Ab3-2 antibody and application thereof

Abstract

The invention relates to the technical field of antibody preparation, in particular to an Ab3-2 antibody and application thereof. The Ab3-2 antibody is obtained by purifying serum after a rabbit is immunized by FHL2K17la modified polypeptide B, and the sequence of the FHL2K17la modified polypeptide B is as shown in SEQ ID No. 1. The ELISA (enzyme-linked immuno sorbent assay) of the Ab3-2 antibody provided by the invention is positive (OD450gt; 1.0) the antibody dilution is greater than 54,000, and the signal of the antibody is 10 times stronger than that of a recognition negative non-modified polypeptide; the Dot blot positive detection limit reaches 1 ng, and the signal is 10 times stronger than the signal for identifying the non-modified polypeptide; other application results show that the Ab3-2 antibody can identify endogenous FHL2 (K17la) polypeptide through WB, and the band size is accurate.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, and in particular to an Ab3-2 antibody and its applications. Background Technology

[0002] FHL2 (Four and a Half LIM Domains 2) is a multifunctional scaffold protein that participates in cell signal transduction and gene expression regulation primarily through interactions with various signaling molecules, transcription factors, and structural proteins. Its pleiotropic nature enables it to play a crucial role in development, tissue homeostasis, and disease progression. In-depth research into the regulatory mechanisms of FHL2 can provide new therapeutic strategies for related diseases.

[0003] Post-translational modifications (PTMs) refer to the processes by which proteins, after synthesis, undergo alterations in structure, function, location, or stability through the covalent addition or removal of chemical groups or peptides. PTMs are a core mechanism for cellular regulation of protein activity and signal transduction, playing a crucial role in the pathogenesis of diseases such as cancer, neurodegenerative diseases, and metabolic diseases.

[0004] Lactic acidification, as a novel post-translational modification of proteins, has been shown to be involved in the development and progression of various diseases. However, the lack of lactation antibodies against FHL2 currently limits its research in the medical field. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an Ab3-2 antibody and its applications. The Ab3-2 antibody provided by this invention exhibits an ELISA-positive (OD450>1.0) antibody dilution greater than 54,000, and is 10 times stronger than the signal recognizing negative unmodified peptides. The Dot blot detection limit reaches 1 ng, and is also 10 times stronger than the signal recognizing unmodified peptides. Other application results indicate that the Ab3-2 antibody can accurately recognize endogenous FHL2 (K17la) peptides via Western blotting.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides an Ab3-2 antibody, which is obtained by immunizing rabbits with FHL2 K17la-modified polypeptide B and then purifying it from the serum. The sequence of the FHL2 K17la-modified polypeptide B is shown in SEQ ID No. 1.

[0008] This invention also provides the application of the Ab3-2 antibody described above in the preparation of reagents that specifically recognize endogenous FHL2 (K17la) peptides.

[0009] The beneficial effects of this invention are:

[0010] 1. ELISA positive (OD450>1.0) antibody dilution greater than 54,000, and 10 times stronger than the signal recognizing negative unmodified peptides;

[0011] 2. The dot blot detection limit reached 1 ng, and was 10 times stronger than the detection of unmodified peptide signals; other application results showed that:

[0012] 3. The Ab3-2 antibody can recognize the endogenous FHL2 (K17la) peptide by Western blotting, with accurate band size.

[0013] This invention verifies the antibody's ability to recognize targets through Western blotting, representing an innovative application scenario: the novel use of antibodies to recognize new lactate modification sites has not been disclosed. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0015] Figure 1 For antibody development roadmap and timeline;

[0016] Figure 2 This is the mass spectrometry detection result of FHL2(K17la) peptide A;

[0017] Figure 3 This is the mass spectrometry detection result of FHL2(K17la) peptide B;

[0018] Figure 4 Mass spectrometry results of FHL2(K18la) modified control peptide C;

[0019] Figure 5 This is the result of mass spectrometry detection of unmodified FHL2 peptides;

[0020] Figure 6 These are the initial screening results for serum WB.

[0021] Figure 7 This is the result of antibody Dot blot detection;

[0022] Figure 8 This refers to the results of antibody Western blot detection;

[0023] Figure 9 This is a purification flowchart. Detailed Implementation

[0024] This invention provides an Ab3-2 antibody, which is obtained by immunizing rabbits with FHL2 K17la-modified polypeptide B and then purifying it from the serum. The sequence of the FHL2 K17la-modified polypeptide B is shown in SEQ ID No. 1.

[0025] This invention also provides the application of the Ab3-2 antibody described above in the preparation of reagents that specifically recognize endogenous FHL2 (K17la) peptides.

[0026] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Instruments and reagents

[0029] 1.1. Instruments: Centrifuge tubes; centrifuge; shaker; electrophoresis equipment; incubation tank;

[0030] 1.2 Reagents and Materials

[0031] Table 1 Reagents and Materials

[0032]

[0033]

[0034] 2. Experimental Procedure and Methods

[0035] 2.1 Preliminary Preparations

[0036] (1) Animals: 4 healthy New Zealand rabbits.

[0037] (2) Antigen design and quality control: Based on the protein information, design and synthesize two modified peptides and one unmodified peptide. The synthesized peptides are detected by mass spectrometry, and the mass spectrometry results are qualified.

[0038] 2.2 Peptide Conjugation

[0039] Two modified peptides were conjugated with KLH and used for rabbit immunization.

[0040] 2.3 Rabbit Immunization

[0041] (1) Prepare immunization materials

[0042] The immunogen was diluted with physiological saline and then mixed with the appropriate adjuvant at a 1:1 volume ratio. After the antigen and adjuvant were thoroughly mixed to form a stable emulsion, the antigen mixture was drawn up with a syringe and injected subcutaneously at two points on both shoulders and intramuscularly at two points on both hind legs of the rabbit. Approximately one-quarter of the volume of immunogen was injected into each area. This allows the immunogen to persist, thereby enhancing the immune response.

[0043] (2) Immunity

[0044] Each rabbit was immunized a total of 6 times, on day 1, day 7, day 14, day 21, day 28, and day 40 respectively;

[0045] (3) Blood collection

[0046] First blood draw: On day 28, 2 mL of blood was collected and centrifuged. The supernatant was then sent to the laboratory for serum screening tests, including ELISA or Dot blot.

[0047] Blood was collected 2 / 3 / 4 times: on day 35, day 47 and day 52, blood was collected 30 mL each time. After centrifugation, the supernatant was collected and sent to the laboratory for serum screening tests, including ELISA or Dot blot.

[0048] The serum that tested positive was purified.

[0049] 2.4 Antibody purification

[0050] (1) Prepare the protein A affinity column:

[0051] Typically, 5 mL or 10 mL of protein A packing material is selected. An equal volume of packing material and PBS buffer solution are mixed and stirred, and air is removed from the packing material by vacuuming.

[0052] Slowly add protein A packing material into the glass column to prepare the chromatography column. Avoid letting the column dry out during this process. After perfusion, equilibrate the column with 10 volumes of pre-cooled PBS buffer.

[0053] (2) Protein A affinity chromatography:

[0054] After filtering the serum, it is loaded onto a pre-equilibrated protein A chromatography column. To detect the binding efficiency between the antiserum and the packing material, the eluent should be retained.

[0055] The column was washed with PBS buffer and then eluted with 150 mM glycine buffer. The eluent was collected and neutralized with neutralizing buffer to adjust the pH to 7.0.

[0056] (3) Enrichment of target antibodies:

[0057] The crude pure IgG obtained after purifying protein A was loaded onto a well-balanced antigen-peptide affinity chromatography column to specifically enrich the target antibody.

[0058] (4) Remove non-specific antibodies:

[0059] The target antibody obtained in the previous step is loaded onto an unmodified affinity chromatography column, and the eluent (FT3) is collected directly to remove non-specific antibody components.

[0060] (5) Antibody preservation:

[0061] The protein content was determined. 10% glycerol was added to preserve the antibody. The purified antibody was aliquoted and stored at -20°C.

[0062] 2.5 Antibody Quality Control

[0063] The antibody was validated using ELISA, Dot blot, and WB.

[0064] 2.5.1 ELISA detection:

[0065] (1) Antigen coating:

[0066] Dilute the antigen with coating buffer and add it to the ELISA plate at a rate of 50 μg / well. Incubate overnight at 4°C or later.

[0067] Incubate in a 37℃ oven for 2 hours.

[0068] (2) Washing the plate:

[0069] Take out the ELISA plate coated the day before and wash it three times with 1×TBST.

[0070] (3) Closed:

[0071] Add 1% BSA blocking solution to the cleaned ELISA plate, incubate at 37°C for 1 hour, and then wash 3 times.

[0072] (4) Primary antibody incubation:

[0073] Perform 3-fold serial dilutions of the antibody, starting at a 1:1K ratio. Adjust the dilution volume as needed. Add the solutions sequentially to the microplate, incubate at 37°C for 1.5 hours, and wash three times.

[0074] (5) Secondary antibody incubation:

[0075] Dilute the secondary antibody to 1:10K with 1% BSA blocking buffer, incubate at room temperature or 37°C for 45 min, and then wash three times.

[0076] (6) Color development:

[0077] After adding TMB colorimetric solution and waiting 10 minutes, the reaction was terminated with 1M sulfuric acid. The data was then read using an ELISA reader.

[0078] 2.5.2 Dot blot detection:

[0079] (1) Spotting:

[0080] Uncrosslinked antigen peptides were spotted onto PVDF membranes in gradients of 1 ng, 4 ng, 16 ng, and 64 ng.

[0081] (2) Closed:

[0082] After the membrane surface dries, add the blocking solution and seal at room temperature for 60 minutes.

[0083] (3) Washing:

[0084] Wash with 1×TBST for 10 minutes.

[0085] (4) Primary antibody incubation:

[0086] Dilute the antibody with 5% skim milk powder, incubate at room temperature for 2 hours, and then wash three times with 1×TBST for 10 minutes each time.

[0087] (5) Secondary antibody incubation:

[0088] Select the corresponding rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 1 hour, and then wash three times with 1×TBST for 10 minutes each time.

[0089] (6) Expose the washed film after adding a color developing substrate.

[0090] 2.5.3 Western blot analysis:

[0091] (1) Cell lysis:

[0092] Cells or tissues are selected for testing according to experimental requirements. Different lysis methods are selected for different samples to obtain the total protein of the sample, and the protein concentration is determined by the BCA method.

[0093] (2) Protein electrophoresis and transfer:

[0094] Protein electrophoresis: Select a separating gel of appropriate concentration according to the molecular weight of the target protein. Add 10% ammonium persulfate and TEMED at the end when preparing the gel. Use 4.7 ml of separating gel per plate. Load 40 μg of sample per well and perform electrophoresis; the electrophoresis conditions are: stacking gel 80 V, separating gel 120 V.

[0095] Wet transfer: The transfer solution is cooled before transfer, and the gel, membrane, and filter paper are laid in the transfer solution in a sandwich structure to avoid air bubble formation. The transfer voltage is 120V.

[0096] (3) Closed:

[0097] Add the transferred membrane to the blocking buffer and incubate at room temperature for 60 minutes. After incubation, wash with 1×TBST for 10 minutes.

[0098] (4) Primary antibody incubation:

[0099] Dilute the antibody with 5% skim milk powder, incubate at room temperature for 2 hours, and then wash three times with 1×TBST for 10 minutes each time.

[0100] (5) Secondary antibody incubation:

[0101] Select the corresponding rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K (i.e., 1:10000), incubate at room temperature for 1 hour, and then wash 3 times with 1×TBST for 10 minutes each time.

[0102] (6) Expose the washed film after adding a color developing substrate.

[0103] Project delivery

[0104] Delivery standards:

[0105] (1) Antibody titer (OD) measured by enzyme-linked immunosorbent assay (ELISA) 450 >1.0) greater than 1:50K.

[0106] (2) Dot blot method: the detection limit of antibody for modified antigen peptides reaches 20ng; the hybridization signal of antibody for modified peptides is 10 times stronger than that for unmodified peptides.

[0107] (3) One of the three methods, Western blot, immunohistochemistry or immnocytochemistry, can detect endogenous target proteins.

[0108] Deliverables:

[0109] (1) Peptides: 1 mg each of 2 modified peptides [FHL2(K17la) peptide A and FHL2(K17la) peptide B]; 1 mg of 1 modified control peptide; and 1 mg of 1 unmodified peptide.

[0110] (2) Antibody:

[0111] Table 2 Antibody Information

[0112]

[0113] For customized antibody development roadmap and expected timeline, please see [link / reference]. Figure 1 .

[0114] 3. Results Report

[0115] 3.1 Immunogen Design

[0116] Based on the protein sequence and modification type, two modified antigen peptides were designed and synthesized for animal immunization, purification, and detection; at the same time, one unmodified control peptide was designed and synthesized for purification and detection.

[0117] Table 3. Peptide Information

[0118]

[0119] Note: (lactyl)K indicates that the amino acid is lactated.

[0120] 3.2 Immunogen Mass Spectrometry Detection

[0121] like Figure 2-5 As shown, the sequences of the three peptides are accurate. The vertical axis represents the intensity of the ion peak, and the horizontal axis represents the mass-to-charge ratio. The difference between the measured and theoretical masses of the three peptides is within 10 ppm, indicating that the sequences are error-free.

[0122] 3.3 Serum screening

[0123] After multiple immunizations, small amounts of serum were collected from four SPF experimental-grade New Zealand white rabbits and subjected to ELISA testing to preliminarily assess the titer and specificity of the antiserum.

[0124] 3.3.1 Serum initial screening ELISA detection

[0125] Rabbits immunized with FHL2(K17la) peptide A were labeled R1 and R2, respectively, while rabbits immunized with FHL2(K17la) peptide B were labeled R3 and R4. The ELISA results are shown in Table 4.

[0126] Table 4. Serum ELISA results of rabbits

[0127]

[0128] ELISA results from four rabbit serum samples showed that the titers of the modified peptides recognized by R1, R2, R3, and R4 were all greater than 1:16K (OD>1).

[0129] 3.3.2 Serum initial screening Western blot (WB) detection

[0130] Endogenous FHL2 (K17la) protein was detected in rabbit serum after immunization using Western blotting. The results are as follows: Figure 6Western blot (WB) results showed that bands with a theoretical molecular weight of approximately 30 kDa of the target protein could be detected at R1, R2, R3, and R4 (Lane 1:R1, Lane 2:R2, Lane 3:R3, Lane 4:R4, Lane+:β-Actin, Lane*:H3; serum dilution: 1 / 100, secondary antibody dilution: 1 / 10000). We selected serum from rabbits with R3 for purification.

[0131] 3.4 Antibody Quality Control

[0132] Sufficient rabbit serum was collected and purified using Protein A and immunogenic peptide column affinity chromatography. The purified antibodies were labeled Ab3-1 and Ab3-2, respectively. The purified antibodies were then analyzed by ELISA, Dot Blot, and Western blotting.

[0133] 3.4.1 Antibody ELISA Detection

[0134] In 96-well ELISA plates coated with antigen-modified peptides and control peptides, antibodies were added and incubated at different dilution ratios (e.g., 1:54K, 1:162K). Then, enzyme-labeled secondary antibody and TMB substrate were applied, and the binding of the peptides and antibodies was detected by colorimetric analysis. ELISA results showed that Ab3-1 and Ab3-2 had a recognition ability for at least one modified peptide at a dilution of 1:50K, and did not recognize unmodified negative peptides (Table 5).

[0135] Table 5. Antibody ELISA Detection Results

[0136]

[0137] 3.4.2 Antibody Dot blot detection

[0138] Different doses (e.g., 4 ng, 16 ng) of modified peptides were compared with those of unmodified peptides. Figure 7 Immobilized on a solid membrane, the peptides were incubated with antibodies, followed by the addition of enzyme-labeled secondary antibody and chemiluminescent substrate. The binding of the peptides and antibodies was then detected. Dot blot results showed that Ab3-1 and Ab3-2 antibodies had virtually no binding to the negative unmodified peptides but exhibited strong binding to the positive peptides (Lane 1: FHL2(K17la) peptide A, Lane 2: FHL2(K17la) peptide B, Lane 3: FHL2(K18la) peptide C, Lane 4: FHL2 unmodified peptide; primary antibody: 1 / 2000, secondary antibody: 1 / 10000). See attached table. Figure 7 .

[0139] 3.4.3 Antibody Western blot detection

[0140] Dot / ELISA positive antibodies Ab3-1 and Ab3-2 were treated with NIH-3T3+Lac Na (this drug can induce increased modification, 25 mM, 24 h), and the resulting lysates and Mouse heart lysates were analyzed by Western blotting. Ab3-1 and Ab3-2 bands of approximately 30 kDa were detectable in both NIH-3T3+Lac Na and Mouse heart lysates, and the band signal changes were significant before and after drug treatment (comparing NIH-3T3+Lac Na with NIH-3T3) (Lane 1: NIH-3T3, Lane 2: NIH-3T3+Lac Na, Lane 3: Mouse heart; primary antibody dilution: 1 / 500, secondary antibody dilution: 1 / 10000). All bands represent the target protein FHL2 (K17la). Results are shown in […]. Figure 8 .

[0141] 3.4.4 Summary of antibody quality control results:

[0142] Based on the antibody detection analysis above, Ab3-2 is recommended as the specific antibody for Anti-FHL2 (K17la). The Ab3-2 antibody results are as follows:

[0143] (1) ELISA positive (OD450>1.0) antibody dilution greater than 54,000 and 10 times stronger than the signal recognizing negative unmodified peptides;

[0144] (2) The positive detection limit of Dot blot reaches 1 ng, which is 10 times stronger than the detection of unmodified peptide signals;

[0145] (3) Other application results show that the Ab3-2 antibody can recognize endogenous FHL2 (K17la) by WB with accurate band size.

[0146] 3.5 Antibody Preservation

[0147] (1) The antibody was stored in PBS buffer (pH 7.4) containing 50% glycerol and 0.01% procolin 300 by volume.

[0148] (2) After receiving the Ab3-2 antibody, centrifuge at 10,000 rpm for 20 seconds and store in a -20 degree freezer at a concentration of 94 mg / ml;

[0149] (3) Antibodies can be dispensed into small tubes to avoid repeated freeze-thaw cycles;

[0150] (4) The antibody is guaranteed for one year of use from the date of receipt.

[0151] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An Ab3-2 antibody, characterized in that, The Ab3-2 antibody is purified from serum of a rabbit immunized with FHL2 K17la modified polypeptide B, and the sequence of the FHL2 K17la modified polypeptide B is shown as SEQ ID No.

1.

2. Use of the Ab3-2 antibody of claim 1 in preparation of a reagent specifically recognizing an endogenous FHL2 (K17la) polypeptide.

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