Preparation method and application of human PGK1 131 site lysine lactic acid modified antibody

Abstract

The invention discloses a preparation method and application of a human PGK1 131-site lysine lactic acid modified antibody, and relates to the technical field of preparation of lactic acid antibodies, the preparation method comprises the following steps: S01, the 131-site lysine in a polypeptide of an amino acid sequence is subjected to lactic acid modification and then is coupled with a carrier protein to obtain an initial antigen, and the carrier protein comprises hemocyanin KLH; s02, injecting the obtained antigen into a living body culture sample for immune culture, and collecting whole blood after a preset period to obtain multi-antibody serum; and S03, purifying to obtain an iterative antibody. According to the present invention, the expression difference in normal cells, tumor cells and tumor tissues can be directly detected by using WesternBlotting, immunofluorescence and immunohistochemistry so as to provide the powerful guarantee for the tumor detection.

Description

Technical Field

[0001] The present invention relates to the preparation of lactated antibodies, and in particular to a method for preparing an antibody modified by lactation of lysine 131 of human PGK1 and its application. Background Art

[0002] Lactylation uses lactic acid, a product of cellular glycolysis, as a substrate to introduce lactoyl groups to lysine residues of specific proteins, thereby affecting the conformation and function of the protein. Therefore, lactylation is a key bridge connecting cell metabolism and gene expression regulation, and plays a regulatory role in many physiological and pathological processes. Studies have found that protein lactylation is closely related to tumor proliferation, invasion, migration, and malignancy. This represents a major breakthrough in understanding the molecular mechanisms of tumor occurrence and development.

[0003] A very important feature of tumors is metabolic abnormalities, which is mainly manifested in the transformation of glucose metabolism from oxidative phosphorylation to aerobic glycolysis, so that tumor cells still give priority to glycolysis for energy production even when there is sufficient oxygen. PGK1 is a kinase that plays an important role in the glycolysis process. Its function is to convert 1,3-bisphosphoglycerate and ADP into 3-phosphoglycerate and ATP, which are the only two processes in glycolysis that release ATP. It is overexpressed or overactivated in many tumors.

[0004] In recent years, more and more protein post-translational modifications have been found on PGK1, including phosphorylation, acetylation, glycosylation and ubiquitination, which plays an important role in different biological processes including various diseases such as tumors. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing an antibody modified by lactic acid at position 131 of human PGK1, comprising the following steps:

[0006] S01, lactating the 131st lysine in the polypeptide of the amino acid sequence and coupling it with a carrier protein to obtain an initial antigen, wherein: the carrier protein includes hemocyanin KLH;

[0007] S02, injecting the obtained antigen into a living culture sample for immune culture, and collecting whole blood after a predetermined period to obtain polyclonal antibody serum;

[0008] S03. After purification, iterative antibodies are obtained.

[0009] Preferably, obtaining the initial antigen in step S01 includes:

[0010] S11, dissolving 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution to obtain a fused KLH solution;

[0011] S12, weigh 8 mg of Sulfo-SMCC and completely dissolve it in 50 μL of LDMSO, then add 150 μL of 1× PBS and mix well to obtain a mixed liquid;

[0012] S13, adding the mixed liquid dropwise into the fused KLH solution, gently shaking while adding, and leaving at room temperature for 1 hour to obtain activated KLH;

[0013] S14, putting the activated KLH solution into a dialysis bag and clamping it with a dialysis clip, and then putting it into 2L of 1×PBS, dialyzing it for 1 hour under magnetic stirring in a 4°C refrigerator, and then replacing it with a new 2L of 1×PBS, and dialyzing it for 2 hours under magnetic stirring in a 4°C refrigerator to obtain activated dialyzed KLH;

[0014] S15, place the activated dialyzed KLH in a 15 mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4°C refrigerator;

[0015] S16, weigh 4 mg of the peptide and dissolve it in 50 μL of DMSO, then add 200 μL of 1×PBS, mix quickly, then add the activated dialyzed KLH according to a predetermined ratio, and react in a refrigerator at 4°C overnight or at room temperature for 2 hours to obtain a KLH-peptide cross-linked complex;

[0016] S17, the KLH-peptide cross-linked complex is placed in a dialysis bag and clamped with a dialysis clamp, and then placed in 4 L of 1×PBS and dialyzed overnight under magnetic stirring in a 4° C. refrigerator to obtain dialyzed KLH-peptide;

[0017] S18. Take out the dialyzed KLH-peptide into a 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator to obtain the initial antigen.

[0018] Preferably, the predetermined ratio in step S16 is 1 mg of polypeptide: 680 μg of the activated dialyzed KLH.

[0019] Preferably, the living culture sample in step S02 is a young and middle-aged New Zealand white rabbit weighing about 2.5 kg.

[0020] Preferably, the immune culture in step S02 comprises the following steps:

[0021] S21, thawing the initial antigen at room temperature, injecting 0.5 ml of the initial antigen into each of the plurality of living culture samples for the first injection, and halving the amount of the initial antigen injected for the second to fourth injections;

[0022] S22. Draw the adjuvant and antigen in a volume ratio of 1:1. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. When drawing, the adjuvant should be fully mixed before being drawn into the syringe.

[0023] S23. The sample liquid extracted in step S22 is then completely emulsified and then injected subcutaneously at multiple points, with 0.2 ml at each point.

[0024] Preferably, the concentration of the initial antigen in the first immunization in step S21 is 1 mg / ml.

[0025] Preferably, the predetermined period in step S02 is: the second immunization is carried out on the 14th day after the first immunization, and the interval between the second and third immunizations is 7 days. The live culture sample is tested by taking a small sample of serum from the middle auricular artery on the 7th day after the third immunization. If the test is qualified, an additional immunization is carried out 7 days later, and whole blood can be collected 7 days after the additional immunization.

[0026] The step of collecting whole blood in step S02 includes:

[0027] S24, using the heart blood collection method, and after the blood collection is completed, the centrifuge tube containing the blood is placed in a 37°C water bath for 15-30 minutes, taken out, cooled and placed in a 4°C refrigerator, waiting for the blood to automatically stratify, and the supernatant is taken out into a clean 50ml centrifuge tube;

[0028] S25, centrifuge at 12000 rpm for 2 min. After centrifugation, add 100 ul of 10% sodium thimerosal solution into the 50 ml centrifuge tube, mix well, and store at -20°C, wherein the final concentration of the 10% sodium thimerosal solution is 0.02%.

[0029] Preferably: before executing the step S03, the obtained polyclonal antibody serum is tested by Elisa to collect positive serum and the unqualified serum is eliminated;

[0030] Preferably, the purification in step S03 comprises:

[0031] S31, the affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1×PBS in sequence at a flow rate of 70 mL / h;

[0032] S32, take 10 mL of serum to be purified into a 50 mL centrifuge tube, and filter it with a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm;

[0033] S33, loading the filtered serum sample at a flow rate of 40 mL / h, repeating once;

[0034] S34, wash the column with 20 mL 1× PBS at a flow rate of 70 mL / h, connect to the protein detector after 10 min, and adjust the instrument transmittance reading to 100 during the washing process;

[0035] S35. Adjust the absorbance reading of the protein detector to 0. At this time, turn on the HD-A computer collector on the computer desktop and adjust the full screen range to 5. Elute the antibody with 0.2M glycine solution at a rate of 40mL / h. At this time, press the green elution record button to start elution. When the reading of the instrument starts to rise, start collecting the antibody;

[0036] S36. During the antibody collection process, the pH value of the antibody was promptly adjusted to about 7 with 1 M sodium bicarbonate, and the highest peak value of the elution peak was recorded;

[0037] S37. After collecting the antibodies, adjust the pH value to about 7, record the volume of eluted antibodies, and then rinse the rubber tube connected to the collector with pure water;

[0038] S38. Wash the affinity chromatography column with 20 mL 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal it, and store it in a refrigerator at 4°C.

[0039] The technical effects and advantages of the present invention are as follows: it can be used to detect the expression differences among normal cells, tumor cells, paracancerous tissues and tumor tissues, which is beneficial to study the role and mechanism of lactic acid modification of human PGK1 protein in the occurrence and development of tumors, and provides potential targets for clinical tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the method proposed in the present invention for detecting the K131la level after mutation of the K131 site in a human intrahepatic cholangiocarcinoma cell line by Western blotting.

[0041] Figure 2 This is a schematic diagram of the immunofluorescence detection of K131la levels in human intrahepatic cholangiocarcinoma cell lines after mutation of the K131 site as proposed in the present invention.

[0042] Figure 3 This is a schematic diagram of the immunohistochemistry method proposed in the present invention for detecting K131la levels in tissues of patients with intrahepatic cholangiocarcinoma. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are provided for the purpose of illustration and description, and are not intended to be exhaustive or to limit the present invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention, and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

[0044] Figure 1 Based on the antibody provided in this example, after the PGK1 131 site was mutated in the human biliary cholangiocarcinoma cell line, Western Blotting experiments detected that the expression level of 131 lactylation was reduced.

[0045] Figure 2 Based on the antibody provided in this example, after the PGK1 131 site was mutated in the human biliary cholangiocarcinoma cell line, the expression level of 131 lactylation was reduced by immunofluorescence experiment.

[0046] Figure 3 Based on the antibody provided in this example, immunohistochemistry was used to stain specimens removed during surgery from patients with cholangiocarcinoma. The results showed that the PGK1-131 lactic acid-modified antibody can be used for immunohistochemistry staining, and that the expression in the patient's cancer and adjacent tissues was different.

[0047] Embodiment 1

[0048] A method for preparing an antibody with lactic acid modification of lysine 131 of human PGK1, comprising:

[0049] S01, lactating the 131st lysine in the polypeptide of the amino acid sequence and coupling it with a carrier protein to obtain an initial antigen, wherein: the carrier protein includes hemocyanin KLH;

[0050] The detailed steps include:

[0051] S11, dissolving 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution to obtain a fused KLH solution;

[0052] S12, weigh 8 mg of Sulfo-SMCC and completely dissolve it in 50 μL of LDMSO, then add 150 μL of 1× PBS and mix well to obtain a mixed liquid;

[0053] S13, adding the mixed liquid dropwise into the fused KLH solution, shaking gently while adding, and leaving at room temperature for 1 hour to obtain activated KLH;

[0054] S14, put the activated KLH solution into a dialysis bag and clamp it with a dialysis clip, then put it into 2L of 1×PBS, dialyze it for 1h under magnetic stirring in a 4℃ refrigerator, then replace it with a new 2L of 1×PBS, and dialyze it for 2h under magnetic stirring in a 4℃ refrigerator to obtain activated dialyzed KLH;

[0055] S15, place the activated dialyzed KLH in a 15 mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4°C refrigerator;

[0056] S16, weigh 4 mg of the peptide and dissolve it in 50 μL of DMSO, then add 200 μL of 1×PBS, mix quickly, then add the activated dialyzed KLH according to the predetermined ratio, and react in a refrigerator at 4°C overnight or at room temperature for 2 hours to obtain a KLH-peptide cross-linked complex (the predetermined ratio is 1 mg of peptide: 680 μg of activated dialyzed KLH);

[0057] S17. The KLH-peptide cross-linked complex was placed in a dialysis bag and clamped with a dialysis clip. Then, the complex was placed in 4 L of 1×PBS and dialyzed overnight under magnetic stirring in a 4°C refrigerator to obtain dialyzed KLH-peptide.

[0058] S18. Take out the dialyzed KLH-peptide into a 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator to obtain the initial antigen.

[0059] S02, injecting the obtained antigen into a living culture sample (a healthy, lively, young and middle-aged New Zealand white rabbit weighing about 2.5 kg) for immune culture, collecting whole blood after a predetermined period (a second immunization is performed on the 14th day after the first immunization, and the interval between the second and third immunizations is 7 days. A small sample of serum is collected from the middle auricular artery of the living culture sample on the 7th day after the third immunization for testing. If the test is qualified, an additional immunization is performed 7 days later, and whole blood can be collected 7 days after the additional immunization) to obtain polyclonal antibody serum;

[0060] The detailed steps include:

[0061] S21, thawing the initial antigen at room temperature, the initial antigen for the first immunization of multiple living culture samples is 0.5 ml / animal (the concentration of the initial antigen for the first immunization is 1 mg / ml), and the amount of the initial antigen injected for the second to fourth immunizations is halved;

[0062] S22. Draw the adjuvant and antigen in a volume ratio of 1:1. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. When drawing, the adjuvant should be fully mixed before being drawn into the syringe.

[0063] S23, then completely emulsify the sample liquid extracted in step S22, and then use multiple points of subcutaneous injection, each point is 0.2ml;

[0064] S24, using the heart blood collection method, and after the blood collection is completed, the centrifuge tube containing the blood is placed in a 37°C water bath for 15-30 minutes, taken out, cooled and placed in a 4°C refrigerator, waiting for the blood to automatically stratify, and the supernatant is taken out into a clean 50ml centrifuge tube;

[0065] S25. Centrifuge at 12000 rpm for 2 min. After centrifugation, add 100 ul of 10% sodium thimerosal solution into a 50 ml centrifuge tube, mix well, and store at -20°C. The final concentration of the 10% sodium thimerosal solution is 0.02%.

[0066] S03. After purification, iterative antibodies are obtained.

[0067] The detailed steps include:

[0068] S31, the affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h;

[0069] S32, take 10 mL of serum to be purified into a 50 mL centrifuge tube, and filter it with a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm;

[0070] S33, loading the filtered serum sample at a flow rate of 40 mL / h, repeating once;

[0071] S34, wash the column with 20 mL 1× PBS (pH 7.4) at a flow rate of 70 mL / h, connect to the protein detector after 10 min, and adjust the instrument transmittance (T gear) to 100 during the washing process;

[0072] S35. Adjust the absorbance of the protein detector (1A gear) to 0. At this time, turn on the HD-A computer collector on the computer desktop and adjust the full screen range to 5. Elute the antibody with 0.2M glycine solution (pH 2.7) at a rate of 40mL / h. At this time, press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody;

[0073] S36. During the antibody collection process, the pH value of the antibody was promptly adjusted to about 7 with 1 M sodium bicarbonate, and the highest peak value of the elution peak was recorded;

[0074] S37. After collecting the antibodies, adjust the pH value to about 7, record the volume of eluted antibodies, and then rinse the rubber tube connected to the collector with pure water;

[0075] S38. Wash the affinity chromatography column with 20 mL 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal it, and store it in a refrigerator at 4°C.

[0076] It should be noted that the sequence number of the polypeptide in the above example is VEEEGK(lac)GKDASGNC.

[0077] Embodiment 2

[0078] This example aims to verify the extracts in the preparation process of the antibody preparation process disclosed in Example 1, and collect positive serum by Elisa test for polyclonal antibody serum, and remove unqualified serum. The details are as follows:

[0079] S41. Dilute the polyclonal antibody serum to 1 μg / ml with a buffer (coating buffer: Na2CO3 and NaHCO3 buffer) and add 50 μl to each reaction well of the polystyrene plate. Incubate overnight at 4°C. The next day, discard the solution in the wells and wash once with 1xPBST washing buffer (180 μl per well).

[0080] S42, add 150 μl of 1% BSA (prepared in PBST) to each well for blocking, incubate at 37°C for 1 hour, and then discard the blocking solution;

[0081] S43, add a certain dilution of polyclonal antibody serum, 50 μl to the blocked reaction wells, set up negative control wells (PBS), incubate at 37°C for 1 hour, then discard the blocking solution, wash three times with 1xPBST washing buffer, 150 μl per well;

[0082] S44, add freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the ELISA plate at 50 μl / well, incubate at 37°C for 45 min, then discard the blocking solution and wash three times with 1xPBST buffer at 150 μl per well;

[0083] S45, add 50 μl of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 min;

[0084] S46, add 50 μl of 1 M sulfuric acid to each reaction well;

[0085] S47. Place the ELISA plate in a preheated ELISA reader (450 nm) for reading to obtain the following table:

[0086]

[0087]

[0088] Embodiment 3

[0089] Based on Example 1, this example aims to detect the iterative antibodies obtained after purification by ELISA method to select qualified iterative antibodies. The detailed steps include:

[0090] S51. After the antibody quantity for delivery is purified and the semi-finished product has passed the titer test, all antibodies are mixed and concentrated using an ultrafiltration concentration tube to reach a certain concentration and volume.

[0091] S51. Place the concentrated antibody in 1L 0.01M PBS (pH 7.4) and dialyze at room temperature. Change the solution every 3 hours, for a total of 3 times (for overnight dialysis, place in a 2-8°C refrigerator).

[0092] S51. Take out the dialyzed antibody into a clean centrifuge tube, filter the antibody using a 0.22mm disposable low-adsorption filter in an ultra-clean workbench, take a small sample for testing, and take another 5mL to test the concentration.

[0093] S51. The concentration was measured on the Protein A280 application of the ultra-micro spectrophotometer (denovix DS-11) to obtain the following table:

[0094]

[0095] In summary, the antibodies provided by the above preparation method can be used to detect the expression differences among normal cells, tumor cells, paracancerous tissues and tumor tissues, which is beneficial to study the role and mechanism of lactic acid modification of human PGK1 protein in the occurrence and development of tumors, and provide potential targets for clinical tumor diagnosis and treatment.

[0096] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field and related fields without creative work should fall within the scope of protection of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention are implemented according to the conventional means in the field unless otherwise specified and limited.

Claims

1. A method for preparing an antibody modified by lactic acid at position 131 of human PGK1, characterized in that: The following steps are involved: S01, lactating the 131st lysine in the polypeptide of the amino acid sequence and coupling it with a carrier protein to obtain an initial antigen, wherein: the carrier protein includes hemocyanin KLH; S02, injecting the obtained antigen into a living culture sample for immune culture, and collecting whole blood after a predetermined period to obtain polyclonal antibody serum; S03. After purification, iterative antibodies are obtained.

2. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 1, characterized in that: The step S01 of obtaining the initial antigen comprises: S11, dissolving 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution to obtain a fused KLH solution; S12, weigh 8 mg of Sulfo-SMCC and completely dissolve it in 50 μL of LDMSO, then add 150 μL of 1× PBS and mix well to obtain a mixed liquid; S13, adding the mixed liquid dropwise into the fused KLH solution, gently shaking while adding, and leaving at room temperature for 1 hour to obtain activated KLH; S14, putting the activated KLH solution into a dialysis bag and clamping it with a dialysis clip, and then putting it into 2L of 1×PBS, dialyzing it for 1 hour under magnetic stirring in a 4°C refrigerator, and then replacing it with a new 2L of 1×PBS, and dialyzing it for 2 hours under magnetic stirring in a 4°C refrigerator to obtain activated dialyzed KLH; S15, place the activated dialyzed KLH in a 15 mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4°C refrigerator; S16, weigh 4 mg of the peptide and dissolve it in 50 μL of DMSO, then add 200 μL of 1×PBS, mix quickly, then add the activated dialyzed KLH according to a predetermined ratio, and react in a refrigerator at 4°C overnight or at room temperature for 2 hours to obtain a KLH-peptide cross-linked complex; S17, the KLH-peptide cross-linked complex is placed in a dialysis bag and clamped with a dialysis clamp, and then placed in 4 L of 1×PBS and dialyzed overnight under magnetic stirring in a 4° C. refrigerator to obtain dialyzed KLH-peptide; S18. Take out the dialyzed KLH-peptide into a 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator to obtain the initial antigen.

3. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 2, characterized in that: The predetermined ratio in step S16 is 1 mg of polypeptide: 680 μg of the activated dialyzed KLH.

4. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 1, characterized in that: The living culture sample in step S02 is a young and middle-aged New Zealand white rabbit weighing about 2.5 kg.

5. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 1, characterized in that: The immune culture in step S02 comprises the following steps: S21, thawing the initial antigen at room temperature, injecting 0.5 ml of the initial antigen into each of the plurality of living culture samples for the first injection, and halving the amount of the initial antigen injected for the second to fourth injections; S22. Draw the adjuvant and antigen in a volume ratio of 1:

1. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. When drawing, the adjuvant should be fully mixed before being drawn into the syringe. S23. The sample liquid extracted in step S22 is then completely emulsified and then injected subcutaneously at multiple points, with 0.2 ml at each point.

6. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 5, characterized in that: The concentration of the initial antigen in the first immunization in step S21 is 1 mg / ml.

7. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 1, characterized in that: The predetermined period in step S02 is: the second immunization is carried out on the 14th day after the first immunization, and the interval between the second and third immunizations is 7 days. The living culture sample is tested by taking a small sample of serum from the middle auricular artery on the 7th day after the third immunization. If the test is qualified, an additional immunization is carried out 7 days later, and whole blood can be collected 7 days after the additional immunization; The step of collecting whole blood in step S02 includes: S24, using the heart blood collection method, and after the blood collection is completed, the centrifuge tube containing the blood is placed in a 37°C water bath for 15-30 minutes, taken out, cooled and placed in a 4°C refrigerator, waiting for the blood to automatically stratify, and the supernatant is taken out into a clean 50ml centrifuge tube; S25, centrifuge at 12000 rpm for 2 min. After centrifugation, add 100 ul of 10% sodium thimerosal solution into the 50 ml centrifuge tube, mix well, and store at -20°C, wherein the final concentration of the 10% sodium thimerosal solution is 0.02%.

8. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 1, characterized in that: The purification in step S03 includes: S31, the affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1×PBS in sequence at a flow rate of 70 mL / h; S32, take 10 mL of serum to be purified into a 50 mL centrifuge tube, and filter it with a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm; S33, loading the filtered serum sample at a flow rate of 40 mL / h, repeating once; S34, wash the column with 20 mL 1× PBS at a flow rate of 70 mL / h, connect to the protein detector after 10 min, and adjust the instrument transmittance reading to 100 during the washing process; S35. Adjust the absorbance reading of the protein detector to 0. At this time, turn on the HD-A computer collector on the computer desktop and adjust the full screen range to 5. Elute the antibody with 0.2M glycine solution at a rate of 40mL / h. At this time, press the green elution record button to start elution. When the reading of the instrument starts to rise, start collecting the antibody; S36. During the antibody collection process, the pH value of the antibody was promptly adjusted to about 7 with 1 M sodium bicarbonate, and the highest peak value of the elution peak was recorded; S37. After collecting the antibodies, adjust the pH value to about 7, record the volume of eluted antibodies, and then rinse the rubber tube connected to the collector with pure water; S38. Wash the affinity chromatography column with 20 mL 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal it, and store it in a refrigerator at 4°C.

9. The method for preparing an antibody modified by lactic acid at position 131 of human PGK1 according to claim 1, characterized in that: Before executing step S03, the obtained polyclonal antibody serum is tested by Elisa to collect positive serum and remove unqualified serum.

10. Use of an antibody wherein the lysine 131 of human PGK1 is lactic acid-modified, which is based on any one of claims 1 to 9 and is characterized in that: It is used for tumor diagnosis of tumor cells, paracancerous tissues and tumor tissues.

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