to a glass slide for fixation and mounting; the solid staining is to transfer cells to a glass slide for fixation, and then carry out operations such as cell permeabilization, antibody incubation and the like.

The key part of the detection method of the present invention is that step S3 of step two uses a combination of antibodies, for which the present invention further comprises performing immunofluorescence detection on the enriched cells against the PD-L1 antibody and the leukocyte common antigen CD45, and performing immunofluorescence detection on PBMC cell smears using antibodies against PD-L1 and CD 8. The core of the immunofluorescence detection method is that an antibody combination aiming at PD-L1+ CD45 is added to detect CTC, antibodies aiming at PD-L1 and CD8 are added to respectively detect PBMC cell smears, and great change is generated in the aspect of detection results, so that the method is completely different from the existing method. These changes are manifested in: 1) the overall state of the metastatic tumor and the level of PD-L1+ CTC of the patient are reflected by the detection of PD-L1 in the CTC and the activation condition of the immune system of the patient is reflected by the detection of PD-L1 and CD8 in PBMC respectively. Combining the detection results of the two, predicting the curative effect of the PD treatment of the patient and guiding the patient to take medicine; 2) eliminating the interference of blood-borne cells by using a CD45 antibody in CTC detection; 3) through analyzing the CTC staining result, PD-L1 positive cells in the CTC can be divided into high, medium and low expression subtypes, and the result is more accurate;

in the second step of the detection method of the present invention, immunofluorescence detection is performed on the enriched CTCs, wherein the detection method is obtained by the present inventors through multiple experiments and data screening, and the screening process of relevant conditions is as follows:

in the step S2, absorbing the redundant liquid on the glass slide, adding CYPP for acting for 5min, adding 100-150 mul of closed liquid, and sealing for 20-30 min at the temperature;

the screening procedure was as follows (breakthrough reagents and blocking time):

in the step S3, 100-150 mul of diluted PD-L1 antibody and CD45 antibody or CD8 antibody are added, and incubation is carried out for 1-2 hours in a room-temperature wet box in the dark;

the screening procedure (antibody incubation, washing conditions) was as follows:

adding 100-150 mu L of diluted fluorescent secondary antibody in step S5 in the second step, incubating in a humid box in the dark at 37 ℃ for 25-30 min or at room temperature for 30-60 min;

the screening procedure was as follows (antibody incubation, washing conditions):

the smear condition and the smear method of the invention are obtained by the screening, and the detection precision and the accuracy of the obtained result are greatly improved.

In order to perform the immunofluorescence assay, the invention designs a kit convenient to operate, wherein the kit comprises main reagents in the immunofluorescence assay step, and specifically comprises the following reagents: buffer solutions CYP1, CYP2, CYPP, a confining liquid, a specific antibody for detecting PD-L1, a corresponding fluorescent secondary antibody, a fluorescent-labeled CD45 antibody, a fluorescent-labeled CD8 antibody, an antibody diluent and a cell nucleus staining solution; the control cells involved in the procedure may or may not be included in the kit.

In a preferred embodiment, the kit of the present invention preferably comprises the following components:

the reagent name terms and the distribution ratio thereof in the kit are detailed in the following preparation method:

CYP 1: fully stirring and dissolving buffer solution, 0.01M PBS and purified water, adjusting the pH value to 7.20-7.40, and subpackaging;

CYP 2: buffer solution, 0.01M PBS contains 0.1% Triton X-100, 0.5% Trehalo, add 0.1% Triton X-100, 0.5% Trehalo in 0.01M PBS solution, split charging after fully stirring and dissolving;

CYPP: buffer solution, 0.01M PBS contains 0.2% Triton X-100, 0.2% Triton X-100 is added into 0.01M PBS solution, and split charging is carried out after fully stirring and dissolving;

sealing liquid: subpackaging the blocking solution containing PBS and BSA;

PD-L1 antibody: 10 times of concentrated solution of PD-L1 antibody is subpackaged in 1.5ml of brown light-avoiding tube;

CD8 antibody: alexa

594 labeled CD8 antibody, dispensed in 1.5ml brown light-avoiding tube;

CD45 antibody: alexa

647 labeled CD45 antibody, dispensed in 1.5ml brown light-avoiding tube;

fluorescent secondary antibody: alexa

488-labeled fluorescent secondary antibody is subpackaged in 1.5ml of brown light avoiding tube;

antibody dilution: an antibody diluent containing a Tris-HCl buffer solution and bovine serum albumin;

DAPI: the staining solution was prepared by diluting DAPI powder (4', 6-diamidino-2-phenylindole, Sigma) with ultrapure water, adding an anti-quencher (ThermoFisher,

) Subpackaging;

the PD-L1 antibody in the kit recognizes a PD-L1 protein sequence, wherein the PD-L1 protein sequence belongs to a known sequence, disclosed in Dong H, Zhu G, Tamada K, Chen L.B7-H1, a third member of the B7family, co-peptides T-cell promotion and interleukin-10 section. Nat Med.1999 Dec; 5(12) 1365-9, its name is B7-H1.

The CD8 antibody in the kit recognizes the CD8 protein sequence, wherein the CD8 protein sequence belongs to a known sequence, disclosed in Littman DR, Thomas Y, Maddon PJ, Chess L, Axel R.the isolation and sequence of the soft gene encoding T8 a molecule defining functional classes of Tymphocycles.cell.1985 Feb; 40(2) 237-46, and the name is T8 in the literature.

The kit is mainly used for CTC, and can also be applied to immunofluorescence detection of cell sheets such as smear/slide, tissues and the like; the kit of the present invention may be suitable for the detection of cells of a plurality of cancers, which may include at least one of: breast cancer, esophageal cancer, gastric cancer, colon cancer, lung cancer, endometrial cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, sarcoma, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphatic cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, kidney cancer, bladder cancer, thyroid cancer, bone cancer, carcinoma, sarcoma, and soft tissue cancer.

The beneficial effects of the invention are described in detail by combining the detection method of the invention with the similarities and differences of the prior art, the specific implementation modes and the experimental data as follows: