Methods for detecting the biological activity of the test substance

Abstract

The present invention discloses a method for detecting the biological activity of a test substance. The detection method comprises: (1) dividing the test substance to be tested into at least two test substance combinations, each combination comprising at least two test substances; (2) co-incubating each combination in (1) with a test cell to obtain biological activity data; (3) co-incubating each test substance corresponding to the combination for which the biological activity data obtains a positive result with the test cell, and the test substance that obtains a positive result is a candidate drug with biological activity. The detection method of the present invention can not only ensure the reliability of the biological activity detection of the test substance, preliminarily screen the target test substance, but also improve the efficiency of the test substance activity detection and reduce the experimental cost in vivo and in vitro.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting the biological activity of a test object. Background Art

[0002] For a long time, cell-based in vitro drug activity testing has been indispensable in pharmacological research, especially for drug screening, where mechanism analysis is an essential technology. When it comes to testing the pharmacological effects of the test substance on cells (such as anti-tumor activity), cells are generally inoculated into cell culture plates, and then the test substances are added to the cells for co-incubation, followed by testing the in vitro anti-tumor activity of the test substance, such as cell activity, tube formation ability, repair and migration ability, etc. When the number of test substances to be tested is as high as hundreds, the amount of cell culture, manual operation and experimental time required for cell-based in vitro activity testing are greatly increased, affecting the efficiency of in vitro activity detection and the control of experimental costs. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art that the amount of cells and cell culture plates used for testing the test substances separately increases several times, resulting in increased operation time, low detection efficiency, and low consistency between batches, and provide a method for detecting the biological activity of the test substance. The detection method of the present invention can not only ensure the reliability of the biological activity detection of the test substance, preliminarily screen the target test substance, but also improve the efficiency of the test substance activity detection and reduce the experimental cost, thereby improving the reliability of the test substance activity screening.

[0004] The present invention solves the above technical problems through the following technical solutions:

[0005] A first aspect of the present invention provides a detection kit for detecting drug activity, wherein the detection kit comprises a cell culture medium.

[0006] In some embodiments of the present invention, the cell culture medium is used to culture and suspend the test cells, and to dilute the test substance to be tested.

[0007] In some embodiments of the present invention, the test cells are human umbilical vein endothelial cells, and the cell culture medium is a complete culture medium for human umbilical vein endothelial cells, such as a complete culture medium for human umbilical vein endothelial cells (HUVEC-90011) from Saiye (Suzhou) Biotechnology Co., Ltd.

[0008] Preferably, the detection kit further comprises a cell digestion agent, and the cell digestion agent preferably comprises 0.05% Trypsin-0.53mM EDTA; the % is the mass percentage.

[0009] Preferably, the detection kit further comprises a cell detergent, and the cell detergent is preferably PBS.

[0010] A second aspect of the present invention provides a method for detecting the biological activity of a test substance, the detection method comprising:

[0011] (1) Dividing the test substances into at least two combinations of test substances, each combination comprising at least two test substances;

[0012] (2) administering each combination in (1) to a test subject to obtain biological activity data;

[0013] (3) administering each test substance corresponding to the combination with positive results in biological activity data to the test subject to obtain biological activity data, and the test substance with positive results is a candidate drug with biological activity;

[0014] The positive result means that for at least one detection indicator, the biological activity data has a significant difference relative to the preset value.

[0015] In the present invention, the test substance refers to a molecule with expected biological activity; preferably, the test substance is selected from polypeptides, protein macromolecules and small molecule compounds, such as polypeptides.

[0016] In the present invention, the subject can be conventional in the art, preferably isolated cells or animal models.

[0017] In the present invention, the preset value may be a reference value set by a person skilled in the art according to the purpose of the experiment, such as a blank control.

[0018] In the present invention, the significant difference may be a significant difference known in the art, for example, a statistically significant difference, P≤0.05.

[0019] In some embodiments of the present invention, in (1), the test substances to be tested are divided into N / n test substance combinations; wherein N is the number of types of all test substances to be tested, N is an integer between 100 and 600, and n is the number of types of test substances to be tested in each combination, wherein n is an integer between 5 and 15.

[0020] In some preferred embodiments of the present invention, N is an integer within the range of 100-500, 100-400, 100-300 or 100-200; n is an integer within the range of 5-12, 5-10 or 5-8.

[0021] In other preferred embodiments of the present invention, in the method, N is an integer within the range of 200-300, 300-400, 400-500 or 500-600; n is an integer within the range of 8-10 or 10-12.

[0022] In some specific embodiments of the present invention, N=100, n=10.

[0023] In some specific embodiments of the present invention, N=100, n=5.

[0024] In some specific embodiments of the present invention, N=300, n=10.

[0025] In some specific embodiments of the present invention, N=150, n=15.

[0026] In some specific embodiments of the present invention, N=440, n=10.

[0027] In some embodiments of the present invention, the test substance is a polypeptide.

[0028] In some specific embodiments of the present invention, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 440; the amino acid sequence of the polypeptide is as shown in any one of SEQ ID NOs: 1 to 440.

[0029] In some specific embodiments of the present invention, the subject is a test cell, and the detection method comprises:

[0030] (1) Dividing the test substances into at least two combinations of test substances, each combination comprising at least two test substances;

[0031] (2) co-incubating each combination in (1) with the test cells to obtain biological activity data;

[0032] (3) Each test substance corresponding to the combination with a positive result in the biological activity data is co-incubated with the test cells to obtain the biological activity data. The test substance with a positive result is a candidate drug with biological activity.

[0033] In some embodiments of the present invention, the test cell is a normal cell or a tumor cell.

[0034] Preferably, the normal cells are epithelial tissue cells or immune cells.

[0035] Preferably, the tumor cells are selected from liver cancer cells, lung cancer cells, ovarian cancer cells, colorectal cancer cells and sarcoma cells.

[0036] More preferably, the epithelial tissue cells are endothelial cells, such as human umbilical vein endothelial cells; and the immune cells are T lymphocytes.

[0037] More preferably, the liver cancer cells are HCCLM-3 cells; the lung cancer cells are NCI-H226 cells; the ovarian cancer cells are A2780 cells; the colorectal cancer cells are Colo205 cells; and the sarcoma cells are HT1080 cells.

[0038] In some embodiments of the present invention, the test cells are subcultured before use.

[0039] Preferably, the subculture operation comprises the following steps: mixing the test cells with a cell culture medium at a volume ratio of 1:2 for subculture; and the subculture conditions are 37° C. and 5% CO 2 .

[0040] More preferably, the passage number of the test cells is P6.

[0041] In some embodiments of the present invention, the co-incubation operation comprises the following steps: culturing cells with a cell culture medium in an environment of 37° C., 5% CO 2 , and saturated humidity.

[0042] In the present invention, the cell culture medium is as described in the first aspect.

[0043] In some embodiments of the present invention, in the co-incubation, the initial concentration of the combination and the test substance is 25-30 μg / mL per test substance, and the initial concentration of the test cells is 1.5-2.5×10 5 cells / mL, for example 2×10 5 cells / mL.

[0044] In the present invention, the initial concentrations of the test substance and the test cells can be set by those skilled in the art according to the expected effective concentration of the test substance.

[0045] In the present invention, the detection index is selected from one or more of the number of nodes, the number of formed tubes, the length of formed tubes and the area of ​​formed tubes.

[0046] In some embodiments of the present invention, the detection index is obtained through a tube forming test.

[0047] Preferably, the operation of the tube forming test includes the following steps:

[0048] S1: Take the test cells in the logarithmic growth phase, remove the cell culture medium, and wash the cells with a cell detergent and digest the cells with a cell digester to obtain dispersed cells;

[0049] S2: centrifuging the dispersed cells obtained in S1, resuspending the cells in a cell culture medium, and counting the cells to obtain the test cells at a working concentration;

[0050] S3: Co-incubate the test cells obtained in S2 with the test substance to be tested, observe the angiogenesis, and take pictures under a microscope to obtain statistical data of the detection index.

[0051] In some embodiments of the present invention, the cell culture medium is a cell culture medium suitable for the proliferation and growth of the test cells.

[0052] In some embodiments of the invention, the cell washing agent is PBS.

[0053] In some embodiments of the present invention, the cell digestion agent is 0.05% Trypsin-0.53 mM EDTA.

[0054] In some embodiments of the present invention, the centrifugation conditions are 300×g, 22-25° C., 5-8 min.

[0055] In some embodiments of the present invention, the working concentration of the test cells is 1.5 to 2.5×10 5 cells / mL, for example 2×10 5 cells / mL.

[0056] In some embodiments of the present invention, the co-incubation conditions are as described above.

[0057] In some embodiments of the present invention, the subject is an animal model, and the detection method comprises:

[0058] (1) Dividing the test substances into at least two combinations of test substances, each combination comprising at least two test substances;

[0059] (2) administering each combination in (1) to the animal model to obtain biological activity data;

[0060] (3) Each test substance corresponding to the combination with a positive result in the biological activity data is co-incubated with the test cells, and test cells without the test substance are set as blank controls to obtain biological activity data. The test substance with a positive result is a candidate drug with biological activity.

[0061] In the present invention, the administration may be any administration method known to those skilled in the art, and preferably the administration method is selected according to the animal model used and the purpose of the experiment, such as subcutaneous injection.

[0062] In some embodiments of the present invention, the administration is carried out in an amount of 8 to 12 mL / kg, and the dose of the test substance is 28 to 32 mg / kg / species; for example, the administration is carried out in an amount of 10 mL / kg, and the dose of the test substance is 30 mg / kg / species.

[0063] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0064] The reagents and raw materials used in the present invention are commercially available.

[0065] The positive and progressive effects of the present invention are:

[0066] In the in vitro detection, the present invention co-incubates the mixed test substance with the cultured cells, and adopts a tube test to screen and evaluate the biological activity of the test substance, such as anti-tumor activity. This not only ensures the reliability of the activity detection of the test substance and preliminarily screens the target test substance, but also improves the in vitro activity detection efficiency of the test substance and reduces the cost of in vitro experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Schematic diagram of the effect of the combination of test substances on the average number of nodes in cell tube formation.

[0068] Figure 2 Schematic diagram of the effect of the combination of test substances on the average number of tubes formed by HUVEC cells.

[0069] Figure 3 Schematic diagram of the effect of the combination of test substances on the total length of HUVEC cell tubes.

[0070] Figure 4 Schematic diagram of the effect of the combination of test substances on the average tube-forming area of ​​HUVEC cells. DETAILED DESCRIPTION

[0071] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0072] The reagent materials used in the examples are shown in Table 1:

[0073] Table 1 Reagents and Materials

[0074]

[0075] The test substances (TB001-TB440) used in the examples have a purity of ≥95% and are stored in the form of lyophilized powder at -85--70°C in the dark.

[0076] The amino acid sequences of the test substances TB001 to TB440 are shown in Table 2:

[0077] Table 2 Amino Acid Sequence Listing

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] The instruments and equipment used in the examples are shown in Table 3:

[0088] Table 3 Instruments and equipment

[0089]

[0090]

[0091] The complete culture medium in the examples is a complete culture medium for human umbilical vein endothelial cells.

[0092] The preparation of the test solution in Examples 1 to 3 is as follows:

[0093] Accurately weigh 2.5 mg of the test substance, add 0.5 mL DMSO to fully dissolve, then add 2.167 mL complete medium, gently shake to mix the test substance, and avoid foaming (if foaming occurs, let it stand until it disappears). Use a hydrophilic sterilizing filter (0.22 μm PES filter membrane) to sterile filter the sample solution and prepare a working solution with a concentration of 1.5 mg / mL, and then dilute it for use.

[0094] All reagents in the examples were prepared and used immediately.

[0095] Example 1 Test on the effect of HUVEC cell tube-forming ability

[0096] In in vitro anti-tumor activity detection, when the number of test substances to be detected is as high as hundreds, cell activity detection is often performed using 96-well plates, and dozens of test substances can be detected at a time. In addition to the increase in cell demand, the detection operation time is only extended by 1-2 hours. However, for tube formation ability detection, such as HUVEC cell tube formation test, the field of view is small when using 96-well plates, and it is difficult to observe the tube formation. Usually, 24-well plates are used for this purpose. Except for the control group, only a few test substances can be detected. The cell dosage and operation time increase several times. Cell repair ability and migration ability experiments are often performed using 12-well plates and 6-well plates. As the number of cells and cell culture plates increases several times, the experimental cost increases accordingly, the operation time increases several times, the detection efficiency is greatly reduced, and it is difficult to ensure that the experiments are carried out in the same batch, making it difficult to maintain consistency before and after the experiment.

[0097] 1. Cell recovery and culture:

[0098] Take 1 tube of frozen HUVEC cells, gently and quickly revive them in a 37°C water bath, centrifuge at 300×g, 4°C for 5 minutes, discard the supernatant, resuspend the cells with an appropriate amount of complete medium, inoculate them in a T25 culture flask, and culture them in a 37°C, 5% CO2 cell culture incubator. When the cell growth confluence reaches about 80% to 90%, digest the cells with 0.05% Trypsin-0.53mM EDTA, subculture the cells at a volume ratio of 1:2, and culture them in a 37°C, 5% CO2 cell culture incubator until the number required for the experiment is 2.16×10 7 Cells, passage number is P6.

[0099] 2. HUVEC cell tube formation test

[0100] (1) Precool the 24-well plate in a -20°C refrigerator for 30 min. Before the experiment, the base gel must be fully thawed on ice for 30 min.

[0101] (2) Place the 24-well plate on ice, add 300 μL of Matrigel to each well, and incubate in a 37°C incubator for 45 min;

[0102] (3) HUVEC cells in the logarithmic growth phase were taken, the culture medium was discarded, and the cells were washed once with PBS; the cells were digested with 0.05% Trypsin-0.53 mM EDTA;

[0103] (4) Centrifuge at 300 × g, 25°C for 5 min and resuspend the cells in 1 mL of complete medium;

[0104] (5) Count cells using the trypan blue method: take 20 μL of cell suspension, add an equal volume of trypan blue and mix thoroughly, take 20 μL and add it to a cell counting plate, and count on a cell counter;

[0105] (6) Use medium containing the test substance to adjust the cell concentration to 2×10 5 cells / mL, 500 μL of cell suspension was added to each well and inoculated on the matrix gel of a 24-well plate, with 2 parallel wells set up for each group;

[0106] (7) The cells were cultured at 5% CO2 and 37°C for 4 h, and the angiogenesis was observed. Photos were taken under a microscope for statistical analysis of the number of nodes, number of tubes, area of ​​tubes, and length of tubes.

[0107] As shown in Table 4, 444 test substances were mixed in equal amounts of ten to prepare 44 test samples. In addition to the experimental results, statistical comparisons of consumables usage, cell usage, experimental time, data statistical time, etc. were added.

[0108] Table 4 Grouping of test substances

[0109] Group Group Abbreviation Concentration (μg / mL) Blank control group (control) Con / Test group 1 to 44 1～44 30μg / mL / species

[0110] 3. Experimental results

[0111] The statistical results of the experimental personnel and reagent materials are shown in Table 5. The conventional detection method is expected to cost 100,000 yuan. The present invention achieves the effect of reducing the experimental cost.

[0112] Table 5 Statistical results of experimental personnel and reagent materials

[0113]

[0114] 3.1 Number of nodes

[0115] The results showed that the number of nodes in groups 31, 36, and 41 were 38.67±15.96, 122.67±102.80, and 160.17±113.22, respectively, which could significantly reduce the number of nodes that formed tubes compared with the blank control group (336.67±66.45) (P<0.05); the number of nodes in groups 5, 20, 21, 37, 40, and 44 were 272.50±74.24, 237.83±59.53, 278.33±37.30, 277.50±46.07, 265.50±38.13, and 277.83±21.58, respectively, which could reduce the number of nodes that formed tubes compared with the blank control group (336.67±66.45), but there was no statistical difference (P>0.05). The levels of the other groups were slightly lower than the blank control or higher than the blank control, and had no significant effect on the number of HUVEC cell tube nodes (P>0.05).

[0116] Effects of each group of test substances on the average number of nodes in HUVEC cell tube formation Figure 1 shown.

[0117] 3.2 Number of tubes

[0118] The HUVEC cell tube formation test showed that the number of HUVEC cell tubes in groups 5, 20, 21, 31, 36, 41, and 44 were 24±10.80, 29.67±9.31, 29.83±6.79, 1.00±1.26, 8.67±9.83, 12.33±11.27, and 31.33±3.93, respectively, which were significantly reduced compared with the blank control group (39.33±9.58) (P<0.05); the number of HUVEC cell tubes in groups 34, 37, 40, and 42 were 34.00±9.17, 33.00±7.48, 29.33±7.89, and 33.67±5.92, respectively, which were reduced compared with the blank control group (39.33±9.58), but there was no statistical difference (P>0.05). The levels of the other groups were slightly lower than the blank control or higher than the blank control, but had no significant effect on the average number of tubes formed by HUVEC cells (P>0.05).

[0119] Effects of each group of test substances on the average number of HUVEC cell tubes Figure 2 shown.

[0120] 3.3 Tube length

[0121] The HUVEC cell tube formation test showed that the lengths of HUVEC tubes in groups 31, 36, and 41 were 4836.17±496.83μm, 6151.67±2346.02μm, and 7048.17±1404.16μm, respectively, which significantly reduced the lengths of HUVEC cell tubes compared with the blank control group (8877.50±794.52μm) (P<0.05); the lengths of HUVEC tubes in groups 5, 20, 21, 37, and 4 The lengths of HUVEC tubes in 4 groups were 8129.50±1169.03μm, 8153.17±919.93μm, 8193.17±590.96μm, 7690.33±829.17μm, and 8076.33±410.81μm, respectively, which were significantly different from the blank control group (8877.50±794.52μm) (P>0.05). The lengths of HUVEC tubes in the other groups were slightly lower than or higher than the blank control group, and had no significant effect on the length of HUVEC tubes (P>0.05).

[0122] Effects of each group of test substances on the total length of HUVEC cell tubes Figure 3 shown.

[0123] 3.4 Pipe area

[0124] The results of the HUVEC cell tube formation test showed that the tube formation area of ​​HUVEC cells in groups 5, 31, 36, and 41 was 181261.00±89064.44μm2 、3252.50±4545.87μm 2 、38033.00±50585.98μm 2 、47339.00±58283.93μm 2 , compared with the blank control group (268386.17±41919.57μm 2 ) can significantly reduce the tube-forming area in the HUVEC cell tube-forming test (P<0.05); the tube-forming area of ​​HUVEC cells treated with the test substance in group 14 was 321441.2±18275.69μm 2 , compared with the blank control group (268386.17±41919.57μm 2 ) can significantly increase the tube-forming area of ​​HUVEC cells in the tube-forming test (P>0.05). The other groups had no significant effect on the tube-forming area of ​​HUVEC cells (P>0.05).

[0125] Effects of each group of test substances on the total length of HUVEC cell tubes Figure 4 shown.

[0126] 4. Conclusion

[0127] Under the conditions of this experiment, the test substances in groups 5, 20, 21, 31, 36, 41, and 44, at a concentration of 30 μg / mL, could inhibit the number of nodes, the number of tubes, the length of tubes, or the area of ​​tubes in HUVEC cells, indicating that they have the effect of inhibiting the angiogenesis of HUVEC cells; while the test substances in the other groups were slightly lower than the blank control or higher than the blank control, and had no obvious inhibitory effect on the tube-forming ability of HUVEC cells.

[0128] Example 2 Test on the effect of HUVEC cell tube-forming ability

[0129] 1. Cell recovery and culture

[0130] Same as Example 1

[0131] 2. HUVEC cell tube formation test

[0132] Take 100 portions of the test substance (TB001-TB100), and prepare 5 portions of each of them as a test sample, as shown in Table 6 below. The experimental operation process is the same as Example 1.

[0133] Table 6 Grouping

[0134]

[0135] 3. Experimental results

[0136] The average values ​​of the number of nodes, tube length, and tube area of ​​HUVEC cells of each combination of two mixed test substances (Group 1 and Group 2 in Example 2 correspond to Group 1 in Example 1) were not significantly different from those in Example 1.

[0137] Example 3 Test on the effect of HUVEC cell tube-forming ability

[0138] 1. Cell recovery and culture

[0139] Same as Example 1

[0140] 2. HUVEC cell tube formation test

[0141] Take 150 portions of the test substance (TB001-TB150), and prepare each 15 portions into one test sample, as shown in Table 7 below. The experimental operation process is the same as Example 1.

[0142] Table 7 Grouping

[0143]

[0144] 3. Experimental results

[0145] The average values ​​of the number of nodes, tube length, and tube area of ​​HUVEC cells in each combination of two mixed test substances (Groups 1-3 in Example 2 correspond to Group 1 in Example 3) were not significantly different from those in Example 2.

Claims

1. A method for detecting the biological activity of a test substance, characterized in that: The detection method comprises: (1) Divide the test substances into at least two combinations of test substances, each combination including at least two test substances; (2) co-incubating each combination in (1) with the test cells to obtain biological activity data; (3) Each test substance corresponding to the combination with positive results in the biological activity data is co-incubated with the test cells, and the test substance with positive results is the candidate drug with biological activity; The positive result means that for at least one detection index, the biological activity data has a significant difference relative to the preset value; (1), the test substances to be tested are divided into N / n test substance combinations; wherein N is the number of all test substances to be tested, and N is an integer between 100 and 440; wherein n is the number of test substances to be tested in each combination, and n is an integer between 5 and 15; The detection indicators include the number of nodes, the number of formed tubes, the length of formed tubes and the area of ​​formed tubes; The test substance is a polypeptide; the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1-440; The biological activity is to inhibit the tube-forming ability of human umbilical vein endothelial cells.

2. The detection method according to claim 1, characterized in that N is an integer between 100 and 400.

3. The detection method according to claim 1, characterized in that N is an integer between 100 and 300.

4. The detection method according to claim 1, characterized in that N is an integer between 100 and 200.

5. The detection method according to any one of claims 2 to 4, characterized in that: n is an integer between 5 and 12.

6. The detection method according to any one of claims 2 to 4, characterized in that: n is an integer between 5 and 10.

7. The detection method according to any one of claims 2 to 4, characterized in that: n is an integer between 5 and 8.

8. The detection method according to claim 1, characterized in that N=100, n=10; Or, N=100, n=5; or, N=300, n=10; Or, N=150, n=15; Or, N=440, n=10.

9. The detection method according to claim 1, characterized in that: The test cells were subcultured before use.

10. The detection method according to claim 9, characterized in that The subculture operation includes the following steps: mixing the test cells with the cell culture medium at a volume ratio of 1:2 for subculture; the subculture conditions are 37°C and 5% CO2.

11. The detection method according to claim 10, characterized in that: The passage number of the tested cells was P6.

12. The detection method according to claim 1, characterized in that: The co-incubation operation includes the following steps: culturing cells with cell culture medium in an environment of 37° C., 5% CO 2 , and saturated humidity.

13. The detection method according to claim 12, characterized in that: The cell culture medium is a cell culture medium suitable for the proliferation and growth of the test cells.

14. The detection method according to claim 12 or 13, characterized in that In the co-incubation, the initial concentration of the combination and the test substance is 25-30 μg / mL for each test substance, and the initial concentration of the test cells is 1.5-2.5×10 5 cells / mL.

15. The detection method according to claim 14, characterized in that: The initial concentration of the tested cells was 2×10 5 cells / mL.

16. The detection method according to claim 1, characterized in that: The detection index is obtained through a tube forming test.

17. The detection method according to claim 16, characterized in that: The operation of the tube forming test comprises the following steps: S1: Take the test cells in the logarithmic growth phase, remove the cell culture medium, and wash the cells with a cell detergent and digest the cells with a cell digester to obtain dispersed cells; S2: centrifuging the dispersed cells obtained in S1, resuspending the cells in a cell culture medium, and counting the cells to obtain the test cells at a working concentration; S3: Co-incubate the test cells obtained in S2 with the test substance, observe the angiogenesis, and take pictures under a microscope to obtain statistical data of the detection index.

18. The detection method according to claim 17, characterized in that: The cell culture medium is a cell culture medium suitable for the proliferation and growth of the test cells; And / or, the cell detergent is PBS; and / or, the cell digestion agent is 0.05% Trypsin-0.53 mM EDTA; And / or, the centrifugation condition is 300×g, 22-25° C., 5-8 min; And / or, the working concentration of the test cells is 1.5-2.5×10 5 cells / mL; And / or, the co-incubation conditions are as described in the co-incubation in the detection method according to any one of claims 12-15.

19. The detection method according to claim 18, characterized in that: The working concentration of the tested cells was 2×10 5 cells / mL.

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