CRTC2 phosphorylated antigenic peptide at amino acid position 433, specific antibody and its application

Abstract

This invention belongs to the field of immunoassay and discloses an antigenic peptide phosphorylated at amino acid position 433 of CRTC2, a specific antibody, and its applications. Mass spectrometry analysis reveals for the first time the role of the CRTC2 Ser433 phosphorylation site in radiotherapy resistance, indicating that the phosphorylation level of CRTC2 Ser433 can serve as an indicator for predicting the radiosensitivity of liver cancer patients. Using the CRTC2 Ser433 phosphorylated antigenic peptide: CPHHRRVPLpSPLS, a specific phosphorylated antibody (CRTC2P-Ser433 antibody) is prepared for the first time. Radiosensitivity can be predicted through immunohistochemical staining scores, demonstrating both specificity and ease of detection.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy, specifically relating to an antigenic polypeptide phosphorylated at amino acid position 433 of CRTC2, a specific antibody, and its application. Background Technology

[0002] Liver cancer is a common and prevalent malignant tumor worldwide, with a generally poor prognosis. Radiotherapy, due to its advantages such as high local control rate and manageable side effects, has become a key method in the comprehensive treatment of liver cancer, especially with the development of technologies such as stereotactic radiotherapy and proton therapy, its clinical application is becoming increasingly widespread. Currently, clinical radiotherapy for liver cancer patients is divided into radical radiotherapy, palliative radiotherapy, and neoadjuvant therapy. Assessment is usually based on the following four aspects: tumor location and size to determine whether it affects surrounding organs; different radiotherapy strategies are used for different tumor sizes and numbers; liver function reserve to ensure that the liver can still maintain normal function after radiotherapy damage; patient's overall assessment to determine the presence of underlying diseases and whether the patient can live independently after radiotherapy; and previous radiotherapy history to assess patient tolerance and efficacy. However, there are significant individual differences in the response of liver cancer to radiation. Studies have shown that only about 18.75% of patients are sensitive to radiotherapy, while about 20% of patients experience disease progression after treatment due to congenital or acquired resistance. This widespread radiotherapy resistance severely limits the clinical benefit for patients. Therefore, finding effective radiosensitization targets to improve radiosensitivity has become an important clinical problem for improving the prognosis of liver cancer. Currently, clinical research often uses the Radiosensitivity Index (RSI), published in 2009, to predict radiosensitivity in various cancers. This index predicts radiosensitivity based on the expression levels of 10 genes (AR, JUN, STAT1, PKC, Rel A, cABL, SUMO1, CDK1, HDAC1, IRF1). It has been validated in cancers such as breast cancer, pancreatic cancer, and glioblastoma, and is also used in conjunction with the GARD (genomic-adjusted radiation dose) score to predict radiation dose. Although these assessment methods have been applied in various cancers, no clear and limited predictive targets or scores have been found in liver cancer. Summary of the Invention

[0003] Current technologies, whether through clinical assessment or gene expression testing to calculate RSI scores, cannot accurately determine the radiosensitivity of liver cancer patients; they can only assess the patient's radiotherapy tolerance. There are currently no clinically effective target genes or gene sets for determining radiosensitivity in liver cancer. Our research explores novel radiosensitization and therapeutic prediction targets, which can provide pathological indicators for pre-radiotherapy prediction in patients.

[0004] Radiotherapy works by causing DNA breaks, leading to tumor cell death. However, excessive repair of DNA damage can result in radioresistance. Cellular and animal experiments have clarified the function of CRTC2 in promoting DNA damage repair. Molecular mechanism exploration revealed that DNA-PKcs phosphorylate the CRTC2 Ser433 site, promoting the transition of CRTC2 from transcriptional regulation to DNA damage repair, thereby accelerating the NHEJ repair process and causing radioresistance. Our mass spectrometry analysis is the first to discover the role of the CRTC2 Ser433 phosphorylation site in radioresistance, indicating that the CRTC2 Ser433 phosphorylation level can serve as an indicator for predicting radiosensitivity in liver cancer patients.

[0005] This invention specifically relates to an antigenic polypeptide phosphorylated at amino acid position 433 of CRTC2, a specific antibody thereof, and its application.

[0006] Specifically,

[0007] This invention provides an antigenic polypeptide with phosphorylated amino acid 433 of CRTC2. Specifically, the amino acid sequence of the antigenic polypeptide is CPHHRRVPLSPLS (SEQ ID No: 1), wherein amino acid S at position 10 is phosphorylated.

[0008] The present invention also provides specific antibodies against the phosphorylated antigenic peptides as described above.

[0009] The present invention also provides articles comprising the phosphorylated antigenic peptides or the specific antibodies described above.

[0010] Furthermore, this invention also provides the use of the phosphorylated antigenic peptides, the specific antibodies, and / or the products described above in the preparation of reagents for predicting radiosensitivity in liver cancer. Specifically, immunohistochemical staining of liver cancer patient tissues that have undergone radiotherapy is performed using purified CRTC2P-Ser433 antibody, and high and low expression levels are distinguished according to a score: low CRTC2P-Ser433 levels indicate radiosensitivity in liver cancer patients, while high CRTC2P-Ser433 levels indicate radioresistance in liver cancer patients.

[0011] Furthermore, the present invention also provides a method for preparing the antibody as described above, comprising the following steps:

[0012] (1) The antigen peptide is coupled with KLH to obtain a KLH-peptide cross-linked complex, wherein the antigen peptide sequence is CPHHRRVPLpSPLS;

[0013] (2) Animal immunization: Immunize animals with the above-mentioned KLH-peptide cross-linked complex;

[0014] (3) Antibody titer detection;

[0015] (4) Antibody purification.

[0016] As described above, step (1) of conjugating the antigen polypeptide to KLH includes:

[0017] A) Dissolve KLH in an EDTA aqueous solution, preferably 10-40 mg, more preferably 20 mg KLH, in 2.5-10 mL, more preferably 5 mM EDTA aqueous solution;

[0018] B) Dissolve Sulfo-SMCC completely in DMSO, then add 1×PBS and mix well to obtain a Sulfo-SMCC solution. Preferably, 4-12 mg, preferably 8 mg of Sulfo-SMCC is completely dissolved in 25-100 μL, preferably 50 μL of DMSO, and then 75-300 μL, preferably 150 μL of 1×PBS is added.

[0019] C) Add the Sulfo-SMCC solution dropwise to KLH while gently shaking, and let it stand at room temperature to obtain an activated KLH solution. Preferably, the solution is left at room temperature for 0.5-3 hours, more preferably 1 hour.

[0020] D) Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clamp, place it in 1×PBS, and dialyze under magnetic stirring at refrigeration. Replace with new 1×PBS and dialyze once. Preferably, dialyze in 1-4L, preferably 2L of 1×PBS, at 4°C under magnetic stirring for 0.5-2h, preferably 1h.

[0021] E) Place the dialyzed KLH in a container and store it under refrigeration, preferably in a refrigerator at 4°C;

[0022] F) Weigh the antigenic peptide CPHHRRVPLpSPLS, dissolve it in DMSO, add 1×PBS, mix quickly, and then immediately add KLH. Perform the cross-linking reaction at refrigeration or room temperature to obtain the KLH-peptide cross-linking complex. Place the cross-linked KLH-peptide cross-linking complex into a dialysis bag, clamp it with a dialysis clip, place it in 1×PBS, and dialyze under magnetic stirring under refrigeration. Take out the dialyzed KLH-peptide into a clean container, aliquot it according to the immunization dose, and freeze it for storage.

[0023] Preferably, 2-8 mg (preferably 4 mg) of the antigenic peptide is weighed and dissolved in 25-100 μL (preferably 50 μL) of DMSO. Then, 100-400 μL (preferably 200 μL) of 1×PBS is added, and the mixture is quickly mixed. Subsequently, KLH is immediately added at a ratio of peptide:KLH = 0.5-2 mg: 340-1360 μg (preferably 1 mg: 680 μg). The mixture is incubated overnight at 4°C or at room temperature for 1-4 hours (preferably 2 hours). The cross-linked KLH-peptide complex is then placed in a dialysis bag, clamped with a dialysis clip, and dialyzed overnight at 4°C with magnetic stirring in 2-8 L (preferably 4 L) of 1×PBS. The dialyzed KLH-peptide is then transferred to clean 1.5 mL centrifuge tubes, aliquoted according to the immunoassay dose, and stored at -20°C.

[0024] As described above, step (2) of animal immunization includes:

[0025] Animal selection: Select healthy animals, preferably New Zealand white rabbits;

[0026] The adjuvant and the KLH-peptide crosslinking complex were thoroughly mixed;

[0027] Immunization: The animals were administered multiple subcutaneous injections of the adjuvant-emulsified KLH-peptide cross-linked complex;

[0028] Blood was collected, and immune serum was separated.

[0029] As described above, the antibody titer detection in step (3) adopts conventional immunoassay methods, such as indirect ELISA, chemiluminescent immunoassay (CLIA), agglutination reaction, immunoblotting, etc.

[0030] As described above, step (4) antibody purification includes:

[0031] The immune serum is first passed through a modified purification column, which is packed with phosphorylated antigenic peptide CPHHRRVPLpSPLS to achieve preliminary screening of phosphorylated antibodies. The eluted antibodies are then passed through a non-modified purification column, which is packed with unphosphorylated antigenic peptide CPHHRRVPLSPLS to remove antibodies that bind to the non-phosphorylated form through negative screening. This yields antibodies that specifically recognize phosphorylated modifications, namely CRTC2 P-Ser433 antibodies.

[0032] This invention utilizes the antigenic peptide: CPHHRRVPLpSPLS (where pS indicates that serine is phosphorylated) to prepare a specific phosphorylated antibody for the first time. This antibody can predict the radiosensitivity of patients through immunohistochemical staining scores, and has both detection specificity and ease of use, thus possessing beneficial technical effects. Attached Figure Description

[0033] Figure 1 Mass spectrometry and conservation analysis of CRTC2 Ser433 phosphorylation site. A. Mass spectrometry analysis of CRTC2 Ser433 site in HCCLM3 cells after radiotherapy. B. Conservation analysis of CRTC2 Ser433 phosphorylation site sequences in different species.

[0034] Figure 2 Specificity and titer of CRTC2 Ser433 phosphorylated antibody.

[0035] Figure 3 CRTC2 Ser433 phosphorylation levels are positively correlated with radiotherapy resistance. A. Immunohistochemical staining for CRTC2 Ser433 phosphorylation. B. Chi-square test for patient prognosis. Detailed Implementation

[0036] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0037] Example 1: Discovery of the role and conservation of CRTC2 Ser433 phosphorylation in radiotherapy

[0038] To investigate the role of CRTC2 in radioresistance, proteomic analysis was used to identify potential phosphorylation sites in the post-radiotherapy DNA damage repair process. DNA-dependent protein kinase catalytic subunits (DNA-PKcs), key proteins interacting with CRTC2, also possess phosphorylation kinase activity. Comparison of phosphorylation mass spectrometry results and potential substrate sequences of DNA-PKcs revealed that DNA-PKcs may phosphorylate the CRTC2 Ser433 site (…). Figure 1 (A), and this site sequence is highly conserved ( Figure 1 (B)

[0039] The specific method is as follows:

[0040] Human hepatocellular carcinoma cells (HCCLM3) were collected after radiotherapy. Pull-down assays were performed using anti-CRTC2 antibody (Proteintech, 12497-1-AP) and Dynabeads magnetic beads (Thermo Fisher, 10006D). Cells were lysed using IP lysis buffer (Beyotime, P0013), followed by the addition of CRTC2 antibody and Dynabeads to pull down CRTC2. The magnetic beads were then resuspended in PBS to obtain a magnetic bead buffer. Subsequently, the magnetic bead buffer was reduced, alkylated, and digested overnight with trypsin. The digested peptides were redissolved in high-performance liquid chromatography (HPLC) solvent A (0.1% (v / v) formic acid aqueous solution) and loaded onto a nano-HPLC system (EASY-nLC 1200, Thermo Fisher Scientific). Each sample was separated using a C18 column (75 μm × 15 cm inner diameter, 3 μm particle size) with a 130-minute HPLC gradient at a flow rate of 300 nL / min. The liquid phase gradient was set as follows: solvent B (0.1% (v / v) formic acid acetonitrile solution) increased from 5% to 7% over 2 minutes; from 7% to 22% over 80 minutes; from 22% to 38% over 38 minutes; from 38% to 100% over 3 minutes; and then held at 100% solvent B for 7 minutes. The liquid phase eluent was electrosprayed into the Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) at a spray voltage of 2.2 kV. Mass spectrometry analysis was performed in data-dependent mode. In the first-stage mass spectrometry scan, the automatic gain control (AGC) target value was 4 × 10⁻⁶. 5 The initial mass spectra were analyzed at a resolution of 60,000; the secondary mass spectra were analyzed at a resolution of 15,000. Raw data were analyzed using Thermo Proteome Discoverer 3.0 software, with the overall false discovery rate of peptides controlled to within 1%. Peptide sequence identification employed trypsin digestion specificity, allowing a maximum of two missed cleavage sites. Cysteine ​​carbamoyl methylation was set as a fixed modification; methionine oxidation and N-terminal acetylation were set as variable modifications. The mass tolerance for precursor ions was set to ±10 ppm, and the mass tolerance for secondary fragment ions was set to ±0.02 Da.

[0041] Example 2: Preparation of CRTC2 Ser433 phosphorylation-specific antibody

[0042] A phosphorylated antibody against CRTC2 Ser433 was prepared. The antigenic polypeptide sequence was: CPHHRRVPLpSPLS. The antibody titer was detected by ELISA, and the final antibody concentration was ( ). Figure 2 0.75 mg / mL.

[0043] The specific method is as follows:

[0044] (1) Antigen-peptide conjugation with KLH

[0045] Dissolve 20 mg KLH in 2 mL of 5 mM EDTA aqueous solution. Weigh 8 mg Sulfo-SMCC and dissolve it completely in 50 μL DMSO, then add 150 μL 1×PBS and mix well. Add the Sulfo-SMCC solution dropwise to the KLH solution while gently shaking (vigorous shaking will produce a precipitate), and let it stand at room temperature for 1 h to obtain an activated KLH solution. Place the activated KLH solution in a dialysis bag, clamp it with a dialysis clamp, and dialyze it in 2 L of 1×PBS at 4°C with magnetic stirring for 1 h. Replace with fresh 1×PBS and dialyze for 2 h, repeating once. Transfer the activated and dialyzed KLH to a 15 mL imported centrifuge tube, label the tube with the reagent name, time, and concentration, and store at 4°C.

[0046] Weigh 4 mg of the antigenic peptide (CPHHRRVPLpSPLS), dissolve it in 50 μL LDMSO, add 200 μL of 1×PBS, mix quickly, and then immediately add KLH at a ratio of peptide:KLH = 1 mg: 680 μg. Incubate overnight at 4°C or at room temperature for 2 h. Place the cross-linked KLH-peptide complex into a dialysis bag, clamp it, and dialyze overnight at 4°C with magnetic stirring in 4 L of 1×PBS. Transfer the dialyzed KLH-peptide into clean 1.5 mL centrifuge tubes, aliquot according to the immunoassay dose, and store at -20°C.

[0047] (2) Animal immunization program

[0048] Animal selection: Choose New Zealand White rabbits with glossy fur and free movement. After selecting the animals, raise them for approximately two weeks. The purpose is to weed out any unsuitable animals to ensure the smooth progress of later experiments.

[0049] Remove the KLH-peptide cross-linked complex from the -20°C freezer and thaw at room temperature, avoiding repeated freeze-thaw cycles. Label the syringes with the project number and animal number. Draw out the KLH-peptide cross-linked complex (ensuring complete mixing). The initial immunization dose is 0.1-1.0 mg (depending on the species and weight of the immunized animal), 0.5 ml / animal. For the second to fourth immunizations, halve the antigen dose. Draw out the adjuvant, with a 1:1 volume ratio of adjuvant to KLH-peptide cross-linked complex. Use complete adjuvant for the initial immunization and incomplete adjuvant for the second to fourth immunizations. Ensure the adjuvant is thoroughly mixed before drawing it into the syringe. Connect the two syringes with the syringe plunger and emulsify completely. The emulsification standard is: the emulsified KLH-peptide cross-linked complex should not disperse when dropped into 37°C water.

[0050] Immunization: Animals were injected subcutaneously at multiple sites with an emulsified KLH-peptide cross-linked complex, 0.2 ml at each site. Immunization schedule: A second immunization was administered 14 days after the first immunization, with a 7-day interval between the second and third immunizations. A small serum sample was collected from the middle ear artery 7 days after the third immunization for testing. If the test was satisfactory, a booster immunization was administered 7 days later, and whole blood could be collected 7 days after the booster immunization.

[0051] After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 15-30 minutes, then remove and cool before placing it in a 4°C refrigerator. Allow the blood to separate automatically, then transfer the supernatant (i.e., immune serum) to a clean 50ml centrifuge tube. Centrifuge at 12000 rpm for 2 minutes, transfer the supernatant to a clean centrifuge tube, add 100ul of 10% sodium thimerosal solution (final concentration 0.02%) to 50ml of the supernatant, mix well, and store at -20°C.

[0052] (3) ELISA detection (indirect method)

[0053] The known antigens (phosphorylated antigenic peptide CPHHRRVPLpSPLS and non-phosphorylated antigenic peptide CPHHRRVPLSPLS) were diluted to 1 μg / ml using coating buffer (Na2CO3 and NaHCO3 buffer). 50 μl of the solution was added to each well of the polystyrene plate and incubated overnight at 4°C. The next day, the solution in the wells was discarded, and the plate was washed once with 180 μl of 1xPBST washing buffer.

[0054] Blocking: Add 150 μl of 1% BSA (prepared with PBST) to each well for blocking, and incubate at 37°C for 1 hour. Then discard the blocking solution.

[0055] Sample loading: Add 50 μl of the diluted test sample (i.e., dilute the phosphorylated antigen peptide CPHHRRVPLpSPLS immune serum according to a certain ratio) to the above-mentioned sealed reaction wells. Also, set up negative control wells (1% BSA). Incubate at 37°C for 30 min, then wash three times with 150 μl of 1xPBST washing buffer per well.

[0056] Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (diluted with 1% BSA) at 50 μl / well to the wells of the ELISA plate, incubate at 37°C for 45 min, and wash 3 times with 1xPBST buffer at 150 μl / well.

[0057] Add substrate solution for color development: Add 50 μl of the temporarily prepared TMB substrate solution to each reaction well and react at 37 °C for 5 min.

[0058] To terminate the reaction, add 50 μl of 1M sulfuric acid to each well.

[0059] Plate reading: Place the ELISA plate in a preheated ELISA reader (450nm) to read the data, save the data, and perform analysis.

[0060] (4) Antibody purification

[0061] The affinity chromatography column was thoroughly washed sequentially with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h. 10 mL of the serum to be purified was placed in a 50 mL centrifuge tube and filtered through a 0.45 μm pore size, 25 mm diameter microporous membrane. The filtered serum sample was loaded onto the column at a flow rate of 40 mL / h, and this process was repeated once. The column was washed with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h for 10 min. The column was then connected to a protein analyzer, and the transmittance (T setting) of the analyzer was adjusted to 100 during the washing process. The absorbance (1A setting) of the protein analyzer was adjusted to 0. The HD-A computer acquisition unit on the computer desktop was then turned on, and the full-screen range was set to 5. The antibody was eluted with glycine solution (pH 2.7, 0.2 M) at a rate of 40 mL / h. The elution recording button was pressed to start elution, and antibody collection began when the instrument reading started to rise. During antibody collection, the pH of the antibody was adjusted to approximately 7 with 1M Tris-HCl, and the peak value of the elution was recorded. After antibody collection, the pH was adjusted to approximately 7, and the volume of eluted antibody was recorded. The rubber tubing connecting the collector was then rinsed with purified water. The affinity chromatography column was washed sequentially with 20 mL of 1×PBS and purified water at a rate of 70 mL / h, followed by the addition of 20% ethanol, sealing, and storage at 4°C.

[0062] During purification, serum is first passed through a modified purification column packed with phosphorylated antigenic peptides (CPHHRRVPLpSPLS) to adsorb phosphorylated antibodies in the serum, achieving preliminary screening for phosphorylated antibodies. The eluted antibodies are then passed through a non-modified purification column packed with unphosphorylated antigenic peptides (CPHHRRVPLSPLS) for negative selection to remove antibodies binding to the non-phosphorylated form, yielding antibodies that specifically recognize phosphorylated antibodies (CRTC2 P-Ser433 antibody). After sufficient antibody quantity for delivery has been purified and the titer of the semi-finished product has passed testing, all antibodies are mixed and concentrated using an ultrafiltration concentrator to achieve a specific concentration and volume. The antibodies are then transferred to clean centrifuge tubes and filtered through a 0.22µm disposable low-adsorption filter in a clean bench. A small sample is sent for testing, and another 5µl is taken for concentration determination. The concentration is then measured using a Protein A280 ultra-micro spectrophotometer (denovix DS-11).

[0063] Example 3: Predicting patient radiosensitivity using CRTC2Ser433 antibody specificity

[0064] To investigate the role of the CRTC2Ser433 site in assessing the radiosensitivity of liver cancer patients, we used the purified CRTC2P-Ser433 antibody from step (4) of Example 2 to perform immunohistochemical staining on the tissues of liver cancer patients who had received radiotherapy, and differentiated between high and low expression levels based on the scoring. Chi-square test revealed that low levels of CRTC2P-Ser433 indicated significant disease remission or no progression, suggesting radiosensitivity, while high levels of CRTC2P-Ser433 mainly indicated disease progression, suggesting radioresistance. Figure 3 China A and Figure 3 (Middle B). The above results indicate that the phosphorylation level of CRTC2Ser433 can be used as an indicator for assessing the radiosensitivity of patients with liver cancer in clinical practice.

[0065] The specific method is as follows:

[0066] (1) Immunohistochemical staining of CRTC2Ser433 phosphorylation

[0067] Twenty-six liver tumor tissue samples were collected from liver cancer patients undergoing radiotherapy at Tianjin Medical University Cancer Hospital. The tissues were collected before radiotherapy, fixed in 4% paraformaldehyde for at least 24 hours, embedded in paraffin, and cut into 5 μm thick sections. Hematoxylin-eosin staining was performed according to the manufacturer's instructions. Immunohistochemical staining was performed according to the established protocol. A brief summary of the steps is as follows: Tissue samples were dewaxed with xylene and rehydrated with graded ethanol to distilled water. After antigen retrieval and blocking, tissue sections were incubated overnight at 4°C with CRTC2 Ser433 phosphorylated antibody, followed by incubation at room temperature for 1 hour with anti-rabbit secondary antibody. Development was performed using DAB substrate solution. Sections were counterstained with hematoxylin, dehydrated, and then mounted.

[0068] (2) Immunohistochemical results were evaluated using a double-blind method, with a score of 0-12. The score was obtained by multiplying the staining intensity score (0: negative; 1: weakly positive; 2: moderately positive; 3: strongly positive) by the percentage of positive cells score (0: no staining; 1: 1%-25% positive cells; 2: 26%-50% positive cells; 3: 51%-75% positive cells; 4: 76%-100% positive cells).

[0069] (3) Based on the examination reports before and after radiotherapy, the prognosis of patients was divided into partial response (PR), stable disease progression (SD), and progressive disease (PD). Patients were grouped according to the phosphorylation level of CRTC2 Ser433, and the disease progression of patients in each group was statistically analyzed and chi-square test was performed.

Claims

1. An antigenic polypeptide phosphorylated at amino acid position 433 of CRTC2, wherein the amino acid sequence of the antigenic polypeptide is CPHHRRVPLSPLS, wherein amino acid position 10, S, is phosphorylated.

2. Specific antibodies against the phosphorylated antigenic polypeptide as described in claim 1.

3. An article comprising the phosphorylated antigenic polypeptide of claim 1 or the specific antibody of claim 2.

4. The use of the phosphorylated antigenic polypeptide of claim 1, the specific antibody of claim 2, or the product of claim 3 in the preparation of a reagent for predicting the radiosensitivity of liver cancer.

5. A method for preparing a specific antibody against an antigenic polypeptide phosphorylated at amino acid position 433 of CRTC2, characterized in that, Includes the following steps: (1) The antigen peptide described in claim 1 is coupled with KLH to obtain a KLH-peptide cross-linked complex; (2) Animal immunization: Immunize animals with the above-mentioned KLH-peptide cross-linked complex; (3) Antibody titer detection; (4) Antibody purification.

6. The method according to claim 5, characterized in that, Step (1), antigen polypeptide conjugation with KLH, includes: A) Dissolve KLH in an EDTA aqueous solution; B) Dissolve Sulfo-SMCC completely in DMSO; C) The Sulfo-SMCC solution is added dropwise to KLH to obtain an activated KLH solution; D) Place the activated KLH solution into a dialysis bag and dialyze under refrigeration with magnetic stirring; E) Weigh the antigenic peptide, dissolve it in DMSO, and then immediately add KLH solution. Perform the cross-linking reaction under refrigeration or at room temperature to obtain the KLH-peptide cross-linking complex. Place the cross-linked KLH-peptide cross-linking complex into a dialysis bag and dialyze it under magnetic stirring under refrigeration. Take out the dialyzed KLH-peptide into a clean container.

7. The method according to claim 5, characterized in that, Step (1), antigen polypeptide conjugation with KLH, includes: A) Dissolve 10-40 mg KLH in 2.5-10 mL of EDTA aqueous solution; B) Dissolve 4-12 mg of Sulfo-SMCC completely in 25-100 μL of DMSO, and add 75-300 μL of 1×PBS; C) Add the Sulfo-SMCC solution dropwise to KLH while gently shaking, and let it stand at room temperature for 0.5-3 hours to obtain an activated KLH solution; D) Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clamp, place it in 1×PBS, and dialyze for 0.5-2 hours under refrigeration with magnetic stirring. Replace with new 1×PBS and repeat once. E) Weigh 2-8 mg of the antigenic peptide, dissolve it in 25-100 μL of DMSO, add 100-400 μL of 1×PBS, mix quickly, then add KLH immediately according to peptide:KLH = 0.5-2 mg: 340-1360 μg, incubate overnight at 4°C or react at room temperature for 1-4 h, place the above cross-linked KLH-peptide complex into a dialysis bag, clamp it with a dialysis clip, and dialyze overnight in 2-8 L of 1×PBS at 4°C with magnetic stirring.

8. The method according to claim 5, characterized in that, Step (2) of animal immunization includes: Healthy New Zealand white rabbits were selected, and the KLH-peptide cross-linked complex was injected subcutaneously at multiple points; blood was collected, and immune serum was separated.

9. The method according to claim 7, characterized in that, The antibody titer detection in step (3) is performed by indirect ELISA, chemiluminescent immunoassay, agglutination reaction or immunoblotting. Step (4) antibody purification includes: The immune serum is first passed through a modified purification column, which is packed with phosphorylated antigenic peptides to achieve preliminary screening of phosphorylated antibodies. The eluted antibodies are then passed through a non-modified purification column, which is packed with unphosphorylated antigenic peptides CPHHRRVPLSPLS to remove antibodies that bind to non-phosphorylated forms through negative screening. This yields antibodies that specifically recognize phosphorylated modifications.

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