Culture medium for in-vitro culture of tumor cells MCF-7 and preparation method of tumor antigen CA15-3

Abstract

The invention discloses a culture medium for culturing a tumor cell MCF-7 in vitro and a preparation method of a tumor antigen CA15-3, and relates to the technical field of biology. The culture medium comprises the following components: a DMEM culture medium, an RPMI-1640 culture medium, FBS, double antibodies, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids. The invention relates to a preparation method of a tumor antigen CA15-3. According to the culture medium provided by the invention, the expression quantity of the antigen CA15-3 can be improved, so that a culture supernatant with the high-concentration CA15-3 antigen is obtained, and then a large number of specific antigens CA15-3 can be obtained through purification.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a culture medium for culturing tumor cells MCF-7 in vitro and a preparation method of tumor antigen CA15-3. Background Art

[0002] Antigen CA15-3 is a glycoprotein encoded by the mucin1 gene, which exists in cancer cells of various adenocarcinomas including breast cancer, lung cancer, ovarian cancer, pancreatic cancer, etc. Its peptide molecular weight is 120-225kDa, and the size after glycosylation is 250-1000kDa. The amino acid sequence and glycosylation degree of CA15-3 in different tissues are different, and there are nearly 80 isomers.

[0003] Under normal circumstances, the antigen CA15-3 only exists in the glandular cell cavity. When the cells become cancerous, the antigen CA15-3 content in the serum increases. The main method for detecting the content of CA15-3 protein in plasma is the immunoassay method. As a calibrator, a protein with high activity and high stability is required. Currently, the commercial sources of CA15-3 protein include recombinant expression and natural extraction. Since the CA15-3 protein contains a large number of variable tandem repeats (VNTRs), the recombinant expression method meets the needs of recombinant expression by reducing the number of VNTRs of the CA15-3 protein. This method is prone to changes in the structure and properties of the protein, and there is a risk of reduced activity. Although the natural extraction method retains the natural structure of the protein, the output CA15-3 protein content is low and the protein structure is uneven.

[0004] Therefore, it is necessary to develop a method for mass production of CA15-3 to meet the needs of kit production. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the antibody CA15-3 prepared by the existing preparation method cannot achieve high content and good activity.

[0006] The present invention is achieved by the following technical scheme: a culture medium for culturing tumor cells MCF-7 in vitro, the components of which include DMEM culture medium, RPMI-1640 culture medium, FBS, double antibody, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids. The culture medium formula can reduce the serum content used in the subsequent purification step and ensure the nutrients for the normal growth of cells.

[0007] Preferably, the concentrations of the components FBS, double antibody, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids in the culture medium are: 1.8-2.2wt%, 0.8-1.2wt%, 0.4-0.6g / L, 0.4-0.6g / L, 8-12μg / mL, 0.8-1.2g / L, 0.8-1.2g / L, 1.8-2.2g / L and 1.8-2.4wt%, respectively.

[0008] Preferably, the mixing ratio of the DMEM culture medium to the RPMI-1640 culture medium is 1:0.8-1.2.

[0009] Preferably, the concentrations of the components FBS, double antibody, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids in the culture medium are: 2wt%, 1wt%, 0.5g / L, 0.5g / L, 10μg / mL, 1g / L, 1g / L, 2g / L and 2wt% respectively;

[0010] The mixing ratio of the DMEM culture medium and the RPMI-1640 culture medium is 1:1.

[0011] In order to better solve the above problems, the present invention also provides a method for preparing tumor antigen CA15-3, comprising the following steps:

[0012] S1: adding the revived MCF-7 cells to CA15-3 medium 1 for culture, and performing subculture when the MCF-7 cells grow to a confluence of 80% to 90%;

[0013] S2: When the MCF-7 cells grow to a confluence of 80% to 90% during the subculture process, the CA15-3 culture medium 1 is replaced with the CA15-3 culture medium 2 to promote CA15-3 antigen expression, wherein the CA15-3 culture medium 2 is the culture medium according to any one of claims 1 to 4;

[0014] S3: After promoting CA15-3 antigen expression, CA15-3 feed was added to MCF-7 cells for culture, and the supernatant was collected; the feed was supplemented with additional nutrients such as carbohydrates and amino acids, which were added as nutrients during the antigen expression process. At the same time, EGF has the effect of promoting CA15-3 antigen expression.

[0015] S4: Repeat step S3 multiple times, and purify the collected supernatant to obtain antigen CA15-3 liquid.

[0016] Preferably, the components of the CA15-3 culture medium 1 include DMEM culture medium, FBS, dual antibodies and insulin, the concentration of the FBS is 8-12wt%, the concentration of the dual antibodies is 0.8-1.2wt%, and the concentration of the insulin is 8-12μg / mL.

[0017] Preferably, the components of the CA15-3 feed include the CA15-3 culture medium 2, and additionally added tryptone, yeast powder, sodium bicarbonate, glucose, galactose, fructose, non-essential amino acids and EGF.

[0018] Preferably, the concentrations of the components of the CA15-3 feed, tryptone, yeast powder, sodium bicarbonate, glucose, galactose, fructose, non-essential amino acids and EGF are 1.8-2.2 g / L, 1.8-2.2 g / L, 1.3-1.8 g / L, 1.8-2.2 g / L, 1.8-2.2 g / L, 3.5-4.5 g / L, 1.8-2.4 wt % and 8-12 ng / mL, respectively. Sodium bicarbonate can be used to adjust the pH, ensure the stability of the environment during cell growth, and prolong the life of the cells.

[0019] Preferably, the purification process in S4 comprises the following steps:

[0020] S41: centrifuging the supernatant to remove residual cell debris to obtain a clarified solution;

[0021] S42: precipitating the antigen CA15-3 in the clear solution by ammonium sulfate precipitation method, and collecting the precipitate by centrifugation;

[0022] S43: The precipitate is redissolved in a buffer solution and dialyzed. After the dialysis is completed, the liquid is collected, and then centrifuged and the supernatant is collected to obtain the antigen CA15-3 liquid.

[0023] Preferably, in the ammonium sulfate precipitation method, the saturation of the ammonium sulfate solution is 60-70 wt%.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) An embodiment of the present invention provides a culture medium for culturing tumor cells MCF-7 in vitro, which can increase the expression level of the antigen CA15-3, thereby obtaining a culture supernatant with a high concentration of CA15-3 antigen, which can then be purified to obtain a large amount of specific antigen CA15-3.

[0026] (2) The embodiment of the present invention screens a culture medium formula that promotes the expression of CA15-3 antigen in MCF-7 cells. The obtained CA15-3 protein improves the antigen expression amount while retaining the natural structure of the CA15-3 antigen. The operation is simple and the supernatant titer is high. The purification process is simple, which is sufficient to meet the needs of kit production and has lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0028] Figure 1 This is a linear detection result diagram of the antigen CA15-3 prepared in an embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0030] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0032] The present invention provides a method for preparing tumor antigen CA15-3, comprising the following steps:

[0033] (1) Cell recovery and passaging: Add the recovered MCF-7 cells to CA15-3 medium 1 and culture them at 4-6wt% CO2 and 35-40°C. Change the medium every 3 days. When the cells grow to a confluence of 80%-90%, pass them at a ratio of 1:3-1:4. Continue culturing and passaging until the cells reach the required number of bottles.

[0034] (2) Antigen expression: When the cells grow to a confluence of 80% to 90%, replace them with CA15-3 medium 2 to promote CA15-3 antigen expression. Three days after the medium change, add CA15-3 supplement to the cells, supplement once every 1 to 3 days, collect the supernatant after 8 to 15 days of culture, add CA15-3 medium 2 to the cells, continue culturing, supplement, collect the supernatant, and repeat 5 to 7 times; the amount of CA15-3 supplement added is 3 to 5 of the volume of the culture medium before addition on the same day.

[0035] (3) Supernatant treatment: The supernatant collected in step (2) is purified and then stored at an ambient temperature of -10°C to -25°C for future use.

[0036] Wherein, the CA15-3 culture medium 1 in step (1) comprises the components shown in Table 1 below:

[0037] Table 1

[0038] Components concentration DMEM medium / FBS 8~12wt% Dual Antibody 0.8~1.2wt% insulin 8–12 μg / mL

[0039] The CA15-3 culture medium 2 in step (2) contains the components shown in Table 2 below:

[0040] Table 2

[0041]

[0042] The CA15-3 feed in step (2) contains the components shown in Table 3 below:

[0043] Table 3

[0044]

[0045]

[0046] The steps of purifying the supernatant in step (3) include the following:

[0047] (4) Supernatant treatment: The cell supernatant obtained in step (3) is centrifuged at 11000-13000 rpm for 10 minutes to remove residual cell debris. It should be noted that before the supernatant treatment, the cell supernatant obtained in step (3) can be subjected to preliminary centrifugation to remove cell debris. The preliminary centrifugation conditions are 5000-6000 rpm for 8 minutes. The supernatant after the preliminary centrifugation is then stored at -10°C to -25°C for use in step (4). Before the centrifugation step in step (4), it can be thawed.

[0048] (5) Ammonium sulfate precipitation: The antigen CA15-3 is precipitated by ammonium sulfate precipitation, and the precipitate is collected by centrifugation. The saturation of the ammonium sulfate solution used is 60-70 wt %; the centrifugation condition in step (5) is 11000-13000 rpm, and the centrifugation time is 15-25 min.

[0049] (6) Redissolution and dialysis: The precipitate obtained in step (5) is re-dissolved in a buffer solution and then dialyzed. After the dialysis is completed, the liquid in the dialysis bag is collected, centrifuged and the supernatant is collected to obtain the antigen CA15-3 liquid, which is stored at -10°C to -25°C. The buffer solution is 18 to 22 mM Tris, with a pH of 8.5 to 9. The pore size of the dialysis bag used for dialysis is 12 to 16 kDa. The centrifugation condition in step (6) is 13000 to 14000 rpm, and the centrifugation time is 8 to 12 min. It should be noted that the dual antibody mentioned in the embodiment of the present invention is a mixed dilution of penicillin and streptomycin, and the dilution multiple is 100 times.

[0050] Example 1

[0051] The present invention provides a method for preparing tumor antigen CA15-3, comprising the following steps:

[0052] 1. Preparation of high concentration CA15-3 cell culture supernatant:

[0053] (1) Cell recovery and subculture: recovery 1×10 6 MCF-7 cells were transferred into T25 culture flasks containing 5 mL CA15-3 medium 1 and cultured at 37°C with 5% CO2. When the cells grew to a confluence of 80%, they were passaged at a ratio of 1:3 for 2 times and transferred into 3 T182 culture flasks, and 25 mL CA15-3 medium 1 was added to each flask.

[0054] (2) Antigen expression: When the cells grow to a confluence of 80%, replace CA15-3 medium 2 to promote antigen expression. Three days after the medium change, start adding CA15-3 feed to the cells, adding 2 mL of CA15-3 feed every 2 days, collect the culture supernatant after 8 to 15 days of culture, add 25 mL of CA15-3 medium 2, continue culturing, feed and collect supernatant, and repeat 6 times.

[0055] (3) Collecting the supernatant: After the collected supernatant was centrifuged at 6000 rpm for 8 minutes, the cell debris was removed, and the supernatant was collected and stored at -10°C to -25°C for future use. A total of 650 mL of supernatant was collected for 7 times.

[0056] 2. CA15-3 antigen purification:

[0057] (4) Supernatant treatment: After the cell supernatant is thawed, it is mixed and centrifuged at 12000 rpm for 10 min to remove residual cell debris.

[0058] (5) Ammonium sulfate precipitation: Weigh 215 g of ammonium sulfate and place the culture supernatant into a beaker twice the volume. Use a magnetic stirrer to slowly add solid ammonium sulfate while stirring (add a portion and wait for it to completely dissolve before continuing to add. Repeat the operation until all ammonium sulfate has been added). After it is completely dissolved, stir overnight at 2-8°C to precipitate. Centrifuge at 12,000 rpm for 20 minutes and collect the precipitate. After ammonium sulfate precipitation, centrifuge and collect the precipitate.

[0059] (6) Redissolution and dialysis: The precipitate obtained in step (5) was re-dissolved using 20 mM Tris, pH 8.8. The re-dissolved solution was placed in a 14 kDa dialysis bag and dialyzed in 100 times 20 mM PBS, pH 7.4 at 2-8°C. The solution was changed every 4-6 hours for a total of 4 times. After the dialysis, the liquid in the dialysis bag was collected and centrifuged at 12000 rpm for 10 minutes. 33 mL of the supernatant was collected as the antigen CA15-3 liquid and stored at -10°C to -25°C.

[0060] Among them, CA15-3 culture medium 1 contains the components shown in Table 4 below:

[0061] Table 4

[0062] Components concentration DMEM medium / FBS 10wt% Dual Antibody 1wt% insulin 10 μg / mL

[0063] CA15-3 medium 2 contains the components shown in Table 5 below:

[0064] Table 5

[0065]

[0066]

[0067] The CA15-3 feed contains the following components as shown in Table 6:

[0068] Table 6

[0069] Components Concentration range CA15-3 Medium 2 / Tryptone 2g / L Yeast powder 2g / L Sodium bicarbonate 1.5g / L glucose 2g / L Galactose 2g / L fructose 4g / L Nonessential amino acids 2wt% EGF 10 ng / mL

[0070] Test results:

[0071] (1) The cell culture supernatant antigen CA15-3 of Example 1 was tested for effective concentration, specifically:

[0072] The cell culture supernatant obtained in step (4) of Example 1 was diluted 4 times and 5 times respectively, and the effective concentration of antigen CA15-3 was determined by chemiluminescent immunoassay.

[0073] Table 7 is the results of determination of effective concentration of antigen CA15-3 in cell culture supernatant:

[0074] Table 7

[0075]

[0076] As shown in Table 7, the titer of antigen CA15-3 in the cell culture supernatant obtained in the present invention was 1197 U / mL.

[0077] (2) Test of effective concentration of finished product antigen CA15-3, specifically:

[0078] The antigen CA15-3 liquid obtained in step (6) of Example 1 was diluted 400 times, 450 times, and 500 times, respectively, and the effective concentration of the antigen CA15-3 was determined by chemiluminescent immunoassay.

[0079] Table 8 Results of determination of effective concentration of finished antigen CA15-3:

[0080] Table 8

[0081]

[0082] As shown in Table 8, the titer of the antigen CA15-3 prepared by the present invention is 120 KU / mL.

[0083] (3) Linearity test, specifically, the antigen CA15-3 liquid obtained in step (6) of Example 1 was diluted 500 times and then diluted according to the ratio in Table 9 below, and the effective concentration of antigen CA15-3 was determined by chemiluminescent immunoassay. The linearity test results are shown in Table 9 below:

[0084] Table 9

[0085] Dilution percentage Concentration (U / mL) 100 235 50 124 25 57 17 42 10 19 5 12

[0086] A standard curve was drawn based on the results in Table 9. Figure 1 As shown in the results, it can be seen that the antigen CA15-3 prepared in the embodiment of the present invention has good linearity, R 2 It can reach 0.998.

[0087] (4) The CA15-3 liquid obtained in step (6) of Example 1 of the present invention was packaged and accelerated at 37°C for 1 day, 4 days, and 7 days, stored at 4°C for 1 month, stored at -20°C for 3 months, 6 months, and 12 months, and frozen and thawed at -20°C for 3 times. The CA15-3 effective concentration was determined by chemiluminescent immunoassay, and the signal retention rate was calculated.

[0088] Table 10 shows the results of the stability test of antigen CA15-3:

[0089] Table 10

[0090] Test conditions Test results (U / mL) Signal retention rate Initial Value 119216 100.0％ 37℃ accelerated for 1 day 120514 101.1％ 37℃ accelerated for 4 days 113765 95.4％ 37℃ accelerated for 7 days 112765 94.6％ Store at 4℃ for 1 month 117285 98.4％ Store at -20℃ for 3 months 118469 99.4％ Store at -20℃ for 6 months 116532 97.7％ Store at -20℃ for 12 months 114546 96.1％ Freeze and thaw 3 times at -20℃ 114213 95.8％

[0091] As can be seen from the results in Table 10, the effective concentration of the antigen CA15-3 prepared in the embodiment of the present invention did not change significantly after being accelerated at 37°C, stored at 4°C for 1 month, stored at -20°C for 12 months, and frozen and thawed three times at -20°C, indicating that the antigen CA15-3 prepared in the embodiment of the present invention has good stability.

[0092] Comparative Example 1

[0093] A method for preparing tumor antigen CA15-3 is provided. The difference from Example 1 is that this method uses CA15-3 culture medium 1 for cell culture, does not use other culture mediums and does not supplement the culture medium, and directly uses CA15-3 culture medium 1 for culture until 5 days, and the cells basically fall off and die. The supernatant is harvested and centrifuged at 6000 rpm for 8 minutes, and then the effective concentration is determined by chemiluminescence immunoassay. The determination results are shown in Table 11 below:

[0094] Table 11

[0095] Dilution multiple Sample concentration U / mL Mother solution concentration U / mL 1 125 125

[0096] As shown in Table 11, when cells were cultured using only CA15-3 medium 1, the cell culture time was short and the effective concentration of antigen CA15-3 in the harvested supernatant was only 125 U / mL, which was too low and not conducive to subsequent purification.

[0097] Comparative Example 2

[0098] A method for preparing tumor antigen CA15-3 is provided. The difference from Example 1 is that after replacing CA15-3 medium 2, the cells are directly cultured to 7 days without adding CA15-3 supplement. At this time, the cells basically fall off and die. The supernatant is harvested and centrifuged at 6000 rpm for 8 minutes, and the effective concentration is determined by chemiluminescent immunoassay. The test results are shown in Table 12:

[0099] Table 12

[0100] Dilution multiple Sample concentration U / mL Mother solution concentration U / mL 1 215 215

[0101] As shown in Table 12, when CA15-3 medium 2 is used for cell culture without CA15-3 supplement, the effective concentration of antigen CA15-3 in the harvested supernatant is only 215 U / mL, and the antigen concentration is too low, which is not conducive to subsequent purification.

[0102] Comparative Example 3

[0103] A method for preparing a tumor antigen CA15-3 is provided. The difference from Example 1 is that the formula of the CA15-3 supplement is as shown in Table 13 below:

[0104] Table 13

[0105] Components concentration CA15-3 Medium 2 / Tryptone 2g / L Yeast powder 2g / L Sodium bicarbonate 1.5g / L EGF 10 ng / mL

[0106] In Comparative Example 3, the medium was changed to CA15-3 medium 2 and then fed after 3 days of culture. The amount and frequency of feeding were the same as those in Example 1. Samples were taken at 9, 12, and 15 days of culture, and the effective concentration was determined by chemiluminescent immunoassay after centrifugation at 6000 rpm for 8 minutes. At 15 days of culture, the cells fell off and died. The results of the determination of the effective concentration of antigen CA15-3 are shown in Table 14:

[0107] Table 14

[0108]

[0109] As shown in Table 14, the use of CA15-3 supplement in this comparative example can prolong the survival time of cells and increase the effective concentration of antigen CA15-3. After culturing for 15 days, the effective concentration of antigen CA15-3 in the culture supernatant can reach 905U / mL, which meets the purification requirements. However, at this time, the cells basically fall off and die, and it is impossible to continue to culture, so it is difficult to carry out large-scale production. It can be seen that only the CA15-3 culture medium 2 provided in Example 1 of the present invention is used in combination with the CA15-3 supplement provided in the embodiment of the present invention to achieve the purpose of high output and high activity of tumor antigen CA15-3.

[0110] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A culture medium for culturing tumor cells MCF-7 in vitro, characterized in that: The components of the culture medium include DMEM culture medium, RPMI-1640 culture medium, FBS, double antibody, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids.

2. A culture medium for culturing tumor cells MCF-7 in vitro according to claim 1, characterized in that: The concentrations of the components FBS, double antibody, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids in the culture medium are respectively: 1.8-2.2wt%, 0.8-1.2wt%, 0.4-0.6g / L, 0.4-0.6g / L, 8-12μg / mL, 0.8-1.2g / L, 0.8-1.2g / L, 1.8-2.2g / L and 1.8-2.4wt%.

3. The method for preparing a tumor antigen CA15-3 according to claim 2, characterized in that: The mixing ratio of the DMEM culture medium to the RPMI-1640 culture medium is 1:0.8-1.

2.

4. The method for preparing a tumor antigen CA15-3 according to claim 3, characterized in that: The concentrations of the components FBS, double antibody, tryptone, yeast powder, insulin, glucose, galactose, fructose and non-essential amino acids in the culture medium are 2wt%, 1wt%, 0.5g / L, 0.5g / L, 10μg / mL, 1g / L, 1g / L, 2g / L and 2wt%, respectively; The mixing ratio of the DMEM culture medium and the RPMI-1640 culture medium is 1:

1.

5. A method for preparing tumor antigen CA15-3, characterized in that: The following steps are involved: S1: adding the revived MCF-7 cells to CA15-3 medium 1 for culture, and performing subculture when the MCF-7 cells grow to a confluence of 80% to 90%; S2: When the MCF-7 cells grow to a confluence of 80% to 90% during the subculture process, the CA15-3 culture medium 1 is replaced with the CA15-3 culture medium 2, and the MCF-7 cells are continued to be cultured, wherein the CA15-3 culture medium 2 is the culture medium according to any one of claims 1 to 4; S3: After promoting CA15-3 antigen expression, CA15-3 feed was added to MCF-7 cells for culture, and the supernatant was collected; S4: Repeat step S3 multiple times, and purify the collected supernatant to obtain the antigen CA15-3.

6. The method for preparing tumor antigen CA15-3 according to claim 5, characterized in that: The components of the CA15-3 culture medium 1 include DMEM culture medium, FBS, dual antibodies and insulin, the concentration of the FBS is 8-12wt%, the concentration of the dual antibodies is 0.8-1.2wt%, and the concentration of the insulin is 8-12μg / mL.

7. The method for preparing tumor antigen CA15-3 according to claim 1, characterized in that: The components of the CA15-3 feed include the CA15-3 culture medium 2, and additionally added tryptone, yeast powder, sodium bicarbonate, glucose, galactose, fructose, non-essential amino acids and EGF.

8. The method for preparing tumor antigen CA15-3 according to claim 7, characterized in that: The concentrations of the components of the CA15-3 feed, namely, tryptone, yeast powder, sodium bicarbonate, glucose, galactose, fructose, non-essential amino acids and EGF, are 1.8-2.2 g / L, 1.8-2.2 g / L, 1.3-1.8 g / L, 1.8-2.2 g / L, 1.8-2.2 g / L, 3.5-4.5 g / L, 1.8-2.4 wt % and 8-12 ng / mL, respectively.

9. The method for preparing tumor antigen CA15-3 according to claim 5, characterized in that: The purification process described in S4 comprises the following steps: S41: centrifuging the supernatant to remove residual cell debris to obtain a clarified solution; S42: precipitating the antigen CA15-3 in the clear solution by ammonium sulfate precipitation method, and collecting the precipitate by centrifugation; S43: The precipitate is redissolved in a buffer solution and dialyzed. After the dialysis is completed, the liquid is collected, and then centrifuged and the supernatant is collected to obtain the antigen CA15-3 liquid.

10. The method for preparing tumor antigen CA15-3 according to claim 9, characterized in that: In the ammonium sulfate precipitation method, the saturation of the ammonium sulfate solution is 60-70 wt%.

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