CN111521789A - Immunofluorescence kit and detection method for detecting expression of peripheral blood circulating tumor cells CA199 of pancreatic cancer patient - Google Patents
Immunofluorescence kit and detection method for detecting expression of peripheral blood circulating tumor cells CA199 of pancreatic cancer patient Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention relates to an immunofluorescence kit and a detection method for detecting the expression of peripheral blood circulating tumor cells CA199 of a pancreatic cancer patient, and belongs to the technical field of molecular biology. The invention provides an immunofluorescence kit and a detection method for detecting the CA199 expression of peripheral blood circulating tumor cells of a pancreatic cancer patient, wherein CTC in peripheral blood of a late stage or recurrent pancreatic cancer patient, which cannot obtain a tissue specimen, is obtained by separation through a membrane filter device, and the CA199 expression of the CTC is further detected by using an immunofluorescence technology. The detection method provided by the invention can detect the CA199 expression condition of a patient with advanced or recurrent pancreatic cancer without taking a tissue sample by a puncture biopsy. The technology belongs to minimally invasive and can detect in real time. Meanwhile, false positive results caused by edge effects possibly generated in the dyeing process can be avoided, the stability is good, the loss of cells is reduced, and the detection accuracy is improved.
Description
Technical Field
The invention relates to an immunofluorescence kit and a detection method for detecting the expression of peripheral blood circulating tumor cells CA199 of a pancreatic cancer patient, and belongs to the technical field of molecular biology.
Background
Pancreatic cancer is a common malignancy of the digestive system and one of the worst prognosis, among deaths caused by malignancies, pancreatic cancer ranks fourth, 90% of patients die within one year after diagnosis, and 5-year survival rate is less than 5%. As early symptoms of pancreatic cancer are hidden, the pancreatic cancer is not specific, the pancreatic cancer is highly invasive, the pancreatic cancer is early in metastasis, the pancreatic cancer is mostly in the middle and late stages when the pancreatic cancer is diagnosed, the surgical resection rate is low, and the pancreatic cancer is diagnosed only by 15%, the selection of the treatment method for pancreatic cancer patients is particularly important, and unnecessary surgical trauma and risks can be avoided for part of patients.
CAl99(carbohydrate antigen 99), also known as tumor associated 199 antigen, gastrointestinal cancer associated antigen or glycoantigen, is a mucin tumor marker that is glycolipids on the cell membrane and exists as mucin in the blood. CAl99 is an antigenic substance highly associated with adenocarcinoma, produced by adenocarcinoma cells, and is introduced into the blood stream via the thoracic duct. The normal reference value of the serum CAl99 is 39 kU/L, the CAl99 concentration in the malignant tumor serum is obviously increased, especially for patients with pancreatic cancer and colon cancer, the increase is several times to dozens of times different, even can reach 1000U/mL, so the method is mainly used for early diagnosis of pancreatic cancer, and in the current clinical practice, the sample detected by the CA199 of the pancreatic cancer patient is mainly tumor tissue, is from operation or puncture biopsy, and is difficult to detect for a plurality of times or in real time.
At present, units such as Shandong province first medical university, Shandong province drug research institute combined with Shandong Qixin Biotechnology limited company, Shandong well-known Biotechnology limited company, Jinan Xin Biotechnology limited company, Shandong discovery biotechnology limited company and the like, carry out industrialized popularization on the key technology of detection and identification of circulating tumor cells, the project is a Shandong province major scientific and technological innovation project, the project takes the Shandong province drug research institute in Jinan school of Shandong first medical university as the core, realizes a registration system, relies on the core diagnostic technology of detection and identification of circulating tumor cells, and further registers, identifies and diagnoses a kit, and comprises PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catendin, E-Cacatenin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, progestin receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A and the like are used as tracers for expression of CTCs, and the identification and diagnosis kit is registered to be an ultrasensitive, ultrafast, high-coverage, low-cost, accurate and specific kit, and industrially popularized by cooperating with Shandong Qicheng Xin Biotech Co., Ltd, Shandong well-known Biotech Co., Ltd, Jinan En Biotech Co., Ltd, Shandong discovery Biotech Co., Ltd registered in Jinan.
Disclosure of Invention
In order to overcome the problem of multiple or real-time detection and simultaneously avoid the risk of bleeding, pancreatic fistula, infection and the like in the process of puncture biopsy, the invention provides an immunofluorescence kit and a detection method for detecting the expression of the CA199 of peripheral blood circulation tumor cells of a pancreatic cancer patient, wherein CTC in peripheral blood of a late stage or recurrent pancreatic cancer patient, which cannot obtain a tissue specimen, is obtained by separation through a membrane filter device, and the CA199 expression condition of the CTC is further detected by using an immunofluorescence technology.
The technical scheme adopted by the invention is as follows:
an immunofluorescence kit for detecting the expression of peripheral blood circulation tumor cells CA199 of a pancreatic cancer patient comprises 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-CA 199, 100 μ L of secondary antibody suspension consisting of a fluorescence-labeled goat anti-mouse, a fluorescence-labeled goat anti-rat and a fluorescence-labeled goat anti-rabbit, and a DAPI blocking tablet;
in the primary anti-suspension, mice anti-CK, rats anti-CD 45 and rabbits anti-CA 199 are respectively diluted according to the proportion of 1:100, 1:400 and 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.
The diluent is 19g/L of potassium chloride, 4.8 g/L of ethylene diamine tetraacetic acid dipotassium, 2.6 g/L of pyridinium sodium, 17g/L of monopotassium phosphate, 9g/L of sodium hydroxide, 0.2g/L of hydroxyethyl piperazine ethanethiosulfonic acid, 2g/L of dihydroxymethyl urea and the balance of purified water.
The destaining solution consists of 0.5 percent hydrochloric acid-70 percent ethanol solution.
The staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.
The method for detecting the expression of the peripheral blood circulating tumor cells CA199 of the pancreatic cancer patient by the immunofluorescence kit in a non-diagnosis purpose comprises the following steps:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by using 45ml of 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 CA199 expression condition of the CTC in the peripheral blood by using an immunofluorescence method.
The specific method for detecting the CA199 expression of the CTC in the step (7) is as follows:
s1 decolorization: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;
and S2 sealing: diluting goat serum with PBS, adding 100 μ l10% goat serum dropwise onto the filter membrane, standing at room temperature for 30min, and removing excessive serum;
s3 primary antibody incubation: dripping 100 μ l primary antibody suspension composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-CA 199 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;
s4 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;
s5, sealing the piece by using a sealing agent containing DAPI, and reading and collecting the picture;
after completion of the S6 sampling, the sections were removed and then stained with Giemsa Richardson, and the results were compared with the IF results.
The device for separating tumor cells by membrane filtration comprises a filter, a blood sample container, a waste liquid tank and an iron stand, wherein the iron stand is provided with a base, a vertical frame and a support, the blood sample container is arranged at the upper part of the iron stand through the support, the filter is arranged below the blood sample container, the filter is communicated to the waste liquid tank through an infusion apparatus, and the waste liquid tank is arranged on the base.
The filter comprises a filter upper opening, a filter membrane carrying platform and a filter lower opening, and the filter membrane is arranged on the filter membrane carrying platform; the upper port of the filter is connected with a blood sample container, and the lower port of the filter is connected with a waste liquid cylinder through an infusion apparatus.
The filter membrane is made of hydrophobic materials, and filter holes with the caliber of 8 microns are uniformly distributed on the filter membrane.
The invention has the beneficial effects that:
(1) the detection method provided by the invention can detect the CA199 expression condition of a patient with advanced or recurrent pancreatic cancer without taking a tissue sample by a puncture biopsy. The technology belongs to minimally invasive and can detect in real time.
(2) The method provided by the invention can avoid false positive results caused by edge effect possibly generated in the dyeing process, has good stability, reduces the loss of cells and improves the detection accuracy.
Drawings
FIG. 1 is a schematic structural view of a membrane filtration apparatus according to the present invention;
FIG. 2 is a schematic sectional view showing the structure of a filter of the membrane filtration apparatus of the present invention;
FIG. 3 is a schematic view showing the structure of a filter membrane of the membrane filtration apparatus of the present invention;
FIG. 4 is an immunofluorescent staining image of peripheral blood circulating tumor cells of a patient with advanced pancreatic cancer.
In the figure: 1 iron stand, 2 blood sample containers, 3 filters, 4 transfusion devices, 5 waste liquid jars, 6 filter upper ports, 7 filter membranes, 8 filter membrane platforms, 9 filter lower ports, 10 filter holes, 11 bases, 12 vertical frames and 13 supports.
Detailed Description
The invention is elucidated below with reference to the figures and embodiments.
The specific specification of the immunofluorescence kit used in the invention is shown in table 1:
TABLE 1 kit Specifications
The primary antibody suspension consists of mouse anti-CK, rat anti-CD 45 and rabbit anti-CA 199, wherein the mouse anti-CK, the rat anti-CD 45 and the rabbit anti-CA 199 are respectively diluted by BD wash buffer according to the ratio of 1:100, the ratio of 1:500 and the ratio of 1:400, and 10 mu L of mouse anti-CK, 50 mu L of rat anti-CD 45 and 40 mu L of rabbit anti-E-Cadherin are taken to form the primary antibody suspension after dilution;
the secondary antibody suspension consists of a fluorescence-labeled goat-Anti mouse, a fluorescence-labeled goat-Anti rat and a fluorescence-labeled goat-Anti rabbit, which are respectively Alexa Fluor 546 gat Anti-mouse, Alexa Fluor 488 gat Anti-rat and Alexa Fluor 647 gat Anti-rabbitt sold in the market, and equal amounts of the three fluorescence-labeled secondary antibodies are respectively diluted by BD washbuffer according to a ratio of 1:500 and mixed uniformly to obtain the secondary antibody suspension.
The technical method is used for separating, obtaining and identifying 15 pancreatic cancer patients (5 normal human samples are simultaneously detected as negative controls) and the peripheral blood circulating tumor cells.
Preparing the diluent: 19g of potassium chloride, 4.8g of ethylene diamine tetraacetic acid dipotassium, 2.6g of pyridinium sodium, 17g of potassium dihydrogen phosphate, 9g of sodium hydroxide, 0.2g of hydroxyethyl piperazine ethanethiosulfonic acid, 2g of dihydroxymethyl urea and 945.4g of purified water.
Example 1
Firstly, separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device, and determining whether the CTCs exist:
collecting 5ml of fasting 8-12 hours fasting blood from the median cubital vein, diluting peripheral blood with 45ml of diluent, and then adding 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;
at fixed intervals, a membrane filtration device was assembled: as shown in fig. 1, 2 and 3, the filter device comprises a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5 and an iron stand 1;
wetting the filter 3 with 10ml of PBS, then adding the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, allowing it to naturally filter by gravity, and the CTC being trapped on the filter membrane 7;
the tumor cells are typically larger than 15 microns in diameter, while the blood cells (including red blood cells, white blood cells) are typically smaller than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells can be filtered by being smaller than filter pores 10, while the CTCs are retained on filter membrane 7 by being larger than filter pores 10.
After the filtration is finished, taking the filter 3 from the filter device, opening and removing the upper opening 6 of the filter, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; filtering the filtrate completely, adding solution B, 1ml, staining for 2min, and pure water 1ml, washing filter 3 with PBS buffer solution, taking down filter membrane 7 with ophthalmic forceps with cell surface facing upwards, and placing on glass slide;
the filters were dried and observed under a microscope to determine the presence of CTCs.
By observation, no CTCs were detected in 5 healthy volunteers; CTCs were detected in 6 patients with recurrent pancreatic cancer and 5 patients with advanced pancreatic cancer (Table 2), except that CTCs were not detected in 3 patients with recurrent pancreatic cancer and 1 patient with advanced pancreatic cancer, and the positive rate of this detection was 73.3%.
It is worth noting that when the diluent is not added with dihydroxymethylurea or hydroxyethylpiperazine ethanethiosulfonic acid, the prepared blood sample has poor stability, a part of the blood sample can be layered, blood cells are easy to aggregate and adhere, and the final detection effect is influenced.
TABLE 2 results of CTC assay in examples
Secondly, detecting the CA199 expression condition of the CTC by using an immunofluorescence technology:
taking down the filter membrane 7 carrying the CTC on the glass slide from the glass slide, soaking in a decoloration solution uniformly mixed by 0.5% hydrochloric acid-70% ethanol solution for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min × 3 times; dripping 100 μ l10% goat serum, standing at room temperature for 30min, sucking off excessive serum, dripping 100 μ l primary antibody suspension onto the filter membrane, incubating at 37 deg.C for 1h, and washing with PBS for 3min × 3 times; then, 100 mul of secondary antibody suspension is dripped on the filter membrane, and the mixture is incubated for 30min at room temperature, and then washed for 2min multiplied by 3 times by PBS; sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing; after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.
The decolorizing solution prepared from 0.5 percent hydrochloric acid-70 percent ethanol solution has better decolorizing effect than the decolorizing solution prepared from 95 percent ethanol and 100 percent dimethylbenzene according to the volume ratio of 1: 1.
FIG. 4 is an immunofluorescence staining image of peripheral blood circulating tumor cells of a patient with advanced pancreatic cancer, and according to the immunological and morphological manifestations, the tumor cells are found to be large in cell size and abnormal in nuclear-to-cytoplasmic ratio, and the immunological manifestations are typical CTCs.
The detected circulating tumor cells use immunofluorescence to verify the expression of CA199, and compare with the expression result of CA199 in pancreatic cancer operation or puncture tissue specimen, and observe the difference, thereby verifying that CTCs used as noninvasive biopsy have great potential for real-time evaluation of pancreatic cancer CA199 expression, and providing important basis for evaluation of pancreatic cancer chemotherapy combined immunotherapy prognosis.
Claims (6)
1. An immunofluorescence kit for detecting the expression of peripheral blood circulation tumor cells CA199 of a pancreatic 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-CA 199, 100 μ L of secondary antibody suspension consisting of a fluorescence-labeled goat anti-mouse, a fluorescence-labeled goat anti-rat and a fluorescence-labeled goat anti-rabbit, and a DAPI blocking tablet;
in the primary anti-suspension, mice anti-CK, rats anti-CD 45 and rabbits anti-CA 199 are respectively diluted according to the proportion of 1:100, 1:400 and 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 immunofluorescence kit according to claim 1, wherein the diluent is 19g/L potassium chloride, 4.8 g/L dipotassium ethylenediamine tetraacetic acid, 2.6 g/L pyridinium sodium, 17g/L potassium dihydrogen phosphate, 9g/L sodium hydroxide, 0.2g/L hydroxyethylpiperazine ethanethiosulfonic acid, 2g/L dihydroxymethylurea, and the balance being purified water.
3. The immunofluorescence kit according to claim 1, wherein the destaining solution consists of a 0.5% hydrochloric acid-70% ethanol solution.
4. The immunofluorescence 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 CA199 expression from peripheral blood circulating tumor cells of a pancreatic cancer patient using the immunofluorescence kit of any one of claims 1-4, comprising the steps of:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent pancreatic cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent pancreatic cancer who cannot obtain tissue specimens: 5ml of peripheral blood of the median cubital vein;
(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by using 45ml of 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 CA199 expression condition of the CTC in the peripheral blood by using an immunofluorescence method.
6. The assay of claim 5, wherein the specific method for detecting CA199 expression of CTCs in step (7) is as follows:
s1 decolorization: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;
and S2 sealing: diluting goat serum with PBS, adding 100 μ l10% goat serum dropwise onto the filter membrane, standing at room temperature for 30min, and removing excessive serum;
s3 primary antibody incubation: dripping 100 μ l primary antibody suspension composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-CA 199 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;
s4 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;
s5, sealing the piece by using a sealing agent containing DAPI, and reading and collecting the picture;
after completion of the S6 sampling, the sections were removed and then stained with Giemsa Richardson, and the results were compared with the IF results.
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| WO2021213298A1 (en) * | 2020-04-20 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting ca199 expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021213298A1 (en) * | 2020-04-20 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | Immunofluorescence kit for detecting ca199 expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
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