A ppp2r5d s80 phosphorylation specific antibody and application thereof

Abstract

The present application relates to a kind of PPP2R5D S80 phosphorylation specificity antibody and its application, belong to the field of biotechnology.The present application provides a kind of PPP2R5D S80 phosphorylation specificity antibody, the PPP2R5D S80 phosphorylation specificity antibody is obtained by using antigen to immunize animal;The antigen is the PPP2R5D S80 phosphorylation peptide segment of amino acid sequence as shown in SEQ ID NO.2.The titer of the PPP2R5D S80 phosphorylation specificity antibody is as high as 1:162K, and S81 polypeptide and PPP2R5D polypeptide are not recognized, with the advantages of high titer and being able to effectively distinguish S80 and S81 phosphorylation modification, in the detection of PPP2R5D S80 site phosphorylation level, it is extremely promising in application.

Description

Technical Field

[0001] This invention relates to a PPP2R5D S80 phosphorylation-specific antibody and its application, belonging to the field of biotechnology. Background Technology

[0002] Protein phosphorylation refers to the process by which protein phosphokinases transfer phosphate groups from adenosine triphosphate (ATP) molecules to specific sites on protein molecules, such as serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. It is the most common and important regulatory mechanism for controlling protein activity and function. Protein phosphorylation participates in various physiological and pathological processes, playing a crucial role in signal transduction, apoptosis, development and differentiation, metabolism, and the occurrence and development of diseases such as cancer. Therefore, detecting protein phosphorylation levels is of great significance for studying cell and protein function and disease mechanisms.

[0003] Protein phosphatase 2A (PP2A) PP2A Serine / threonine phosphatase (SPF) is one of the four major serine / threonine phosphatases. It is involved in key cellular processes such as cell growth and division, muscle contraction, and transcriptional regulation. It has a variety of substrate-specific physiological functions in different tissues. It consists of a catalytic subunit, a structural subunit, and a regulatory subunit. It plays an important role in maintaining neurons and regulating neuronal signal transduction and insulin signal transduction. PPP2R5D The gene encodes the B regulatory subunit of PP2A. As a regulatory subunit, PPP2R5D can be phosphorylated by protein kinases, influencing the PP2A-PPP2R5D holoenzyme's negative regulation of cell growth, chromatin remodeling, and transcriptional regulation by regulating PP2A activity, localization, and binding to target substrates. Previous studies have shown that decreased phosphorylation at the PPP2R5D S80 site in mouse brown adipose tissue leads to increased PP2A activity and increased PP2A-AKT interaction, thereby affecting the insulin signaling pathway and glucose uptake. This indicates that the PPP2R5D S80 site plays an important role in PP2A-PPP2R5D function and is a potential target for prevention and treatment. Therefore, detecting the phosphorylation level of the PPP2R5D S80 site will help elucidate the role of PP2A-PPP2R5D and provide new targets for the development of therapeutics for diseases related to PP2A-PPP2R5D dysfunction.

[0004] Mass spectrometry (MS) is a highly sensitive and high-throughput protein analysis technique commonly used to identify protein phosphorylation sites and quantify phosphorylation levels. However, current methods for detecting PPP2R5D S80 phosphorylation levels still have several limitations. First, phosphorylated peptides detected by MS are typically weak and poorly ionized, making it difficult to detect low-abundance phosphorylated protein signals against a high background of abundant non-phosphorylated proteins. Therefore, purification and enrichment of phosphorylated peptides in the sample are necessary to avoid these limitations, increasing experimental complexity. Furthermore, MS analysis is relatively expensive, requiring specific equipment and demanding detection conditions. Most importantly, the S80 and S81 phosphorylation sites of PPP2R5D are adjacent, and MS cannot distinguish between S80 and S81 phosphorylation modifications. Therefore, MS cannot specifically detect PPP2R5D S80 phosphorylation levels.

[0005] Western blotting is a hybridization technique combining high-resolution gel electrophoresis and immunochemical analysis. It offers advantages such as large analytical capacity, high sensitivity, and high specificity, making it a commonly used method for detecting protein and phosphorylated protein levels. It has been widely applied in various fields, including gene expression studies at the protein level, antibody activity detection, and early disease diagnosis. This technique utilizes the principle of specific antigen-antibody binding, using phosphorylation-specific antibodies as probes to identify the phosphorylation level of the target protein. Western blotting is the most feasible, economical, and convenient method for detecting proteins and phosphorylated proteins in the laboratory. However, there are currently no commercially available phosphorylation-specific antibodies for PPP2R5D S80, which severely hinders the application of Western blotting in detecting PPP2R5D S80 phosphorylation.

[0006] Enzyme-linked immunosorbent assay (ELISA) is an immunoassay method commonly used to measure antibodies or antigens, including proteins or glycoproteins, in biological samples. Like other immunoassays, it relies on the binding of antibodies to the target protein to facilitate detection. It offers advantages such as high specificity, high sensitivity, simplicity, large analytical capacity, and low cost. It generally does not require expensive equipment, simplifies or eliminates pretreatment steps, and offers a range of commercially available technologies. It possesses unparalleled selectivity and high sensitivity compared to conventional physicochemical analysis techniques, making it highly suitable for analyzing specific components in complex matrices. Compared to Western blot, phosphorylation-specific ELISA technology has significant advantages. First, it allows for easy quantification using calibrated standards; second, the use of two proteins-specific antibodies in a sandwich configuration ensures high specificity; third, ELISA offers higher sensitivity, allowing for the use of smaller sample sizes to detect low-abundance proteins; and finally, the microplate format offers higher throughput. Currently, rapid ELISA kits are a commonly used technique for detecting protein phosphorylation levels. However, there are currently no kits on the market for detecting PPP2R5D S80 phosphorylation levels. Similar to the challenges faced by Western blot, the most significant obstacle to the development of PPP2R5D S80 phosphorylation level detection kits is the lack of specific antibodies for PPP2R5D S80 phosphorylation.

[0007] Besides Western blotting and enzyme-linked immunosorbent assay (ELISA), the lack of specific antibodies targeting PPP2R5D S80 phosphorylation has limited the development of other techniques for detecting PPP2R5D S80 phosphorylation levels based on the principle of antigen-antibody specific binding (e.g., flow cytometry, immunocytochemistry, and immunohistochemistry). Therefore, there is an urgent need to develop specific antibodies targeting the PPP2R5D S80 phosphorylation site for detecting phosphorylation levels at this site in biological samples or for developing antibody-dependent experimental methods (e.g., Western blotting, ELISA, flow cytometry, immunocytochemistry, and immunohistochemistry) to elucidate the role of PP2A-PPP2R5D and advance research into related diseases and novel drug targets. Summary of the Invention

[0008] To address the above problems, the present invention provides an antigen for preparing PPP2R5D S80 phosphorylated specific antibodies, wherein the antigen is a PPP2R5D S80 phosphorylated peptide (C KERRQ-(phospho)S-SFPFNLN) with the amino acid sequence shown in SEQ ID NO.2.

[0009] In one embodiment of the present invention, the antigen is coupled with a carrier protein; the carrier protein includes hemocyanin, bovine serum albumin, rabbit serum albumin, human serum albumin, chicken ovalbumin, glutathione S-transferase and / or multivalent antigenic peptides.

[0010] The present invention also provides a PPP2R5D S80 phosphorylation-specific antibody, which can specifically bind to the above-mentioned antigen.

[0011] In one embodiment of the present invention, the antibody is obtained by immunizing an animal with the above-mentioned antigen.

[0012] In one embodiment of the present invention, the PPP2R5D S80 phosphorylation-specific antibody is a polyclonal antibody.

[0013] In one embodiment of the present invention, the animal is a rabbit, mouse, sheep, monkey, horse, or chicken.

[0014] The present invention also provides a method for preparing the above-mentioned PPP2R5D S80 phosphorylated specific antibody, the method comprising: immunizing an animal with the above-mentioned antigen to obtain an immunized animal; and extracting the above-mentioned PPP2R5D S80 phosphorylated specific antibody from the serum of the immunized animal.

[0015] In one embodiment of the present invention, the immunization is performed as follows: six immunizations are administered on days 1, 7, 14, 21, 28, and 40, respectively; blood is collected and serum is separated on days 35 and 47, respectively. For the first immunization, physiological saline and the antigen are mixed at a ratio of 1 mL:(0.5~1.5) mg to dilute the antigen, obtaining an antigen dilution. The antigen dilution and adjuvant are mixed at a volume ratio of 1:0.5~1.5, and then injected subcutaneously at two points on both shoulders and at two points in the muscles of both hind legs of the animal, with an immunization dose of 0.25 mg / animal to 1.5 mg / animal. For the second to sixth immunizations, the immunization dose is 0.1 mg / animal to 0.75 mg / animal. The remaining steps are the same as the first immunization. The adjuvant is a non-specific immune enhancer; when injected together with the antigen or pre-injected into the body, it can enhance the body's immune response to the antigen or change the type of immune response.

[0016] The present invention also provides the application of the above-described antigen, or the above-described PPP2R5D S80 phosphorylation-specific antibody, or the above-described method in the preparation of products for detecting the phosphorylation level of the PPP2R5D S80 site.

[0017] In one embodiment of the present invention, the product is a test kit.

[0018] In one embodiment of the present invention, the product is a detection kit based on the principle of antigen-antibody specific binding.

[0019] In one embodiment of the present invention, the product is a detection kit based on Western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunocytochemistry (ICC), immunohistochemistry (IHC), or immunoelectron microscopy (IEM).

[0020] The present invention also provides a product for detecting the phosphorylation level of the PPP2R5D S80 site, the product comprising a PPP2R5D S80 phosphorylation-specific antibody obtained by immunizing an animal with the above-mentioned antigen; or, the product comprising the above-mentioned PPP2R5D S80 phosphorylation-specific antibody.

[0021] In one embodiment of the present invention, the product is a test kit.

[0022] In one embodiment of the present invention, the product is a detection kit based on the principle of antigen-antibody specific binding.

[0023] In one embodiment of the present invention, the product is a detection kit based on Western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunocytochemistry (ICC), immunohistochemistry (IHC), or immunoelectron microscopy (IEM).

[0024] In one embodiment of the present invention, when the product is a detection kit based on enzyme-linked immunosorbent assay (ELISA), the product comprises a primary antibody and a secondary antibody; the primary antibody is a PPP2R5D S80 phosphorylated specific antibody obtained by immunizing animals with the above-mentioned antigen or the above-mentioned PPP2R5D S80 phosphorylated specific antibody; the secondary antibody is an antibody that can specifically bind to the primary antibody.

[0025] The present invention also provides a method for detecting the phosphorylation level of the PPP2R5D S80 site, the method being for non-disease diagnosis and treatment purposes, the method comprising: using the above-mentioned product to detect the sample to be tested.

[0026] In one embodiment of the present invention, the method is based on Western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunocytochemistry (ICC), immunohistochemistry (IHC), or immunoelectron microscopy (IEM).

[0027] The present invention also provides the application of the above-mentioned antigen or the above-mentioned PPP2R5D S80 phosphorylation-specific antibody or the above-mentioned product or the above-mentioned method in the detection of phosphorylation level at the PPP2R5D S80 site, wherein the application is not for the purpose of disease diagnosis and treatment.

[0028] The technical solution of this invention has the following advantages: 1. This invention provides an antigen for preparing PPP2R5D S80 phosphorylation-specific antibodies, wherein the antigen is a PPP2R5D S80 phosphorylated peptide (C KERRQ-(phospho)S-SFPFNLN) with the amino acid sequence shown in SEQ ID NO.2. Immunizing animals with this antigen yields PPP2R5D S80 phosphorylation-specific antibodies. The PPP2R5D S80 phosphorylation-specific antibodies obtained by immunizing animals with this antigen have a high titer of 1:162K, exhibiting high sensitivity and easily detecting phosphorylated proteins in routine samples without the need for enrichment, significantly reducing sample requirements. Furthermore, the PPP2R5D S80 phosphorylation-specific antibodies obtained by immunizing animals with this antigen have excellent specificity, showing no binding reaction with the PPP2R5DS81 phosphorylation site, effectively solving the problem of cross-recognition between S80 and S81 (a problem that cannot be solved by other detection methods such as mass spectrometry). Therefore, the antigen has great application potential in the detection of phosphorylation level at the PPP2R5D S80 site. The PPP2R5D S80 phosphorylation-specific antibody obtained by immunizing animals with the antigen can not only effectively broaden the detection technology of PPP2R5D phosphorylation level based on phosphorylation-specific antibody (such as flow cytometry, immunocytochemistry, immunohistochemistry, and immunoelectron microscopy staining), but also has the advantages of simpler operation and significantly reduced experimental costs. In addition, it may also enable intracellular localization of phosphorylated proteins, which will help to study the function of PPP2R5D in greater depth.

[0029] 2. This invention provides a PPP2R5D S80 phosphorylation-specific antibody, which is obtained by immunizing animals with an antigen; the antigen is a PPP2R5D S80 phosphorylated peptide with an amino acid sequence as shown in SEQ ID NO.2. The PPP2R5D S80 phosphorylation-specific antibody has a high titer of 1:162K, exhibiting high sensitivity and easily detecting phosphorylated proteins in routine samples without the need for enrichment, significantly reducing sample requirements; furthermore, the PPP2R5D S80 phosphorylation-specific antibody has excellent specificity, showing no binding reaction with the PPP2R5D S81 phosphorylation site, effectively solving the problem of cross-recognition between S80 and S81 (a problem that cannot be solved by other detection methods such as mass spectrometry). Therefore, the PPP2R5D S80 phosphorylation-specific antibody has great application potential in the detection of phosphorylation levels at the PPP2R5D S80 site. It can not only effectively broaden the detection technology of PPP2R5D phosphorylation level based on phosphorylation-specific antibodies (such as flow cytometry, immunocytochemistry, immunohistochemistry, and immunoelectron microscopy staining), but also has the advantages of simpler operation and significantly reduced experimental costs. In addition, it may also enable intracellular localization of phosphorylated proteins, which will help to study the function of PPP2R5D in greater depth. Attached Figure Description

[0030] Figure 1 Western blot analysis of R1, R2, R3 and R4 in NIH / 3T3 and F9 cell lines of immunized rabbit serum.

[0031] Figure 2 Western blot results of R1, R2, R3 and R4 in the heart and kidney of mice after immunization of rabbit serum.

[0032] Figure 3 Dot Blot results of PPP2R5D S80 phosphorylated specific antibodies Ab2 and Ab4.

[0033] Figure 4 Western blot analysis of PPP2R5D S80 phosphorylated specific antibody Ab4 in mouse brown adipose tissue and primary brown adipocytes.

[0034] Figure 5 Western blot results of PPP2R5D S80 phosphorylated specific antibody Ab4 in NIH / 3T3 cell line. Detailed Implementation

[0035] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0036] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0037] Example 1: Preparation, purification and identification of PPP2R5D S80 phosphorylated specific antibody 1. Antigen synthesis and animal immunization Based on PPP2R5D 73-85aa and PPP2R5D 75-87aa, two PPP2R5D S80ph modified peptides were designed and synthesized in vitro using bioinformatics methods, labeled as S80 peptide A (amino acid sequence as shown in SEQ ID NO.1) and S80 peptide B (amino acid sequence as shown in SEQ ID NO.2), respectively. Based on PPP2R5D 73-85aa, one PPP2R5D S81ph modified control peptide and one PPP2R5D unmodified peptide were designed and synthesized using bioinformatics methods, labeled as S81 peptide (amino acid sequence as shown in SEQ ID NO.3) and PPP2R5D peptide (amino acid sequence as shown in SEQ ID NO.4), respectively. The peptide sequences are shown in Table 1.

[0038] Using the Imject™ Maleimide-Activated mcKLH Centrifugation Kit (Thermo Scientific), S80 peptides A and B, containing thiol groups, were conjugated to highly immunogenic keyhole hemocyanin (KLH), and the conjugates were sterile filtered to obtain S80 peptides A and B conjugated to keyhole hemocyanin for animal immunization.

[0039] Two female New Zealand rabbits (weighing 2-3 kg, purchased from Yuhang Kelian Rabbit Farm) were immunized with S80 peptide A and S80 peptide B, conjugated with keyhole cyanin, as antigens. The immunization program was as follows: six immunizations were administered on days 1, 7, 14, 21, 28, and 40. Blood samples were collected on days 35 and 47, and serum was separated. 30 mL of blood was collected each time, and the supernatant was obtained by centrifugation. For the first immunization, 0.5 mg of the antigen was diluted with 0.5 mL of physiological saline (purchased from Sigma) to obtain an antigen dilution. The antigen dilution was mixed with an equal volume of adjuvant (stimune, purchased from Sigma) and injected subcutaneously at two points on both shoulders and intramuscularly at two points on both hind legs. The immunization dose was 0.5 mg per rabbit, with 1 / 4 volume of immunogen in each area. For the second to sixth immunizations, the immunization dose was 0.25 mg per rabbit, and the remaining steps were the same as the first immunization. The serum obtained by immunizing the first New Zealand rabbit with S80 polypeptide A was named R1, the serum obtained by immunizing the second New Zealand rabbit with S80 polypeptide A was named R2, the serum obtained by immunizing the first New Zealand rabbit with S80 polypeptide B was named R3, and the serum obtained by immunizing the second New Zealand rabbit with S80 polypeptide B was named R4.

[0040] Table 1. Peptide Information

[0041] 2. Serum screening 2.1 ELISA Detection ELISA assays were performed on R1, R2, R3, and R4 respectively to preliminarily assess the titer and specificity of the antiserum; the ELISA assay steps are as follows: Coating: S80 peptide A, S80 peptide B, S81 peptide, and PPP2R5D peptide were diluted with 1×TBST buffer to a protein concentration of 1 ng / μL, resulting in S80 peptide A dilution, S80 peptide B dilution, S81 peptide dilution, and PPP2R5D peptide dilution. Each of these dilutions was added to a 96-well microplate at a concentration of 50 μg / well, and the plates were incubated at 4°C for 16 h for coating. After coating, the plates were washed three times with 1×TBST buffer. After washing, 200 μL of 1×TBST buffer containing 1% (v / v) BSA (purchased from Sigma) was added to each well of the 96-well microplate, and the plates were incubated at 37°C for 1 h for blocking. After blocking, the plates were washed three times with 1×TBST buffer. Serum incubation: The obtained serum R1, R2, R3, and R4 were diluted with 1×TBST buffer at dilution factors of 1:250, 1:1K, 1:4K, 1:16K, 1:64K, 1:256K, 1:1024K, and 1:4096K, respectively, to obtain serum dilutions of different concentrations. Each serum dilution of different concentrations was added to a 96-well microplate at a rate of 50 μL / well and incubated at 37°C for 1.5 h. After incubation, the plates were washed three times with 1×TBST buffer. Secondary antibody incubation: Dilute the secondary antibody (HRP goat anti-rabbit, purchased from CST) with 1×TBST buffer containing 1% (v / v) BSA (purchased from Sigma) at a dilution factor of 1:10K to obtain the secondary antibody dilution solution; add 50 μL of the secondary antibody dilution solution to each well of a 96-well microplate and incubate at 37°C for 45 min; after incubation, wash the plate 3 times with 1×TBST buffer; Color development: 50 μL of TMB chromogenic solution (purchased from Invitrogen) was added to each well of a 96-well microplate, and the plates were incubated at 37°C in the dark for 10 min to develop color. After color development, 50 μL of 1M sulfuric acid (purchased from Sigma) aqueous solution was added to each well to terminate the reaction. After termination of the reaction, the 96-well microplate was placed in a microplate reader at 450 nm for reading, and the titers of serum R1, R2, R3, and R4 were obtained based on the readings at different dilutions.

[0042] The ELISA results are shown in Tables 2 and 3. As can be seen from Tables 2 and 3, the titers of the modified peptides recognized by R1, R2, R3, and R4 are all greater than 1:10K (OD>1).

[0043] Table 2. ELISA results of serum R1 and R2 generated by S80 polypeptide A immunization.

[0044] Table 3. ELISA results of serum R3 and R4 generated by S80 polypeptide B immunization.

[0045] 2.2 Western blot detection Serum samples R1, R2, R3, and R4 were collected and Western blot was used to detect endogenous PPP2R5D (S80ph) protein, with Anti-HSP70 Rabbit mAb (purchased from Abcam) added as a control. Western blot results are shown below. Figures 1-2 As shown. By Figures 1-2It was found that serum samples R1, R2, R3, and R4 could detect bands of the target protein with a theoretical molecular weight of approximately 70 kDa. Based on the combined results of ELISA and Western blot analysis, serum samples R2 and R4 were selected for further purification.

[0046] 3. Antibody purification R2 and R4 were purified by affinity chromatography to obtain PPP2R5D S80 phosphorylated specific antibodies Ab2 and Ab4 (antibody purification was performed by Hangzhou Jingjie Biotechnology Co., Ltd.). The PPP2R5D S80 phosphorylated specific antibodies are polyclonal antibodies. The protein content of PPP2R5D S80 phosphorylated specific antibodies Ab2 and Ab4 in the eluent obtained from affinity chromatography purification was detected using NanoDrop (purchased from Thermo Scientific), and the measured protein contents were 3.03 mg / mL and 2.16 mg / mL, respectively. The purified PPP2R5D S80 phosphorylated specific antibodies were aliquoted and stored at -20°C.

[0047] 4. Antibody identification 4.1 Identification by ELISA The purified PPP2R5D S80 phosphorylated specific antibodies Ab2 and Ab4 were identified using ELISA; the steps of the ELISA method are as follows: Coating: S80 peptide A, S80 peptide B, S81 peptide, and PPP2R5D peptide were diluted with 1×TBST buffer to a protein concentration of 1 ng / μL, resulting in S80 peptide A dilution, S80 peptide B dilution, S81 peptide dilution, and PPP2R5D peptide dilution. Each of these dilutions was added to a 96-well microplate at a concentration of 50 μg / well, and the plates were incubated at 4°C for 16 h for coating. After coating, the plates were washed three times with 1×TBST buffer. After washing, 200 μL of 1×TBST buffer containing 1% (v / v) BSA (purchased from Sigma) was added to each well of the 96-well microplate, and the plates were incubated at 37°C for 1 h for blocking. After blocking, the plates were washed three times with 1×TBST buffer. Primary antibody incubation: The purified PPP2R5D S80 phosphorylated specific antibody was diluted with 1×TBST buffer at dilution factors of 1:2K, 1:6K, 1:18K, 1:54K, 1:162K, 1:486K, 1:1458K, and 1:4374K to obtain different concentrations of primary antibody dilutions. Each concentration of primary antibody dilution was added to a 96-well microplate at a rate of 50 μL / well and incubated at 37℃ for 1.5 h. After incubation, the plate was washed three times with 1×TBST buffer. Secondary antibody incubation: Dilute the secondary antibody (HRP goat anti-rabbit, purchased from CST) with 1×TBST buffer containing 1% (v / v) BSA (purchased from Sigma) at a dilution factor of 1:10K to obtain the secondary antibody dilution solution; add 50 μL of the secondary antibody dilution solution to each well of a 96-well microplate and incubate at 37°C for 45 min; after incubation, wash the plate 3 times with 1×TBST buffer; Color development: 50 μL of TMB chromogenic solution (purchased from Invitrogen) was added to each well of a 96-well microplate, and the plate was incubated at 37°C in the dark for 10 min to develop color. After color development, 50 μL of 1M sulfuric acid (purchased from Sigma) aqueous solution was added to each well to terminate the reaction. After termination of the reaction, the 96-well microplate was placed in a microplate reader at 450 nm for reading, and the titer of the phosphorylated specific antibody was obtained based on the readings at different dilutions.

[0048] The titer results of the PPP2R5D S80 phosphorylation-specific antibodies Ab2 and Ab4 are shown in Tables 4 and 5. A positive result was indicated by an A450 > 1. As shown in Tables 4 and 5, Ab2 had a potency value of 1:54K against both S80 peptides and recognized the two negative control peptides, S81 peptide and PPP2R5D peptide; while Ab4 had a potency value of 1:162K against S80 peptide B and did not recognize the two negative control peptides.

[0049] Table 4. ELISA detection results of antibody Ab2

[0050] Table 5. ELISA detection results of antibody Ab4

[0051] 4.2 Identification by Dot blot method The purified PPP2R5D S80 phosphorylated specific antibodies Ab2 and Ab4 were identified using Dot blot; the steps of the Dot blot method are as follows: Fixation: After activating the PVDF membrane with methanol (purchased from Merck) for 1 min, the activated PVDF membrane was placed in ddH2O and washed on a decolorizing shaker for 15 min. Then, it was dried at room temperature (25℃) until the water film disappeared. 1, 4, 16, and 64 ng of uncrosslinked S80, S81, and PPP2R5D peptides were respectively spotted onto the dried PVDF membrane and air-dried until completely dry. The completely dried PVDF membrane was placed in 1×TBST buffer containing 5% (w / v, g / 100 mL) skim milk powder and incubated at room temperature (25℃) for 1 h for blocking. After blocking, the PVDF membrane was washed with 1×TBST buffer for 10 min. Primary antibody incubation: The purified PPP2R5D S80 phosphorylated specific antibody was diluted with 1×TBST buffer containing 5% (w / v, g / 100mL) skim milk powder at a dilution factor of 1:2K to obtain the primary antibody dilution solution; 1mL of the primary antibody dilution solution was added to the PVDF membrane and incubated at room temperature (25℃) for 1h; after incubation, the PVDF membrane was washed 3 times with 1×TBST buffer for 10min each time; Secondary antibody incubation: The secondary antibody (HRP goat anti-rabbit, purchased from CST) was diluted with 1×TBST buffer containing 5% (w / v, g / 100mL) skim milk powder at a dilution factor of 1:10K to obtain the secondary antibody dilution solution; 2mL of the secondary antibody dilution solution was added dropwise to the PVDF membrane and incubated at room temperature (25℃) for 45min; after incubation, the PVDF membrane was washed 3 times with 1×TBST buffer for 10min each time; Exposure: After blotting the 1×TBST buffer on the PVDF membrane with filter paper, add 500μL of ECL developing solution (A solution: B solution = 1:1, v / v, purchased from Biosharp) to the PVDF membrane, expose for 30s, and detect the binding of peptides and antibodies.

[0052] The results of the Dot blot positive detection limit are shown in [the table]. Figure 3 .like Figure 3 As shown, the positive detection limits of both Ab2 and Ab4 can reach 4 ng. Furthermore, Ab2 and Ab4 have strong binding to S80 peptide A and S80 peptide B. Among them, Ab4 has almost no binding to PPP2R5D peptide and S81 peptide, while Ab2 binds to these two negative peptides.

[0053] 4.3 Identification by Western blot The purified PPP2R5D S80 phosphorylated specific antibody Ab4 was identified using Western blot; the steps of the Western blot method are as follows: Preparation of cell lysis buffer: Add 10 μL of 100× protease inhibitor (purchased from Sigma) and 10 μL of 100× phosphatase inhibitor (purchased from MCE) to each 1 mL of RIPA lysis buffer (purchased from Beyotime), mix thoroughly, and place on ice for later use to obtain cell lysis buffer; Cell lysis: Primary brown preadipocytes (mouse primary brown preadipocytes, obtained from C57BL / 6 mice purchased from Guangdong Provincial Medical Laboratory Animal Center; isolation method as described in the literature "Klein J, Fasshauer M, Ito M, Lowell BB, Benito M, Kahn CR. beta(3)-adrenergic stimulation differentially inhibits insulin signaling and decreases insulin-induced glucose uptake in brown adipocytes. J Biol Chem. 1999 Dec 3;274(49):34795-802.") isolated from mouse brown adipose tissue were lysed at 1×10⁻⁶. 6Inoculate 10 cells / well into 6-well plates (each well contains 2 mL of DMEM medium, purchased from Gibco) and incubate at 37°C in a 5% (v / v) CO2 incubator. After confluence, brown adipocytes are induced to differentiate into adipocytes (the induction method is described in the literature "Klein J, Fasshauer M, Ito M, Lowell BB, Benito M, Kahn CR. beta(3)-adrenergic stimulation differentially inhibits insulin signaling and decreases insulin-induced glucose uptake in brown adipocytes. J Biol Chem. 1999 Dec"). 3;274(49):34795-802.”); After induction of differentiation, the culture medium was discarded, and the wells were washed three times with PBS buffer (purchased from Gibco) pre-cooled to 4°C; after washing, the cell lysis buffer was added to the 6-well plate at a rate of 200 μL / well, and the plate was incubated on ice for 5 min to fully lyse the cells; after lysis, adherent cells were scraped off with a cell scraper, and all cells and cell lysis buffer were transferred to a clean EP tube, vortexed and incubated on ice for 20 min to fully lyse the cells and obtain the lysis product; after centrifugation of the lysis product at 4°C and 12000 rpm for 15 min, the supernatant was transferred to a new EP tube; Tissue lysis: 50 mg of mouse brown adipose tissue (BAT, from C57BL / 6 mice purchased from Guangdong Provincial Medical Laboratory Animal Center) was placed in a 2 mL grinding tube; 400 μL of cell lysis buffer and 3 grinding beads (purchased from Servicebio) were added to the grinding tube, and the mixture was homogenized in a cryogenic homogenizer (75 Hz, -75 ℃) for 150 s until no visible debris was found in the tissue, thus obtaining the homogenate product; the homogenate product was centrifuged at 4 ℃ and 12000 rpm for 15 min, and the supernatant was transferred to a new EP tube; Sample preparation: After determining the BCA protein concentration in the supernatant (purchased from Beyotime), all samples were adjusted to the same concentration (2.2 mg / mL) using lysis buffer (purchased from Beyotime). Then, 4× loading buffer (purchased from Takara) was added to the samples and incubated in a metal bath at 100℃ for 10 min to obtain the protein sample to be tested. Protein electrophoresis and transfer: The protein sample and protein marker (Thermo Scientific) were sequentially added to the wells of a 10% (w / v, g / 100mL) SDS-polyacrylamide pre-prepared gel. Electrophoresis was performed at a constant voltage of 80V for 30 min, followed by a constant voltage of 100V for 1 h. Electrophoresis was stopped when the protein marker showed the appropriate molecular weight interval for the target protein. The PVDF membrane (Merck) was activated by immersing it in methanol for 1 min. The pre-prepared gel plate was removed from the electrophoresis tank and pried open, and the gel was temporarily stored in the electrotransfer buffer. On the transfer apparatus, the following components were placed sequentially from negative to positive: sponge pad, filter paper, gel, membrane, filter paper, sponge pad. Air bubbles were removed during placement, and the PVDF membrane completely covered the gel. The electrotransfer apparatus was connected, and the electrotransfer parameters were constant current 200mA for 120 min. The electrophoresis tank was covered with crushed ice to cool it down. Blocking: Wash the PVDF membrane once with 1×TBST buffer; after washing, place the PVDF membrane in 1×TBST buffer containing 5% (w / v, g / 100mL) skim milk powder and incubate at room temperature (25℃) for 1h for blocking; after blocking, wash the PVDF membrane 3 times with 1×TBST buffer for 5min each time. PPP2R5D S80 antibody incubation: The purified PPP2R5DS80 phosphorylated specific antibody was diluted with primary antibody dilution buffer (purchased from Beyotime) at a dilution factor of 1:1K to obtain the primary antibody dilution buffer; 5 mL of the primary antibody dilution buffer was added to the PVDF membrane and incubated at 4℃ for 16 h; after incubation, the PVDF membrane was washed 3 times with 1×TBST buffer for 5 min each time; Secondary antibody incubation: The secondary antibody (HRP goat anti-rabbit, purchased from CST) was diluted with 1×TBST buffer containing 5% (w / v, g / 100mL) skim milk powder at a dilution factor of 1:5K to obtain the secondary antibody dilution solution; 5mL of the secondary antibody dilution solution was added dropwise to the PVDF membrane and incubated at room temperature (25℃) and 40rpm for 1h; after incubation, the PVDF membrane was washed 3 times with 1×TBST buffer for 5min each time; Exposure of PPP2R5D S80 phosphorylated protein: After laying the PVDF membrane flat on the developing tray, add 500 μL of ECL developer (A solution: B solution = 1:1, v / v, purchased from Biosharp) to the PVDF membrane and expose for 10 seconds; Antibody stripping: Rinse the PVDF membrane exposed to PPP2R5D S80 phosphorylated protein three times with 1×TBST buffer, 5 min each time; after rinsing, first put the PVDF membrane into antibody stripping solution (purchased from Thermo) and rinse on a shaker at 300 rpm for 30 min, then rinse three times with 1×TBST buffer, 5 min each time. Blocking: Place the PVDF membrane in 1×TBST buffer containing 5% (w / v, g / 100mL) skim milk powder and incubate at room temperature (25℃) for 1 h for blocking; after blocking, wash the PVDF membrane 3 times with 1×TBST buffer for 5 min each time; PPP2R5D total protein antibody incubation: The PPP2R5D antibody (purchased from Proteintech) was diluted with primary antibody dilution buffer (purchased from Beyotime) at a dilution factor of 1:1K to obtain the primary antibody dilution buffer; 5 mL of the primary antibody dilution buffer was added to the PVDF membrane and incubated at 4°C for 16 h; after incubation, the PVDF membrane was washed 3 times with 1×TBST buffer for 5 min each time; Secondary antibody incubation: The secondary antibody (HRP goat anti-rabbit, purchased from CST) was diluted with 1×TBST buffer containing 5% (w / v, g / 100mL) skim milk powder at a dilution factor of 1:5K to obtain the secondary antibody dilution solution; 5mL of the secondary antibody dilution solution was added dropwise to the PVDF membrane and incubated at room temperature (25℃) and 40rpm for 1h; after incubation, the PVDF membrane was washed 3 times with 1×TBST buffer for 5min each time; PPP2R5D total protein exposure: After laying the PVDF membrane flat on the developing plate, add 500 μL of ECL developing solution (A solution: B solution = 1:1, v / v, purchased from Biosharp) to the PVDF membrane and expose it in the Bio-Rad imager (20s exposure for brown adipose tissue samples and 10s exposure for brown adipocyte samples).

[0054] The results of Western blot detection of antigen-antibody binding are shown below. Figure 4 .like Figure 4 As shown, the PPP2R5D S80 phosphorylation-specific antibody Ab4 can recognize endogenous PPP2R5D (S80ph) by Western blot, with accurate band size.

[0055] Example 2: Application of PPP2R5D S80 phosphorylation-specific antibody in the detection of phosphorylation level at PPP2R5D S80 site 1. Establishment of detection methods The phosphorylation level of the PPP2R5D S80 site in the test sample was detected using the PPP2R5D S80 phosphorylation-specific antibody prepared in Example 1. The detection method is described in section 4.3 of Example 1. The difference is that the primary antibody was diluted 1:500 and incubated at room temperature (25°C) for 2 hours, and the secondary antibody was diluted 1:10K and incubated at room temperature (25°C) for 45 minutes. Furthermore, the steps of tissue lysis, antibody stripping, and subsequent antibody stripping were not required.

[0056] 2. Validation of the detection method Western blotting was performed on NIH-3T3 cells (purchased from ATCC) and NIH-3T3 cells treated with phorbol 12-tetradecanoate 13-acetate (PMA, CAS: 16561-29-8) using the detection method in step 1 to detect the phosphorylation level of PPP2R5D S80 site. The results are shown in [Figure 1]. Figure 5 NIH-3T3 cells were cultured in DMEM medium (purchased from Gibco). The PMA treatment concentration was 200 mM and the treatment time was 10 min. PMA treatment was performed by adding PMA to the culture medium of NIH-3T3 cells and incubating at 37°C.

[0057] Western blot analysis of PPP2R5D S80 phosphorylation level results are shown below. Figure 5 . Figure 5 The results show that the PPP2R5D S80 phosphorylation-specific antibody Ab4 prepared in Example 1 can be used to detect the phosphorylation level of the PPP2R5D S80 site with good specificity and high accuracy.

[0058] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antigen for preparing PPP2R5D S80 phosphorylation-specific antibodies, characterized in that, The antigen has the amino acid sequence shown in SEQ ID NO. 2: C KERRQ-(phospho)S-SFPFNLN.

2. The antigen as described in claim 1, characterized in that, The antigen is coupled with a carrier protein; the carrier protein is: hemocyanin, bovine serum albumin, rabbit serum albumin, human serum albumin, chicken ovalbumin and / or glutathione S-transferase.

3. A method for preparing PPP2R5D S80 phosphorylated specific antibody, characterized in that, The method is as follows: immunizing an animal with the antigen described in claim 1 or 2 to obtain an immunized animal; and extracting the PPP2R5D S80 phosphorylated specific antibody from the serum of the immunized animal.

4. The method as described in claim 3, characterized in that, The immunization process is as follows: six immunizations are administered on days 1, 7, 14, 21, 28, and 40, respectively. Blood samples are collected and serum is separated on days 35 and 47. For the first immunization, physiological saline and the antigen described in claim 1 or 2 are mixed at a ratio of 1 mL: 0.5 to 1.5 mg to dilute the antigen described in claim 1 or 2, resulting in an antigen dilution. The antigen dilution and adjuvant are mixed at a volume ratio of 1:0.5 to 1.5 and injected subcutaneously at two points on both shoulders and at two points in the muscles of both hind legs of the animal. The immunization dose is 0.25 mg / animal to 1.5 mg / animal. For the second to sixth immunizations, the immunization dose is 0.1 mg / animal to 0.75 mg / animal. The remaining steps are the same as for the first immunization.

5. A PPP2R5D S80 phosphorylation-specific antibody, characterized in that, The PPP2R5D S80 phosphorylated specific antibody is prepared by the method of claim 3 or 4.

6. The use of the antigen of claim 1 or 2 or the PPP2R5D S80 phosphorylation-specific antibody of claim 5 in the preparation of a product for detecting the phosphorylation level of the PPP2R5D S80 site.

7. A product for detecting phosphorylation levels at the PPP2R5D S80 site, characterized in that, The product comprises a PPP2R5D S80 phosphorylated specific antibody obtained by immunizing an animal with the antigen of claim 1 or 2; or, the product comprises the PPP2R5D S80 phosphorylated specific antibody of claim 5.

8. A method for detecting phosphorylation level at the PPP2R5D S80 site, characterized in that, The method is not for the purpose of diagnosing or treating diseases; the method is to use the product of claim 7 to test the sample to be tested.

9. The application of the antigen of claim 1 or 2, the PPP2R5D S80 phosphorylation-specific antibody of claim 5, or the product of claim 7 in the detection of phosphorylation levels at the PPP2R5D S80 site, characterized in that, The application is for purposes other than disease diagnosis and treatment.

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