CN111521796A - Immunofluorescence kit and detection method for detecting expression of peripheral blood circulating tumor cells PD-L1 of renal cancer patient - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and in particular relates to an immunofluorescence kit and a detection method for detecting PD-L1 expression in peripheral blood circulating tumor cells of renal cancer patients. Kit includes diluent 45mL, destaining solution 1mL, staining solution A 0.5mL, staining solution B 1mL, 200μl methanol, 200μl 2% PFA, 100μl 10% goat serum, mouse anti-CK, rat anti-CD45 and rabbit anti-PD‑L1 100 μl of primary antibody suspension composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, fluorescently labeled goat anti-rabbit composed of 100 μl of secondary antibody suspension, DAPI mounting medium; Methods: PD-L1 expression in patients with advanced or recurrent renal cancer can be detected without biopsy of tissue samples. The technique is minimally invasive and enables real-time detection.

Description

An immunofluorescence assay for detecting PD-L1 expression in peripheral blood circulating tumor cells of patients with renal cancer Optical kit and detection method

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to an immunofluorescence kit and a detection method for detecting PD-L1 expression in peripheral blood circulating tumor cells of renal cancer patients.

Background technique

Renal cell carcinoma (RCC) is one of the most common malignant tumors in the urinary system, and its morbidity and mortality account for about 2-3% of all malignant tumors in the whole body. Radical surgery is an effective method for the treatment of renal cancer, but about 20% to 30% of renal cancer patients have developed distant metastasis when they visit a doctor. Even after radical surgery for renal cancer, 20% to 40% of patients will experience recurrence and metastasis. Previous studies have shown that epithelial-mesenchymal transition (EMT) plays an important role in the recurrence and metastasis of renal cancer. Therefore, monitoring EMT can be used to judge the efficacy and prognosis of renal cancer.

Circulating tumor cells (CTCs) are tumor cells that are shed from solid tumors and enter the peripheral blood circulation. Since their discovery in 1989, various methods have been used to detect circulating tumor cells in peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of tumor patients, especially those with advanced tumors, and selecting appropriate individualized therapy. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.

Immunosuppression and immune escape are closely related to the overexpression of PD-L1 in tumor cells. Tumor cells can bind to PD-1 on the surface of immune cells T cells through PD-L1 on their surface to transmit inhibitory signals, so that T cells cannot recognize, Attacks tumor cells, leading to immune escape of tumor cells. Based on this theory, it is hypothesized that the apoptosis, immune clearance or survival, and metastasis outcome of circulating tumor cells (CTCs) shed from the primary tumor into the circulation are closely related to their PD-L1 expression levels. Previous studies have confirmed that the efficacy of PD-1 or PD-L1 immunotherapy is mostly related to the expression level of PD-L1 in tumor tissue, suggesting that PD-L1 may be a biomarker for predicting the efficacy of immunotherapy. The high expression of PD-L1 in renal cancer tissue was positively correlated with tumor aggressiveness.

Immunofluorescence analysis technology combines immunological methods (antigen-antibody specific binding) with fluorescent labeling technology to study the distribution of specific protein antigens in cells. Since the fluorescence emitted by fluorescein can be detected under a fluorescence microscope, fluorescein emits bright fluorescence (yellow-green or orange-red) when irradiated by excitation light, and the cells or tissues where the fluorescence is located can be seen. This allows for cellular characterization and localization analysis of the antigen.

In view of the current clinical practice, the specimens for PD-L1 detection in renal cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection. Therefore, the detection of CTC expression in circulating tumor cells is of great value for the prognosis of renal cancer and the evaluation of the efficacy of immunotherapy.

At present, Shandong First Medical University, Shandong Pharmaceutical Research Institute, Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd., etc. Research on the industrialization of key technologies for tumor cell detection and identification. This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Pharmaceutical Research Institute of Shandong First Medical University Jinan Campus as the core, implement the registrant system, rely on Circulating tumor cells detect and identify core diagnostic technologies, and further register and identify diagnostic kits to include PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, TKI-EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, pregnancy Hormone receptors, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc. are used as tracers for CTCs expression, with ultra-sensitive, ultra-rapid, high-coverage, low-cost, accurate and specific registration The identification and diagnosis kits of the company have been industrialized through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd. registered in Jinan.

SUMMARY OF THE INVENTION

In view of the shortcomings in the prior art that the detection of advanced or recurrent renal cancer patients cannot obtain tissue samples in real time or repeatedly, and thus cannot evaluate the real-time dynamic status of PD-L1 in patients, and the existing detection methods are prone to false positives and false negatives. The invention provides an immunofluorescence kit and detection method for PD-L1 expression in peripheral blood circulating tumor cells of patients with renal cancer. A membrane filtration device is used to separate and obtain circulating tumor cells (CTCs) in peripheral blood of patients with advanced renal cancer. technology to detect PD-L1 expression on CTCs.

The technical scheme adopted in the present invention is as follows:

A kit for detecting PD-L1 gene mutation in peripheral blood circulating tumor cells of renal cancer patients, including 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 200μl methanol, 200μl 2% PFA, 100μl 10% goat Serum, 100 μl of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1, fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, fluorescently labeled goat anti-rabbit Secondary antibody suspension 100μL, DAPI mounting medium;

Mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1 were diluted 1:100, 1:400 and 1:500 in the primary antibody suspension, respectively, with a total volume of 100 μL;

Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit were diluted 1:500 in the secondary antibody suspension.

Preferably, the diluent is composed of 1 mmol/L EDTA+0.1% BSA+0.1% trehalose+0.2% laureth.

Preferably, the decolorizing solution is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.

Preferably, the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.

A method for detecting the expression of PD-L1 in peripheral blood circulating tumor cells of renal cancer patients for the non-diagnostic purpose of the above-mentioned kit, comprising the following steps:

(1) Use a membrane filtration device to separate and obtain peripheral blood from patients with advanced or recurrent renal cancer for which tissue samples cannot be obtained: collect 5ml of peripheral blood from the median cubital vein of patients with advanced or recurrent renal cancer for which tissue samples cannot be obtained;

(2) Pretreatment of peripheral blood samples: The collected peripheral blood samples were diluted 10 times with diluent, and after dilution, paraformaldehyde was added to fix the peripheral blood samples for 10 minutes, and the final concentration was 0.25%;

(3) Filter peripheral blood samples using membrane filtration device for separating tumor cells, and separate and obtain peripheral blood CTCs: add the pretreated peripheral blood sample to the blood sample container of the device for membrane filtration and separate tumor cells, and let it filter naturally by gravity;

(4) After filtration, remove the filter from the device for separating tumor cells by membrane filtration, add 0.5 ml of circulating tumor cell staining solution A to the filter, stain for 3 minutes, and rinse with PBS buffer; after the filtrate is completely filtered, add staining solution 1ml of solution B, dyed for 2min, rinsed twice with 1ml of pure water;

(5) Add 200 μl of 2% PFA to the filter, fix it at room temperature for 5 min, and rinse with 0.5 ml of PBS for 3 times, 2 min each time;

(6) Add 200 μl of pre-cooled methanol to the filter, fix it at 4°C for 15 minutes, remove the filter membrane, place it on a glass slide, and observe it under a microscope after drying to determine whether there is CTC;

(7) The expression of PD-L1 in peripheral blood CTCs was detected by immunofluorescence method.

Further, the specific method for detecting PD-L1 expression in peripheral blood CTCs described in step (7) is as follows:

(1) Decolorization: Remove the filter membrane with CTC from the glass slide, soak it in the decolorizing solution for 4-6 hours, remove the CTC staining solution, and wash with PBS for 2min×3 times;

(2) Blocking: drop 100 μl of 10% goat serum onto the filter membrane, leave it at room temperature for 30 minutes, and remove excess serum after completion (note: goat serum is diluted with PBS);

(3) Primary antibody incubation: drop 100 μl of primary antibody suspension consisting of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1 on the filter, incubate at 37°C for 1 h or 4°C overnight, and wash with PBS after completion. 3min×3 times;

(4) Secondary antibody incubation: drop 100 μl of a secondary antibody suspension consisting of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit onto the filter membrane, and incubate at room temperature for 30 minutes. Wash with PBS for 2min×3 times;

(5) Mount the slides with DAPI-containing mounting media, read the slides, and take pictures;

(6) After the image collection is completed, Wright-Giemsa staining is performed after removing the slices, and the results are compared with the IF results.

The membrane filtration device for separating circulating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid tank and an iron stand. The iron stand is provided with a base, a stand and a bracket, and the blood sample container is arranged on the upper part of the iron stand through the stand. , the lower part of the blood sample container is a filter, the filter is connected to the waste liquid tank through the infusion device, and the waste liquid tank is arranged on the base.

The filter includes an upper filter port, a filter membrane, a filter-carrying membrane platform and a filter lower port, the filter membrane is placed on the filter-carrying membrane platform; the upper filter port is connected to the blood sample container, and the lower port of the filter is connected to a waste liquid tank through an infusion device.

The filter membrane is made of hydrophobic material, and it is evenly covered with filter pores with a diameter of 8 microns; the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. After peripheral blood is filtered, blood cells can be filtered because the diameter is smaller than the filter hole, while CTCs are trapped on the filter membrane because the diameter is larger than the filter hole.

The beneficial effects of the present invention are:

(1) The detection method provided by the present invention can detect the expression of PD-L1 in patients with advanced or recurrent renal cancer without the need for biopsy to obtain tissue samples. The technique is minimally invasive and enables real-time detection.

(2) The method provided by the present invention has good separation of circulating tumor cells, can avoid the interference of blood cells, can avoid false positive results caused by edge effects that may occur in the staining process, has good stability, reduces cell loss, and improves detection accuracy. sex.

Description of drawings

1 is a schematic structural diagram of a membrane filtration device of the present invention;

2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention;

Fig. 3 is the structural representation of the filter membrane of the membrane filtration device of the present invention;

Figure 4 is an image of PD-L1 immunofluorescence staining of peripheral blood circulating tumor cells in patients with advanced renal cancer.

In the picture: 1 iron stand, 2 blood sample container, 3 filter, 4 infusion set, 5 waste liquid tank, 6 filter upper port, 7 filter membrane, 8 filter membrane platform, 9 filter lower port, 10 filter hole, 11 base, 12 stand, 13 stand.

Detailed ways

The present invention is described below with reference to the accompanying drawings and embodiments.

The specific specifications of the immunofluorescence kit used in the embodiment of the present invention are shown in Table 1:

Table 1

The primary antibody suspension is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1. Mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1 are respectively used in BD wash buffer at 1:100. , 1:500 and 1:400 dilution, after dilution, take 10 μL mouse anti-CK, 50 μL rat anti-CD45 and 40 μL rabbit anti-PD-L1 to form a primary antibody suspension;

The secondary antibody suspension consists of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit, which are commercially available Alexa Fluor 546 goat Anti-mouse, Alexa Fluor 488 goat Anti -rat and Alexa Fluor 647 goat Anti-rabbit, take equal amounts of the above three fluorescently labeled secondary antibodies, dilute them with BD washbuffer at 1:500 and mix well to obtain a secondary antibody suspension.

An example of using this technique to isolate, obtain and identify circulating tumor cells in peripheral blood from 10 patients with renal cancer (at the same time, 10 normal human samples were detected as negative controls).

Example 1

1. Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent renal cancer for which no tissue specimens can be obtained, and determine whether CTCs exist:

Collect 5ml of fasting blood from the median cubital vein for 8-12 hours, dilute the peripheral blood with 45ml of diluent (component: 1mmol/L EDTA+0.1%BSA+0.1%trehalose+0.2% laureth), and then Add 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;

In a fixed interval, assemble the membrane filtration device: as shown in Figure 1, Figure 2, Figure 3, the filter device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;

Wet the filter 3 with 10ml PBS, then add the fixed peripheral blood sample to the blood sample container 2 of the membrane filtration device, so that it is naturally filtered by gravity, and CTC is trapped on the filter membrane 7;

The diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, when the peripheral blood containing CTCs is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, while the diameter of CTCs is larger than The filter pores 10 are trapped on the filter membrane 7 .

After filtration, remove the filter 3 from the filter device, open and remove the upper port 6 of the filter, add 0.5 ml of circulating tumor cell staining solution A to the filter, stain for 3 min, and rinse with PBS buffer; the filtrate is completely filtered Then add B solution, 1ml, stain for 2min, pure water 1ml, PBS buffer to rinse filter 3, remove filter 7 with ophthalmic tweezers, place the cell side up on a glass slide;

The filter membrane was dried and observed under a microscope to determine whether there was CTC. The test results are shown in Table 2.

Through observation, no CTCs were detected in 10 healthy volunteers; except for 2 patients with advanced renal cancer and 1 patient with recurrent renal cancer, CTCs were detected in the remaining 7 patients (Table 1). The test was positive. The rate is 70%. It is worth noting that when the diluent does not add 0.1% trehalose or does not add 0.2% lauryl alcohol polyoxyethylene ether, single use 0.3% trehalose or 0.3% lauryl alcohol polyoxyethylene ether to prepare The stability of the blood samples is poor, and some blood samples will form layers, and blood cells are prone to aggregation and adhesion, which affects the final detection effect.

Table 2 Embodiment CTC detection results

2. Using immunofluorescence technique to detect the expression of PD-L1 in CTCs:

Remove the CTC-loaded filter 7 from the glass slide and soak it in a decolorizing solution of 95% alcohol and 100% xylene in a volume ratio of 1:1 for 4-6 hours to remove the CTC staining. solution, washed 2min×3 times with PBS; add 100μl 10% goat serum dropwise, leave at room temperature for 30min, after completion, remove excess serum, add 100μl primary antibody suspension dropwise to the filter, incubate at 37°C for 1h, after completion, PBS Wash for 3 min × 3 times; then drop 100 μl of secondary antibody suspension on the filter membrane, incubate at room temperature for 30 min, and wash with PBS for 2 min × 3 times after completion; use DAPI-containing mounting medium to mount the slides, read the slides, take pictures, and collect After the figure is completed, the Wright-Giemsa staining is performed after decapsulation to compare with the IF results.

Figure 4 shows the PD-L1 immunofluorescence staining images of peripheral blood circulating tumor cells in patients with advanced renal cancer. According to the immunological and morphological manifestations, it was found that the tumor cells were large in size and abnormal in nuclear-cytoplasmic ratio. The immunological manifestations were typical CTCs. Among them, A is merge; B is PD-L1; C is CK; D is CD45.

The detected circulating tumor cells were confirmed by immunohistochemistry to confirm the expression of PD-L1 and compared with the results of PD-L1 in the gross specimens of patients with advanced renal cancer to observe the differences. patients, guide targeted therapy for patients with advanced renal cancer, and provide new ideas for targeted therapy for patients with advanced renal cancer.

Claims (6)

1. A kit for detecting the gene mutation of peripheral blood circulation tumor cells PD-L1 of a renal cancer patient is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 200 μ L of methanol, 200 μ L of 2% PFA, 100 μ L of 10% goat serum, 100 μ L of primary antibody suspension consisting of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1, 100 μ L of secondary antibody suspension consisting of fluorescence-labeled goat anti-mouse, fluorescence-labeled goat anti-rat and fluorescence-labeled goat anti-rabbit, and a DAPI encapsulated tablet;

in the primary anti-suspension, the mouse anti-CK, the rat anti-CD 45 and the rabbit anti-PD-L1 are respectively diluted according to the ratio of 1:100, the ratio of 1:400 and the ratio of 1:500, and the total volume is 100 mu L;

and diluting the secondary antibody suspension with fluorescence-labeled goat-anti mouse, fluorescence-labeled goat-anti rat and fluorescence-labeled goat-anti rabbit at a ratio of 1: 500.

2. The kit according to claim 1, characterized in that the diluent consists of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% laureth alcohol.

3. The kit of claim 1, wherein the destaining solution is comprised of 95% alcohol to 100% xylene in a volume ratio of 1: 1.

4. The kit according to claim 1, wherein the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.

5. A method for non-diagnostic detection of the expression of PD-L1 in circulating tumor cells in the peripheral blood of a patient with renal cancer using the kit of any one of claims 1 to 4, comprising the steps of:

(1) and (3) separating and obtaining peripheral blood of a patient with advanced or recurrent renal cancer who cannot obtain a tissue specimen by using a membrane filtration device: collecting 5ml of peripheral blood of the median elbow vein of a patient with late stage or recurrent renal cancer who cannot obtain a tissue specimen;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; after the filtrate is completely filtered, 1ml of staining solution B is added, the dyeing is carried out for 2min, and 1ml of pure water is used for washing for 2 times;

(5) adding 200 μ l of 2% PFA into the filter, fixing at room temperature for 5min, and rinsing with 0.5ml PBS for 3 times, each for 2 min;

(6) adding 200 μ l of precooled methanol into the filter, fixing at 4 ℃ for 15min, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(7) and detecting the expression condition of PD-L1 of the CTC in the peripheral blood by using an immunofluorescence method.

6. The method of claim 5, wherein the specific method for detecting PD-L1 expression in peripheral blood CTC in step (7) is as follows:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min × 3 times;

(2) and (3) sealing: dropping 100 μ l10% goat serum onto the filter membrane, standing at room temperature for 30min, and removing excess serum (diluting goat serum with PBS);

(3) primary antibody incubation: dripping 100 μ L primary antibody suspension composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 onto the filter membrane, incubating at 37 deg.C for 1h or overnight at 4 deg.C, and washing with PBS for 3min × 3 times;

(4) and (3) secondary antibody incubation: dripping 100 μ l of secondary antibody suspension composed of fluorescence labeled goat-anti mouse, fluorescence labeled goat-anti rat, and fluorescence labeled goat-anti rabbit onto the filter membrane, incubating at room temperature for 30min, and washing with PBS for 2min × 3 times;

(5) sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing;

(6) after the image collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

Images