A method for preparing and applying antibodies modified by lactation of lysine at position 131 of human PGK1

Abstract

This invention discloses a method for preparing and applying antibodies modified by lactation of lysine at position 131 of human PGK1, relating to the field of lactated antibody preparation technology. The method includes the following steps: S01, lactating lysine at position 131 of a polypeptide and conjugating it with a carrier protein to obtain an initial antigen, wherein the carrier protein includes hemocyanin KLH; S02, injecting the obtained antigen into a live culture sample for immunoculture, collecting whole blood after a predetermined period to obtain polyclonal antibody serum; S03, purifying the antigen to obtain an iterative antibody. This invention can directly apply Western blotting, immunofluorescence, and immunohistochemistry to detect expression differences in normal cells, tumor cells, and tumor tissues, thus providing a strong guarantee for tumor detection.

Description

Technical Field

[0001] This invention relates to the preparation of lactated antibodies, specifically to a method and application for preparing antibodies modified by lactation of lysine at position 131 of human PGK1. Background Technology

[0002] Lactation, using lactate, a product of cellular glycolysis, as a substrate, introduces lactyl groups onto the lysine residues of specific proteins, thereby affecting protein conformation and function. Therefore, lactation is a crucial bridge connecting cellular metabolism and gene expression regulation, playing a regulatory role in numerous physiological and pathological processes. Studies have found that protein lactation is closely related to tumor proliferation, invasion, migration, and malignancy. This represents a significant breakthrough in understanding the molecular mechanisms of tumorigenesis and development.

[0003] A key characteristic of tumors is metabolic abnormality, primarily manifested as a shift in glucose metabolism from oxidative phosphorylation to aerobic glycolysis. This allows tumor cells to preferentially utilize glycolysis for energy production even under oxygen-rich conditions. PGK1 is a crucial kinase in glycolysis, converting 1,3-bisphosphoglycerate and ADP into 3-phosphoglycerate and ATP—the only two processes in glycolysis that release ATP. It is overexpressed or excessively activated in various tumors.

[0004] In recent years, an increasing number of protein post-translational modifications, including phosphorylation, acetylation, glycosylation, and ubiquitination, have been discovered on PGK1, which play important roles in various biological processes and a variety of diseases, including tumors. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1, comprising the following steps:

[0006] S01. The lysine at position 131 of the polypeptide in the amino acid sequence is lactated and then coupled with a carrier protein to obtain the initial antigen, wherein: the carrier protein includes hemocyanin KLH;

[0007] S02. The obtained antigen is injected into a live culture sample for immune culture. After a predetermined period, whole blood is collected to obtain polyclonal antibody serum.

[0008] S03, after purification, yields iterative antibodies.

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

[0010] S11. Dissolve 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 dissolve it completely in 50 μL of LDMSO. Then add 150 μL of 1×PBS and mix well to obtain a mixed liquid.

[0012] S13. Add the mixed liquid dropwise to the fused KLH solution while gently shaking, and let it stand at room temperature for 1 hour to obtain activated KLH;

[0013] S14. Place the activated KLH solution into a dialysis bag and clamp it with a dialysis clamp. After completion, place it into 2L of 1×PBS and dialyze for 1h under magnetic stirring at 4℃. Then replace it with a new 2L of 1×PBS and dialyze for 2h under magnetic stirring at 4℃ to obtain activated and dialyzed KLH.

[0014] S15. Place the activated and dialyzed KLH into a 15mL imported centrifuge tube, and label the tube with the reagent name, time and concentration. Store at 4℃.

[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, and then add the activated and dialyzed KLH according to the predetermined ratio. Incubate overnight at 4°C or react at room temperature for 2 h to obtain the KLH-peptide cross-linked complex.

[0016] S17. The KLH-peptide crosslinking complex is placed in a dialysis bag and clamped with a dialysis clamp. Then it is placed in 4L of 1×PBS and dialyzed overnight at 4°C with magnetic stirring to obtain the dialyzed KLH-peptide.

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

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

[0019] Preferably, the live 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 includes the following steps:

[0021] S21. The initial antigen, which is thawed at room temperature, is injected into the first immunization of multiple live culture samples at a rate of 0.5 ml / sample, and the amount of the initial antigen injected into the second to fourth immunization is halved.

[0022] S22. The adjuvant and antigen are drawn in a 1:1 volume ratio. The first immunization uses complete adjuvant, and the second to fourth immunizations use incomplete adjuvant. When drawing, the adjuvant must be fully mixed before being drawn into the syringe.

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

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

[0025] Preferably, the predetermined cycle in step S02 is as follows: the second immunization is performed 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 collecting a small sample of serum from the middle ear artery on the 7th day after the third immunization. If the test is qualified, the immunization is repeated 7 days later. Whole blood can be collected 7 days after the immunization is completed.

[0026] The steps for collecting whole blood described in step S02 include:

[0027] S24. Use the cardiac blood collection method. After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 15-30 minutes, then remove it, cool it, and place it in a 4°C refrigerator. Wait for the blood to automatically separate into layers, and then transfer the supernatant into a clean 50ml centrifuge tube.

[0028] S25. The centrifuge is set at 12000 rpm for 2 min. After centrifugation, 100 μL of 10% sodium thimerosal solution is added to the 50 ml centrifuge tube and mixed well. The mixture is then stored at -20 °C. The final concentration of the 10% sodium thimerosal solution is 0.02%.

[0029] Preferably, before performing step S03, the obtained polyclonal antibody serum is collected using ELISA detection, and unqualified serum is discarded;

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

[0031] S31. Wash the affinity chromatography column thoroughly with 20 mL of pure water and 1×PBS at a flow rate of 70 mL / h.

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

[0033] S33. Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once.

[0034] S34. Wash the column with 20 mL of 1×PBS at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance reading to 100.

[0035] S35. When the absorbance reading of the protein detector is 0, turn on the HD-A computer acquisition device on the computer desktop and adjust the full-screen range to 5. Use 0.2M glycine solution to elute the antibody at a rate of 40mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0036] S36. During antibody collection, adjust the pH of the antibody to around 7 with 1M sodium bicarbonate in a timely manner, and record the highest peak value of the elution peak.

[0037] S37. After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tube connected to the collector with purified water.

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

[0039] The technical effects and advantages of this invention are: it can be used to detect expression differences among normal cells, tumor cells, adjacent normal tissues and tumor tissues, which is beneficial for studying the role and mechanism of human PGK1 protein lactation modification in the process of tumor development and provides potential targets for clinical tumor diagnosis and treatment. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the K131la level after detecting the K131 site in the mutant human intrahepatic cholangiocarcinoma cell line using Western blotting, as proposed in this invention.

[0041] Figure 2 This is a schematic diagram illustrating the K131la level after detecting the K131 site in the mutant human intrahepatic cholangiocarcinoma cell line using immunofluorescence, as proposed in this invention.

[0042] Figure 3 This is a schematic diagram illustrating the immunohistochemical detection of K131la levels in tissues of patients with intrahepatic cholangiocarcinoma, as proposed in this invention. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose.

[0044] Figure 1 Based on the antibody provided in this example, after mutating PGK1 at site 131 in human biliary cholangiocarcinoma cell lines, Western blotting experiments showed a decrease in the expression level of 131 lactation.

[0045] Figure 2 Based on the antibody provided in this example, after mutating PGK1 at site 131 in human biliary cholangiocarcinoma cell lines, immunofluorescence assays showed a decrease in the expression level of 131 lactation.

[0046] Figure 3 Based on the antibody provided in this example, immunohistochemistry was used to stain specimens taken during surgery from patients with cholangiocarcinoma. The results showed that the PGK1-131 lactation-modified antibody can be used for immunohistochemical staining, and that the expression of the antibody differed between the cancer and adjacent normal tissues in the patients.

[0047] Example 1

[0048] A method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1, comprising:

[0049] S01. The lysine at position 131 of the polypeptide in the amino acid sequence is lactated and then coupled with a carrier protein to obtain the initial antigen, wherein the carrier protein includes hemocyanin KLH.

[0050] Detailed steps include:

[0051] S11. Dissolve 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 dissolve it completely in 50 μL of LDMSO. Then add 150 μL of 1×PBS and mix well to obtain a mixed liquid.

[0053] S13. Add the mixed liquid dropwise to the fused KLH solution while gently shaking, and let it stand at room temperature for 1 hour to obtain activated KLH.

[0054] S14. Place the activated KLH solution into a dialysis bag and clamp it with a dialysis clamp. After that, place it into 2L of 1×PBS and dialyze for 1 hour under magnetic stirring at 4°C. Then replace it with a new 2L of 1×PBS and dialyze for 2 hours under magnetic stirring at 4°C to obtain activated and dialyzed KLH.

[0055] S15. Place the activated and dialyzed KLH into a 15mL imported centrifuge tube, and label the tube with the reagent name, time and concentration. Store at 4℃.

[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, and then add activated and dialyzed KLH in a predetermined ratio. Incubate overnight at 4°C or react at room temperature for 2 h to obtain the KLH-peptide cross-linked complex (the predetermined ratio is 1 mg of peptide to 680 μg of activated and dialyzed KLH).

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

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

[0059] S02. The obtained antigen is injected into a live culture sample (a healthy, active, young New Zealand white rabbit weighing about 2.5 kg) for immune culture. Whole blood is collected after the predetermined cycle (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 ear artery of the live culture sample on the 7th day after the third immunization. If the test is qualified, a booster immunization is performed 7 days later. Whole blood can be collected 7 days after the booster immunization) to obtain polyclonal antibody serum.

[0060] Detailed steps include:

[0061] S21. The initial antigen thawed at room temperature is injected into multiple live culture samples for the first immunization at a rate of 0.5 ml / sample (the concentration of the initial antigen for the first immunization is 1 mg / ml). The amount of initial antigen injected for the second to fourth immunizations is halved.

[0062] S22. The adjuvant and antigen are drawn in a 1:1 volume ratio. The first immunization uses complete adjuvant, and the second to fourth immunizations use incomplete adjuvant. When drawing, the adjuvant must be fully mixed before being drawn into the syringe.

[0063] S23. Then, the sample solution extracted in step S22 is completely emulsified and then injected subcutaneously at multiple points, with 0.2 ml at each point.

[0064] S24. Use the cardiac blood collection method. After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 15-30 minutes, then remove it, cool it, and place it in a 4°C refrigerator. Wait for the blood to automatically separate into layers, and then transfer the supernatant into a clean 50ml centrifuge tube.

[0065] S25. Centrifuge at 12000 rpm for 2 min. After centrifugation, add 100 μL of 10% sodium thimerosal solution to 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, yields iterative antibodies.

[0067] Detailed steps include:

[0068] S31. The affinity chromatography column was thoroughly washed 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 the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0070] S33. Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once.

[0071] S34. Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance (T setting) reading to 100.

[0072] S35. When the absorbance of the protein detector (1A range) is 0, turn on the HD-A computer acquisition device on the computer desktop and adjust the full screen range to 5. Use 0.2M glycine solution (pH 2.7) to elute the antibody at a rate of 40mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0073] S36. During antibody collection, adjust the pH of the antibody to around 7 with 1M sodium bicarbonate in a timely manner, and record the highest peak value of the elution peak.

[0074] S37. After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tube connected to the collector with purified water.

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

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

[0077] Example 2

[0078] This embodiment aims to verify the extract in the antibody preparation process disclosed in Example 1, and to collect positive serum using ELISA detection of polyclonal antibody serum, discarding unqualified serum. Details are as follows:

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

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

[0081] S43. Add a certain amount of diluted polyclonal antibody serum, 50 μl, to the above-mentioned blocked reaction wells, and set up negative control wells (PBS). Incubate at 37°C for 1 hour, then discard the blocking solution and wash 3 times with 1xPBST washing buffer at 150 μl per well.

[0082] S44. Add 50 μl of freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to each well of the microplate and incubate at 37°C for 45 min. Then discard the blocking solution and wash three times with 150 μl of 1xPBST buffer 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 1M sulfuric acid to each reaction well;

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

[0086]

[0087]

[0088] Example 3

[0089] Based on Example 1, this example aims to select qualified iterative antibodies by detecting the purified iterative antibodies using an ELISA method. The detailed steps include:

[0090] S51. After purifying enough antibodies to meet the delivery requirements, and after the titer of the semi-finished product is qualified, mix all the antibodies and concentrate them using an ultrafiltration concentrator to achieve a certain concentration and volume.

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

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

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

[0094]

[0095] In summary, the antibodies prepared by the above methods can be used to detect expression differences in normal cells, tumor cells, adjacent normal tissues, and tumor tissues. This is beneficial for studying the role and mechanism of human PGK1 protein lactation modification in tumor development and provides potential targets for clinical tumor diagnosis and treatment.

[0096] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention, unless otherwise specified or limited, shall be implemented according to conventional means in the art.

Claims

1. A method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1, characterized in that, Includes the following steps: S01. A polypeptide with the amino acid sequence VEEEGK(lac)GKDASGNC is coupled to a carrier protein to obtain an initial antigen, wherein the carrier protein includes hemocyanin KLH; S02. The obtained initial antigen is injected into a live culture sample for immune culture. After a predetermined period, whole blood is collected to obtain polyclonal antibody serum. S03, Obtain purified antibody.

2. The method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1 according to claim 1, characterized in that, The initial antigen obtained in step S01 includes: S11. Dissolve 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 dissolve it completely in 50 μL of LDMSO. Then add 150 μL of 1×PBS and mix well to obtain a mixed liquid. S13. Add the mixed liquid dropwise to the fused KLH solution while gently shaking, and let it stand at room temperature for 1 hour to obtain activated KLH; S14. Place the activated KLH solution into a dialysis bag and clamp it with a dialysis clamp. After completion, place it into 2L of 1×PBS and dialyze for 1h under magnetic stirring at 4℃. Then replace it with a new 2L of 1×PBS and dialyze for 2h under magnetic stirring at 4℃ to obtain activated and dialyzed KLH. S15. Place the activated and dialyzed KLH into a 15mL imported centrifuge tube, and label the tube with the reagent name, time and concentration. Store at 4℃. S16. Weigh 4 mg of the peptide with the amino acid sequence VEEEGK(lac)GKDASGNC and dissolve it in 50 μL of DMSO. Then add 200 μL of 1×PBS, mix quickly, and then add the activated and dialyzed KLH according to the predetermined ratio. Incubate overnight at 4°C or react at room temperature for 2 h to obtain the KLH-peptide cross-linked complex. S17. The KLH-peptide crosslinking complex is placed in a dialysis bag and clamped with a dialysis clamp. Then it is placed in 4L of 1×PBS and dialyzed overnight at 4°C with magnetic stirring to obtain the dialyzed KLH-peptide. S18. Take the dialyzed KLH-peptide into a 1.5mL centrifuge tube, aliquot it according to the immunization dose, and store it in a -20℃ refrigerator to obtain the initial antigen.

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

4. The method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1 according to claim 1, characterized in that, The live 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 lactation of lysine at position 131 of human PGK1 according to claim 1, characterized in that, The immune culture in step S02 includes the following steps: S21. The initial antigen thawed at room temperature, the initial antigen injected into the first immunization of multiple live culture samples is 0.5 ml / animal, and the amount of the initial antigen injected into the second to fourth immunization is halved; S22. The adjuvant and antigen are drawn in a 1:1 volume ratio. The first immunization uses complete adjuvant, and the second to fourth immunizations use incomplete adjuvant. When drawing, the adjuvant must be fully mixed before being drawn into the syringe. S23. Then, the sample solution extracted in step S22 is completely emulsified and then injected subcutaneously at multiple points, with each point containing 0.2 ml.

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

7. The method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1 according to claim 1, characterized in that, The predetermined cycle in step S02 is as follows: the second immunization is performed 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 collecting a small sample of serum from the middle ear artery on the 7th day after the third immunization. If the test is qualified, the immunization is repeated 7 days later. Whole blood can be collected 7 days after the immunization is completed. The steps for collecting whole blood described in step S02 include: S24. Use the cardiac blood collection method. After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 15-30 minutes, then remove it, cool it, and place it in a 4°C refrigerator. Wait for the blood to automatically separate into layers, and then transfer the supernatant into a clean 50ml centrifuge tube. S25. The centrifuge is set at 12000 rpm for 2 min. After centrifugation, 100 μL of 10% sodium thimerosal solution is added to the 50 ml centrifuge tube and mixed well. The mixture is then stored at -20 °C. The final concentration of the 10% sodium thimerosal solution is 0.02%.

8. The method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1 according to claim 1, characterized in that, The purification in step S03 includes: S31. Wash the affinity chromatography column thoroughly with 20 mL of pure water and 1×PBS at a flow rate of 70 mL / h. S32. Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm. S33. Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once. S34. Wash the column with 20 mL of 1×PBS at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance reading to 100. S35. When the absorbance reading of the protein detector is 0, turn on the HD-A computer acquisition device on the computer desktop and adjust the full-screen range to 5. Use 0.2M glycine solution to elute the antibody at a rate of 40mL / h. Press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody. S36. During antibody collection, adjust the pH of the antibody to around 7 with 1M sodium bicarbonate in a timely manner, and record the highest peak value of the elution peak. S37. After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tube connected to the collector with purified water. S38. Wash the affinity chromatography column sequentially with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store at 4°C.

9. The method for preparing an antibody modified by lactation of lysine at position 131 of human PGK1 according to claim 1, characterized in that, Before performing step S03, positive serum is collected by ELISA detection of the obtained polyclonal antibody serum, and unqualified serum is discarded.

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