CN113049344B - Preparation method of quality control product for cell staining - Google Patents
Preparation method of quality control product for cell staining Download PDFInfo
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- CN113049344B CN113049344B CN202110429098.2A CN202110429098A CN113049344B CN 113049344 B CN113049344 B CN 113049344B CN 202110429098 A CN202110429098 A CN 202110429098A CN 113049344 B CN113049344 B CN 113049344B
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- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention discloses a preparation method of a quality control product for cell staining, in the preparation method, E-Cadherin immunomagnetic beads and CEA immunomagnetic beads are specifically combined with lung cancer circulating tumor cells, and CD45 immunomagnetic beads are specifically combined with white blood cells; the staining kit can control the lung cancer circulating tumor cells by staining the enriched lung cancer circulating tumor cells and leukocytes. The preparation method of the quality control product for cell staining has high reliability of the quality control result through the specific combination of the antigen and the antibody.
Description
Technical Field
The invention relates to the field of cell staining detection, in particular to a preparation method of a quality control product for cell staining.
Background
Cancer cells shed from the primary site and enter the peripheral blood of the human body, and are called Circulating Tumor Cells (CTCs). Lung cancer mainly originates from the mucosal epithelium of the bronchi, and has become the cancer with the highest incidence and the highest death number worldwide. In lung cancer patients, the metastasis of circulating tumor cells of lung cancer to other organs of the body by blood transmission is a major cause of death in patients. Therefore, the method for detecting the content change of the lung cancer circulating tumor cells in the peripheral blood has positive significance for monitoring the dynamic state of the lung cancer cells in the body of a patient in real time, evaluating the treatment effect and the like.
However, the existing lung cancer circulating tumor cell staining kit has low detection accuracy on lung cancer circulating tumor cells in peripheral blood, and results such as false positive/false negative and non-specificity are easy to appear, so that doctors cannot make accurate judgment, and great influence is brought to subsequent analysis, diagnosis and the like. Although the chinese patent CN107153021B discloses a method for preparing quality control products for staining circulating tumor cells, the effect of enriching circulating tumor cells of lung cancer is not good.
The above is only for the purpose of assisting understanding of the technical solutions of the present invention, and does not represent an admission that the above is the prior art.
Disclosure of Invention
The invention mainly aims to provide a preparation method of a quality control product for cell staining, and aims to solve the technical problem that the accuracy of the existing staining kit for detecting circulating tumor cells of lung cancer is not high.
In order to achieve the purpose, the invention provides a preparation method of a quality control product for cell staining, which comprises the following steps:
(1) mixing E-Cadherin immunomagnetic beads with a binding buffer solution to obtain E-Cadherin immunomagnetic bead working solution, mixing CEA immunomagnetic beads with the binding buffer solution to obtain CEA immunomagnetic bead working solution, and mixing CD45 immunomagnetic beads with the binding buffer solution to obtain CD45 immunomagnetic bead working solution;
(2) mixing the E-Cadherin immunomagnetic bead working solution, the CEA immunomagnetic bead working solution, the CD45 immunomagnetic bead working solution, the white blood cells and the lung cancer circulating tumor cells to obtain a first mixed solution;
(3) removing the liquid in the first mixed solution, and adding a stationary liquid for fixation to obtain the quality control product of the lung cancer circulating tumor cells.
Optionally, the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is greater than or equal to 3: 4, and less than or equal to 4: 3; the ratio of the number of the E-Cadherin immunomagnetic beads, the number of the CEA immunomagnetic beads and the number of the CD45 immunomagnetic beads is greater than or equal to 4: 3, and less than or equal to 5: 2; the number ratio of the leukocytes to the lung cancer circulating tumor cells is greater than or equal to 1:10, and less than or equal to 10: 1.
in a second aspect, the present invention provides another method for preparing a quality control material for cell staining, comprising the following steps:
(1) mixing E-Cadherin immunomagnetic beads with a binding buffer solution to obtain E-Cadherin immunomagnetic bead working solution, mixing CEA immunomagnetic beads with the binding buffer solution to obtain CEA immunomagnetic bead working solution, and mixing CD45 immunomagnetic beads with the binding buffer solution to obtain CD45 immunomagnetic bead working solution;
(2) mixing the E-Cadherin immunomagnetic bead working solution, the CEA immunomagnetic bead working solution and lung cancer circulating tumor cells to obtain a second mixed solution, and mixing the CD45 immunomagnetic bead working solution and white blood cells to obtain a third mixed solution;
(3) and respectively removing the liquid in the second mixed liquid and the third mixed liquid, and adding a fixing liquid for fixation to obtain the quality control product of the lung cancer circulating tumor cells.
Optionally, the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is greater than or equal to 3: 4, and less than or equal to 4: 3.
in the preparation method, the E-Cadherin immunomagnetic beads are specifically combined with the surface antigen of the lung cancer circulating tumor cells through an E-Cadherin antibody, the CEA immunomagnetic beads are specifically combined with the surface antigen of the lung cancer circulating tumor cells through a CEA antibody, the E-Cadherin immunomagnetic beads, the CEA immunomagnetic beads and the lung cancer circulating tumor cells form a magnetic bead-antibody-cell compound, and a detector dyes the compound to obtain a specifically combined dyeing result; the CD45 immunomagnetic beads are specifically bound with leukocyte surface antigen through CD45 antibody and serve as a negative control of the staining result of lung cancer circulating tumor cells. By adopting the preparation method of the quality control product for cell staining, quality control can be performed on different lung cancer circulating tumor cell staining kits, for example, indexes such as precision, accuracy and reagent change of the staining kit are evaluated, so that whether the staining kit is abnormal or not is monitored; the preparation method of the quality control product for cell staining has high reliability of the quality control result through the specific combination of the antigen and the antibody.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a screenshot of the control results of one week of cell preservation according to the embodiment;
FIG. 2 is a screenshot of the control results of one month of cell preservation according to the embodiment;
FIG. 3 is a screenshot of the control results of one embodiment of three months after cell preservation;
FIG. 4 is a screenshot of the quality control results of three cells of the example stored for one week;
FIG. 5 is a screenshot of the quality control results of three cells stored for one month in the example;
FIG. 6 is a screenshot of the quality control results of three-month three-cell storage according to the example;
FIG. 7 is a screenshot of the quality control results of comparative example one cell stored for one week;
FIG. 8 is a screenshot of the quality control results of comparative example one cell stored for one month;
FIG. 9 is a screenshot of the quality control results of comparative example one cell stored for three months;
FIG. 10 is a screenshot of the quality control results of five cells of comparative example stored for one week;
FIG. 11 is a screenshot of the quality control results of five cells of comparative example stored for one month;
FIG. 12 is a screenshot of the control results of five cells of the comparative example stored for three months.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
It should be noted that if the description of "first", "second", etc. is provided in the embodiment of the present invention, the description of "first", "second", etc. is only for descriptive purposes and is not to be construed as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" appearing throughout is to include three juxtapositions, exemplified by "A and/or B" including either scheme A, or scheme B, or a scheme in which both A and B are satisfied.
The invention provides a preparation method of a quality control product for cell staining, which comprises the following steps: (1) mixing E-Cadherin immunomagnetic beads with a binding buffer solution to obtain E-Cadherin immunomagnetic bead working solution, mixing CEA immunomagnetic beads with the binding buffer solution to obtain CEA immunomagnetic bead working solution, and mixing CD45 immunomagnetic beads with the binding buffer solution to obtain CD45 immunomagnetic bead working solution; (2) mixing the E-Cadherin immunomagnetic bead working solution, the CEA immunomagnetic bead working solution, the CD45 immunomagnetic bead working solution, the white blood cells and the lung cancer circulating tumor cells to obtain a first mixed solution; (3) removing the liquid in the first mixed solution, and adding a stationary liquid for fixation to obtain the quality control product of the lung cancer circulating tumor cells.
In the preparation method of the invention, E-Cadherin (E-Cadherin) and Carcinoembryonic Antigen (CEA) are common tumor markers, and can be combined with the lung cancer circulating tumor cells for monitoring the treatment and identification of lung cancer. Since the surface of some circulating tumor cells of lung cancer may express only E-Cadherin antigen or CEA antigen, the E-Cadherin antibody and the CEA antibody cannot bind 100% of all circulating tumor cells of lung cancer. The two antibodies are combined with the lung cancer circulating tumor cells together, so that the specific enrichment of the lung cancer circulating tumor cells is enhanced, and the quality control effect is improved. The CD45 antibody can specifically bind to leukocyte surface antigen, and is a commonly used antibody for detecting and enriching leukocytes.
In the preparation method, the E-Cadherin immunomagnetic beads are specifically combined with lung cancer circulating tumor cells through an E-Cadherin antibody, the CEA immunomagnetic beads are specifically combined with the lung cancer circulating tumor cells through a CEA antibody, the E-Cadherin immunomagnetic beads, the CEA immunomagnetic beads and the lung cancer circulating tumor cells form a magnetic bead-antibody-cell compound, and a detector dyes the double magnetic bead-antibody-cell compound to obtain a specifically combined dyeing result; the CD45 immunomagnetic beads are specifically bound with leukocyte surface antigen through CD45 antibody and serve as a negative control of the staining result of lung cancer circulating tumor cells. By adopting the preparation method of the quality control product for cell staining, quality control can be performed on different lung cancer circulating tumor cell staining kits, for example, indexes such as precision, accuracy and reagent change of the staining kit are evaluated, so that whether the staining kit is abnormal or not is monitored; the kit is simple to operate, and the reliability of a quality control result is high through the specific combination of the antigen and the antibody.
Optionally, the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is greater than or equal to 3: 4, and less than or equal to 4: 3; the ratio of the number of the E-Cadherin immunomagnetic beads, the number of the CEA immunomagnetic beads and the number of the CD45 immunomagnetic beads is greater than or equal to 4: 3, and less than or equal to 5: 2; the number ratio of the leukocytes to the lung cancer circulating tumor cells is greater than or equal to 1:10, and less than or equal to 10: 1.
when the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is less than 3: and 4, the content of the E-Cadherin immunomagnetic beads is low, and the capacity of competing with the CEA immunomagnetic beads for binding the lung cancer circulating tumor cells is low, so that the number of the lung cancer circulating tumor cells finally enriched on the E-Cadherin immunomagnetic beads and the CEA immunomagnetic beads is reduced, and the quality control result is influenced. When the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is more than 4: 3, the content of the CEA immunomagnetic beads is low, and the ability of the CEA immunomagnetic beads to compete with the E-Cadherin immunomagnetic beads to bind the lung cancer circulating tumor cells is weak, so that the number of the lung cancer circulating tumor cells finally enriched on the E-Cadherin immunomagnetic beads and the CEA immunomagnetic beads is reduced, and the quality control result is influenced.
When the ratio of the number of the E-Cadherin immunomagnetic beads, the CEA immunomagnetic beads and the number of the CD45 immunomagnetic beads is less than 4: 3, the content of CD45 immunomagnetic beads is low, and the effect of binding leukocytes in mixed cells (leukocytes and lung cancer circulating tumor cells) is poor, resulting in a low number of enriched leukocytes and a poor control effect. When the ratio of the number of the E-Cadherin immunomagnetic beads, the number of the CEA immunomagnetic beads and the number of the CD45 immunomagnetic beads is more than 5: 2, the content of the CD45 immunomagnetic beads is excessive, so that the combination of the E-Cadherin and CEA immunomagnetic beads and lung cancer circulating tumor cells in mixed cells (white blood cells and lung cancer circulating tumor cells) is influenced, and the quality control result is further influenced.
When the ratio of the number of the white blood cells to the number of the lung cancer circulating tumor cells is less than 1: at 10, the number of leukocytes was small, and the control effect was insufficient. When the number ratio of the white blood cells to the lung cancer circulating tumor cells is more than 10:1, the number of the lung cancer circulating tumor cells is small, and the quality control result is also influenced. Preferably, the ratio of the number of said leukocytes to the number of said circulating tumor cells of lung cancer is 1: 1.
the invention provides a second preparation method of a quality control product for cell staining, which comprises the following steps: (1) mixing E-Cadherin immunomagnetic beads with a binding buffer solution to obtain E-Cadherin immunomagnetic bead working solution, mixing CEA immunomagnetic beads with the binding buffer solution to obtain CEA immunomagnetic bead working solution, and mixing CD45 immunomagnetic beads with the binding buffer solution to obtain CD45 immunomagnetic bead working solution; (2) mixing the E-Cadherin immunomagnetic bead working solution, the CEA immunomagnetic bead working solution and lung cancer circulating tumor cells to obtain a second mixed solution, and mixing the CD45 immunomagnetic bead working solution and white blood cells to obtain a third mixed solution; (3) and respectively removing the liquid in the second mixed liquid and the third mixed liquid, and adding a fixing liquid for fixation to obtain the quality control product of the lung cancer circulating tumor cells.
The beneficial effects of the second preparation method are consistent with those of the first preparation method, and are not described herein again.
The two preparation methods of the invention also can comprise the collection of the white blood cells and the preparation process of the lung cancer circulating tumor cells. The leucocyte collection can be carried out by collecting peripheral blood of a subject in a conventional arm vein blood collection mode. The collection of leukocytes mainly includes the steps of preparation of lymphocyte separation liquid (Ficoll), washing of buffer solution, centrifugation, suction and separation of leukocytes, and the like, and the specific steps refer to the prior art. In the step of collecting the white blood cells, the centrifuge can adopt a low-gear rotating speed and is set to slow down; sucking the leucocytes in the layering liquid gently and slowly; the supernatant can be centrifuged again and the leukocytes can be aspirated, which all contribute to an increased leukocyte collection rate. The preparation of the lung cancer circulating tumor cells comprises the processes of culturing cells, cleaning cells, digesting cells (trypsin can be adopted), suspending cells, diluting cells and the like, and the specific steps are also referred to in the prior art. In the preparation work of the lung cancer circulating tumor cells, the operation is also gentle and slow, so that the cells are prevented from being broken and the subsequent experiment is prevented from being influenced. The lung cancer circulating tumor cell may comprise at least one of a plurality of lung cancer cell lines, such as a human non-small cell lung cancer adenocarcinoma cell line (NCI-H1993), a human lung adenocarcinoma cell line (NCI-H1975), a human lung cancer epidermal cell (Calu-1), or a human lung cancer cell line (GLC-15), and the like.
Example one
1. Mixing leukocytes with NCI-H1993 cells (number ratio 1:10) in a total volume of 700 ul;
2. taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
3. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 2 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
4. adding 30ul of the E-Cadherin immunomagnetic bead working solution, 40ul of the CEA immunomagnetic bead working solution and 50ul of the CD45 immunomagnetic bead working solution into the cell mixed solution in the step 1, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifugal tube in a refrigerator at 4 ℃ for 2 hours at a rotating speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L1.6% fixing solution for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 200. mu.L of binding buffer was added, and the mixture was stored at 4 ℃ until use.
In the above steps, in order to avoid the damage of the cells after the damage, which affects the subsequent verification results, the operations such as mixing the solution are gentle and slow. The concentration of the fixative solution and the fixation time can be adjusted according to different experimental conditions and cell states.
And taking the quality control products which are prepared, stored for one week, one month and three months on the same day as experimental objects, and dyeing the quality control products according to the instruction of the dyeing kit. The staining rate of the cells is nearly 100%. FIG. 1 is a screenshot of a quality control result stored for one week; FIG. 2 is a screenshot of the quality control result stored for one month; fig. 3 is a screenshot of the quality control result stored for three months. And respectively calculating the number ratio of the stained cells to the original cells in the experimental group, thereby obtaining the binding rate of the immunomagnetic beads combined with the cells. See table 1 for the calculation results.
TABLE 1 quantitative ratio of stained cells to primary cells in example one
The results in Table 1 show that, in the first method for preparing a quality control product for cell staining of the present invention, when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 3: 4, the ratio of the number of E-Cadherin immunomagnetic beads to the number of CEA immunomagnetic beads to the number of CD45 immunomagnetic beads is 7: 5, the number ratio of the white blood cells to the lung cancer circulating tumor cells is 1: when 10 hours, the binding rate of the immunomagnetic beads and the cells can reach 99 percent, which shows that the first preparation method of the invention has high reliability; the stable storage life of the quality control product is up to three months, which shows that the quality control product prepared by the first preparation method has stable property.
Example two
1. Mixing leukocytes with NCI-H1993 cells (cell number ratio of 10:1) in a total volume of 700 ul;
2. taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
3. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 2 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
4. adding 40ul of the E-Cadherin immunomagnetic bead working solution, 30ul of the CEA immunomagnetic bead working solution and 28ul of the CD45 immunomagnetic bead working solution into the cell mixed solution in the step 1, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifugal tube in a refrigerator at 4 ℃ for 2 hours at a rotating speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L1.6% fixing solution for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 200. mu.L of binding buffer was added, and the mixture was stored at 4 ℃ until use.
And taking the quality control products which are prepared, stored for one week, one month and three months on the same day as experimental objects, and dyeing the quality control products according to the instruction of the dyeing kit. The staining results were consistent with those of example one and are not shown. The results of the number ratio of stained cells to primary cells are shown in Table 2.
TABLE 2 quantitative ratio of stained cells to primary cells in example two
The above results show that, in the first method for preparing a quality control product for cell staining of the present invention, when the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is 4: 3, the ratio of the number of E-Cadherin immunomagnetic beads to the number of CEA immunomagnetic beads to the number of CD45 immunomagnetic beads is 5: 2, the number ratio of the white blood cells to the lung cancer circulating tumor cells is 10:1, the binding rate of the immunomagnetic beads and the cells can also reach 99%, which indicates that the first preparation method of the invention has high reliability; the stable storage life of the quality control product is up to three months, and the quality control product prepared by the first preparation method of the invention is proved to have stable property.
EXAMPLE III
1. Taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
2. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 1 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
3. taking 30ul of the E-Cadherin immunomagnetic bead working solution and 40ul of the CEA immunomagnetic bead working solution, adding 350ul of NCI-H1993 cell liquid, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
4. taking 30ul of the CD45 immunomagnetic bead working solution, adding 350ul of leukocyte solution, reversing and uniformly mixing, discarding supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifuge tubes obtained in the steps 3 and 4 in a refrigerator at 4 ℃ for 2 hours at a rotation speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L of 1.6% fixative for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 100. mu.L of each binding buffer was added, and the mixture was stored at 4 ℃ until use.
FIG. 4 is a screenshot of the quality control results stored for one week; FIG. 5 is a screenshot of the quality control result stored for one month; fig. 6 is a screenshot of the quality control result stored for three months. The results of the number ratio of stained cells to primary cells are shown in Table 3.
TABLE 3 quantitative ratio of stained cells to primary cells in example III
The results in Table 3 show that, in the second method for preparing a quality control material for cell staining of the present invention, when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 3: 4, the binding rate of the immunomagnetic beads and the cells reaches 99%, which shows that the second preparation method has high reliability; the stable storage life of the quality control product is up to three months, which shows that the quality control product prepared by the second preparation method has stable property.
Example four
1. Taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
2. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 1 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
3. taking 40ul of the E-Cadherin immunomagnetic bead working solution and 30ul of the CEA immunomagnetic bead working solution, adding 350ul of NCI-H1993 cell liquid, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
4. taking 30ul of the CD45 immunomagnetic bead working solution, adding 350ul of leukocyte solution, reversing and uniformly mixing, discarding supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifuge tubes obtained in the steps 3 and 4 in a refrigerator at 4 ℃ for 2 hours at a rotation speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L of 1.6% fixative for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 100. mu.L of each binding buffer was added, and the mixture was stored at 4 ℃ until use.
The dyeing results of the quality control products prepared, stored for one week, one month and three months on the same day are similar to those of the third example and are not shown. The results of the number ratio of stained cells to primary cells are shown in Table 4.
TABLE 4 quantitative ratio of stained cells to primary cells in example four
As shown in Table 4, in the second method for preparing a quality control product for cell staining of the present invention, when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 4: 3, the binding rate of the immunomagnetic beads and the cells can reach 99%, which indicates that the second preparation method has high reliability; the stable storage life of the quality control product is up to three months, and the quality control product prepared by the second preparation method is stable in property.
Comparative example 1
1. Mixing leukocytes with NCI-H1993 cells (cell number ratio 1:1) to make total volume 700 ul;
2. taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
3. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 2 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
4. adding 30ul of the E-Cadherin immunomagnetic bead working solution, 40ul of the CEA immunomagnetic bead working solution and 60ul of the CD45 immunomagnetic bead working solution into the cell mixed solution in the step 1, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifugal tube in a refrigerator at 4 ℃ for 2 hours at a rotating speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L1.6% fixing solution for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 200. mu.L of binding buffer was added, and the mixture was stored at 4 ℃ until use.
The dyeing results of the quality control products prepared, stored for one week, one month and three months on the same day are shown in fig. 7 to 9. FIG. 7 is a screenshot of the quality control results stored for one week; FIG. 8 is a screenshot of the quality control result stored for one month; fig. 9 is a screenshot of the quality control result stored for three months. The results of the number ratio of stained cells to primary cells are shown in Table 5.
TABLE 5 quantitative ratio of stained cells to primitive cells in comparative example one
The results in Table 5 show that when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 3: 4, the ratio of the number of E-Cadherin immunomagnetic beads to the number of CEA immunomagnetic beads to the number of CD45 immunomagnetic beads is 7: when 6, the binding rate of immunomagnetic beads to cells was about 93%, and the binding effect was inferior to that of examples one and two.
Comparative example No. two
1. Mixing leukocytes with NCI-H1993 cells (cell number ratio 1:1) to make total volume 700 ul;
2. taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
3. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 2 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
4. adding 40ul of the E-Cadherin immunomagnetic bead working solution, 30ul of the CEA immunomagnetic bead working solution and 20ul of the CD45 immunomagnetic bead working solution into the cell mixed solution in the step 1, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifugal tube in a refrigerator at 4 ℃ for 2 hours at a rotating speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L1.6% fixing solution for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 200. mu.L of binding buffer was added, and the mixture was stored at 4 ℃ until use.
The dyeing results of the quality control products prepared, stored for one week, one month and three months on the same day are similar to those of the first comparative example. The results of the quantitative ratio of the stained cells to the original cells are shown in Table 6.
TABLE 6 quantitative ratio of stained cells to primitive cells in comparative example II
The results in Table 6 show that when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 4: 3, the ratio of the number of E-Cadherin immunomagnetic beads to the number of CEA immunomagnetic beads to the number of CD45 immunomagnetic beads is 7: 2, the binding rate of immunomagnetic beads to cells was about 90%, and the binding effect was inferior to that of the first and second examples.
Comparative example No. three
1. Taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
2. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 1 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
3. taking 30ul of the E-Cadherin immunomagnetic bead working solution and 50ul of the CEA immunomagnetic bead working solution, adding 350ul of NCI-H1993 cell liquid, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
4. taking 30ul of the CD45 immunomagnetic bead working solution, adding 350ul of leukocyte solution, reversing and uniformly mixing, discarding supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifuge tubes obtained in the steps 3 and 4 in a refrigerator at 4 ℃ for 2 hours at a rotation speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L of 1.6% fixative for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 100. mu.L of each binding buffer was added, and the mixture was stored at 4 ℃ until use.
And taking the quality control products which are prepared, stored for one week, one month and three months on the same day as experimental objects, and dyeing the quality control products according to the instruction of the dyeing kit. The dyeing results of this example are similar to those of comparative example one. The results of the quantitative ratio of the stained cells to the original cells are shown in Table 7.
TABLE 7 quantitative ratio of stained cells to primitive cells in comparative example three
In Table 7, when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 3: at 5, the binding rate of immunomagnetic beads to cells was about 94%, indicating that the binding effect was not as good as in the third and fourth examples.
Comparative example No. four
1. Taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
2. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 1 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
3. taking 40ul of the E-Cadherin immunomagnetic bead working solution and 20ul of the CEA immunomagnetic bead working solution, adding 350ul of NCI-H1993 cell liquid, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
4. taking 30ul of the CD45 immunomagnetic bead working solution, adding 350ul of leukocyte solution, reversing and uniformly mixing, discarding supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifuge tubes obtained in the steps 3 and 4 in a refrigerator at 4 ℃ for 2 hours at a rotation speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L of 1.6% fixative for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 100. mu.L of each binding buffer was added, and the mixture was stored at 4 ℃ until use.
And taking the quality control products which are prepared, stored for one week, one month and three months on the same day as experimental objects, and dyeing the quality control products according to the instruction of the dyeing kit. The picture of the staining results is similar to the comparative example one and is not shown. See table 8 for the results of the number ratio of stained cells to primary cells.
TABLE 8 quantitative ratio of stained cells to primitive cells in comparative example four
The results in Table 8 show that when the number ratio of E-Cadherin immunomagnetic beads to CEA immunomagnetic beads is 2: 1, the binding rate of immunomagnetic beads to cells was about 93%, and the binding effect was inferior to that of examples three and four.
Comparative example five
1. White blood cells were compared with NCI-H1993 cells (cell number ratio 1:10)2) Mixing, the total volume is 700 ul;
2. taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
3. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 2 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
4. adding 30ul of the E-Cadherin immunomagnetic bead working solution, 40ul of the CEA immunomagnetic bead working solution and 50ul of the CD45 immunomagnetic bead working solution into the cell mixed solution in the step 1, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifugal tube in a refrigerator at 4 ℃ for 2 hours at a rotating speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L1.6% fixing solution for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 200. mu.L of binding buffer was added, and the mixture was stored at 4 ℃ until use.
And dyeing the quality control products which are prepared, stored for one week, one month and three months on the same day according to the instruction of the dyeing kit. FIG. 10 is a screenshot of the quality control results stored for one week; FIG. 11 is a screenshot of the quality control result stored for one month; fig. 12 is a screenshot of the quality control result stored for three months. The results of the quantitative ratio of the stained cells to the original cells are shown in Table 9.
TABLE 9 quantitative ratio of stained cells to primitive cells in comparative example five
The above results show that when the ratio of the number of the white blood cells to the number of the circulating tumor cells of the lung cancer is 1:102The enrichment rate of the cells was about 95%, and the results were inferior to those of example one.
Comparative example six
1. White blood cells were compared with NCI-H1993 cells (cell number ratio 10)2:1) mixing, the total volume is 700 ul;
2. taking 30ul of E-Cadherin immunomagnetic beads, adding 1ml of binding buffer solution, discarding supernatant, and resuspending the beads with 1ml of binding buffer solution to prepare E-Cadherin immunomagnetic bead working solution;
3. processing the CEA immunomagnetic beads and the CD45 immunomagnetic beads by the same method in the step 2 to prepare a CEA immunomagnetic bead working solution and a CD45 immunomagnetic bead working solution;
4. adding 40ul of the E-Cadherin immunomagnetic bead working solution, 30ul of the CEA immunomagnetic bead working solution and 28ul of the CD45 immunomagnetic bead working solution into the cell mixed solution in the step 1, reversing and uniformly mixing, discarding the supernatant, and repeating the operation for 3 times;
5. uniformly mixing the centrifugal tube in a refrigerator at 4 ℃ for 2 hours at a rotating speed of 15 revolutions per minute;
6. rotating, mixing, removing supernatant, fixing with 50 μ L1.6% fixing solution for 30min, and resuspending once every 10 min;
7. the supernatant was discarded, 200. mu.L of binding buffer was added, and the mixture was stored at 4 ℃ until use.
The dyeing results of the quality control products prepared, stored for one week, one month and three months on the same day are similar to those of the fifth comparative example and are not shown. The results of the number ratio of stained cells to primary cells are shown in Table 10.
TABLE 10 quantitative ratio of stained cells to primitive cells in comparative example six
The results in Table 10 show that the ratio of the number of the white blood cells to the number of the circulating tumor cells of lung cancer is 102:1, the enrichment rate of the cells is about 92%, and the enrichment effect is not good.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (2)
1. A preparation method of a quality control product for cell staining is characterized by comprising the following steps:
(1) mixing E-Cadherin immunomagnetic beads with a binding buffer solution to obtain E-Cadherin immunomagnetic bead working solution, mixing CEA immunomagnetic beads with the binding buffer solution to obtain CEA immunomagnetic bead working solution, and mixing CD45 immunomagnetic beads with the binding buffer solution to obtain CD45 immunomagnetic bead working solution;
(2) mixing the E-Cadherin immunomagnetic bead working solution, the CEA immunomagnetic bead working solution, the CD45 immunomagnetic bead working solution, the white blood cells and the lung cancer circulating tumor cells to obtain a first mixed solution;
(3) removing the liquid in the first mixed solution, and adding a stationary liquid for fixation to obtain a quality control product of the lung cancer circulating tumor cells;
the E-Cadherin immunomagnetic beads specifically bind to lung cancer circulating tumor cells via an E-Cadherin antibody, the CEA immunomagnetic beads specifically bind to lung cancer circulating tumor cells via a CEA antibody, the E-Cadherin immunomagnetic beads, the CEA immunomagnetic beads and the lung cancer circulating tumor cells forming bead-antibody-cell complexes;
the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is more than or equal to 3: 4, and less than or equal to 4: 3; the ratio of the number of the E-Cadherin immunomagnetic beads, the number of the CEA immunomagnetic beads and the number of the CD45 immunomagnetic beads is greater than or equal to 4: 3, and less than or equal to 5: 2; the number ratio of the leukocytes to the lung cancer circulating tumor cells is greater than or equal to 1:10, and less than or equal to 10: 1.
2. a preparation method of a quality control product for cell staining is characterized by comprising the following steps:
(1) mixing E-Cadherin immunomagnetic beads with a binding buffer solution to obtain E-Cadherin immunomagnetic bead working solution, mixing CEA immunomagnetic beads with the binding buffer solution to obtain CEA immunomagnetic bead working solution, and mixing CD45 immunomagnetic beads with the binding buffer solution to obtain CD45 immunomagnetic bead working solution;
(2) mixing the E-Cadherin immunomagnetic bead working solution, the CEA immunomagnetic bead working solution and lung cancer circulating tumor cells to obtain a second mixed solution, and mixing the CD45 immunomagnetic bead working solution and white blood cells to obtain a third mixed solution;
(3) respectively removing the liquid in the second mixed liquid and the third mixed liquid, and adding a stationary liquid for fixation to obtain a quality control product of the lung cancer circulating tumor cells;
the E-Cadherin immunomagnetic beads specifically bind to lung cancer circulating tumor cells via an E-Cadherin antibody, the CEA immunomagnetic beads specifically bind to lung cancer circulating tumor cells via a CEA antibody, the E-Cadherin immunomagnetic beads, the CEA immunomagnetic beads and the lung cancer circulating tumor cells forming bead-antibody-cell complexes;
the number ratio of the E-Cadherin immunomagnetic beads to the CEA immunomagnetic beads is more than or equal to 3: 4, and less than or equal to 4: 3.
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