Galectin-3 antigenic epitope peptides, antigens, antibodies and chemiluminescent kits
Antibodies were prepared by conjugating Galectin-3 antigenic epitope peptides with carrier proteins and detected by chemiluminescence, which solved the problem of insufficient detection sensitivity of Galectin-3 and achieved detection results with high sensitivity, stability and convenience.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2024-12-17
- Publication Date
- 2026-05-15
AI Technical Summary
Existing Galectin-3 detection methods lack sufficient sensitivity, making it difficult to achieve early disease diagnosis and personalized treatment plans. Furthermore, the test results are easily affected by interfering substances, and there is a lack of convenient and stable detection methods.
An antigen was prepared by conjugating the epitope peptide of Galectin-3 with a carrier protein, and a highly sensitive antibody was prepared by combining it with chemiluminescence. The content of Galectin-3 was detected by a competitive method and the detection was performed using a chemiluminescence kit.
It achieves highly sensitive Galectin-3 detection, capable of detecting extremely low concentrations of target substances, ensuring the accuracy and stability of detection results, facilitating large-scale sample screening and on-site testing, and improving detection efficiency and accuracy.
Smart Images
Figure CN119751640B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biodetection technology, and more particularly to Galectin-3 antigenic epitope peptides, antigens, antibodies, and chemiluminescent reagent kits. Background Technology
[0002] Galectin-3 (Gal-3) detection has shown broad application prospects and significant clinical importance in the medical field. As a protein that plays a key role in various pathological processes, Gal-3 detection is of great value for the diagnosis and treatment of cardiovascular diseases, kidney diseases, tumors, and nervous system diseases.
[0003] In cardiovascular diseases, particularly heart failure, Gal-3 serves as a biomarker that can accurately predict patients' hospitalization and mortality risks, providing strong evidence for clinical decision-making. Simultaneously, in kidney diseases, Gal-3 level detection aids in the diagnosis of idiopathic membranous nephropathy, lupus nephritis, and other conditions, and can assess the degree of kidney damage, providing important reference for early intervention and treatment of kidney diseases.
[0004] Furthermore, the overexpression of Gal-3 in various malignant tumors makes it a potential tool for tumor diagnosis, staging, and prognostic assessment. In neurological diseases, such as Alzheimer's disease, Gal-3 detection also aids in disease diagnosis and treatment monitoring.
[0005] Galectin-3 testing enables early disease diagnosis, allowing for timely intervention and treatment. Furthermore, Galectin-3 levels are closely related to the prognosis of many diseases; regular testing can assess patient outcomes and provide a basis for developing personalized treatment plans. During treatment, monitoring changes in Galectin-3 levels can also reflect the effectiveness of treatment, allowing for timely adjustments to the treatment plan to improve outcomes. Therefore, Galectin-3 testing and its sensitivity are particularly important. Summary of the Invention
[0006] The purpose of this application is to provide a new antigen, antibody, and related kit for the detection of Galectin-3.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] An antigenic epitope peptide of Galectin3, wherein the antigenic epitope peptide is the antigenic epitope sequence of Galectin-3, amino acids 196-225, and the amino acid sequence is shown in SEQ ID NO: 1.
[0009] Preferably, the steps of the method for synthesizing the antigenic epitope peptide of Galectin3 are as follows:
[0010] S1: Deprotection:
[0011] Using resin as a solid support, the resin was swelled in dimethylformamide (DMF), and the first amino acid (i.e., C-terminal amino acid H) in the antigenic epitope sequence 196-225 of Galectin-3 was coupled to the solid support by the linker diisopropylcarbazolium.
[0012] S2: Cleaning:
[0013] DMF containing 20% piperidine was used as a solution protectant to remove the amino protecting group, which was an Fmoc protecting group; DMF was then used to wash the resin to ensure that all deprotecting agents were removed. The next amino acid (the next monomer at the N-terminus) must first have a protecting group (such as Fmoc);
[0014] S3: Amino acid coupling
[0015] The amino acid is coupled to the deprotected amino acid using a coupling agent, and repeated washing is performed to remove unreacted reagents and byproducts.
[0016] Repeat steps S2 and S3 to couple all amino acids sequentially until the entire polypeptide sequence is synthesized.
[0017] S4: Cutting and removing protection:
[0018] The synthesized peptide chains are cut from the solid support using a cleavage solution, and all remaining protecting groups are removed.
[0019] The cutting solution used was a mixture of trifluoroacetic acid (TFA) and other reagents.
[0020] S5: Separation and purification:
[0021] The peptides were separated by cold ether precipitation; the peptides were further purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to ensure that they met the required purity requirements.
[0022] Preferably, the coupling agent is N,N'-diisopropylcarbodiamide (DIC).
[0023] Preferably, the cutting fluid has a formulation of TFA:water:TIPS:phenol = 95:2.5:2.5:1.
[0024] This application also provides an antigen of Galectin-3, obtained by conjugating the antigenic epitope peptide of Galectin-3 described above with a carrier protein.
[0025] Preferably, the carrier protein is hemocyanin (KLH); and during the coupling process, the cross-linking agent glutaraldehyde is used.
[0026] This application also provides an antibody against Galectin-3, the preparation method of which is shown below:
[0027] Mouse immunization:
[0028] For four immunizations, for the first immunization, 100 μg of immunogen was mixed with Freund's complete adjuvant in a 1:1 ratio and the immunogen was injected into 6-8 week old female BALB / C mice using a multi-point injection method on the back.
[0029] From the second to the fourth immunization, 50 μg of immunogen was injected intraperitoneally into mice.
[0030] The immunogen is the Galectin-3 antigen described above;
[0031] Antibody preparation:
[0032] BALB / c mice that achieved the target serum titer were subjected to an immunization shock after the fourth immunization. Serum from the immunized mice was collected, and antibodies were purified using a protein A / G column.
[0033] Preferably, the first to fourth immunizations are administered two weeks apart.
[0034] This application also provides the application of a Galectin-3 antibody in the preparation of a Galectin-3 detection kit, wherein the Galectin-3 antibody is the aforementioned Galectin-3 antibody, and the detection kit is a chemiluminescent detection kit.
[0035] The aforementioned chemiluminescence immunoassay kit for detecting Galectin-3 content offers several advantages over conventional kits. Firstly, it boasts high sensitivity, capable of detecting extremely low concentrations of the target substance, making it suitable for trace analysis and ensuring accurate results. Secondly, its reaction system is simple and easy to operate; only the addition of appropriate reagents is required to complete the luminescence reaction, significantly improving detection efficiency. Furthermore, the chemiluminescence kit exhibits high specificity, effectively eliminating interfering substances and guaranteeing accurate results. Thirdly, the kit is stable, reliable, and reproducible, maintaining high sensitivity over extended periods, making it suitable for screening large batches of samples. Finally, the chemiluminescence kit is highly portable, facilitating on-site testing in various locations and enabling rapid acquisition of accurate results. In conclusion, the chemiluminescence immunoassay kit outperforms conventional kits in terms of sensitivity, ease of operation, specificity, stability, and portability. Attached Figure Description
[0036] Figure 1This invention provides an embodiment for screening the antigenic epitopes 196-225 of Galectin-3.
[0037] Figure 2 This invention provides a correlation comparison between the Galectin-3 chemiluminescence detection kit and the control kit from Roche on clinical specimens. Detailed Implementation
[0038] The present invention will be further described in detail below with reference to specific embodiments.
[0039] An antigenic epitope peptide of Galectin-3, please refer to [link / reference]. Figure 1 The antigenic epitope peptide is the antigenic epitope sequence of Galectin-3, amino acids 196-225, as shown in SEQ ID NO: 1.
[0040] The amino acid sequence of the antigen epitope peptide is as follows:
[0041] KPFKIQVLVEPDHFKVAVNDAHLLQYNHRV.
[0042] This application provides a method for synthesizing antigenic epitope peptides, the steps of which are as follows:
[0043] S1: Deprotection:
[0044] Specifically, using resin as a solid-phase carrier, the resin is swelled in dimethylformamide (DMF), and the first amino acid (i.e., C-terminal amino acid H) in the antigenic epitope sequence 196-225 of Galectin-3 is coupled to the solid-phase carrier using the linker diisopropylcarbazolium.
[0045] S2: Cleaning:
[0046] In one embodiment, DMF containing 20% piperidine is used as a solution protectant to remove the amino protecting group, wherein the amino protecting group is an Fmoc protecting group; DMF is used to wash the resin to ensure that all deprotecting agents are removed. The next amino acid (the next monomer at the N-terminus) must first be equipped with a protecting group (such as Fmoc).
[0047] S3: Amino acid coupling
[0048] The amino acid is coupled to the deprotected amino acid using a coupling agent, and repeated washing is performed to remove unreacted reagents and byproducts.
[0049] The coupling reagent is N,N'-diisopropylcarbodiamide (DIC).
[0050] Repeat steps S2 and S3 to couple all amino acids sequentially until the entire polypeptide sequence is synthesized.
[0051] S4: Cutting and removing protection:
[0052] The synthesized peptide chains are cut from the solid support using a cleavage solution, and all remaining protecting groups are removed.
[0053] In one embodiment, the cutting solution used is a mixture of trifluoroacetic acid (TFA) and other reagents (such as water, triisopropylsilane (TIPS) and phenol), with a preferred formulation of TFA:water:TIPS:phenol = 95:2.5:2.5:1.
[0054] S5: Separation and purification:
[0055] The peptides were separated by cold ether precipitation. The peptides were further purified using reversed-phase high-performance liquid chromatography (RP-HPLC) to ensure they met the required purity requirements.
[0056] An antigen of Galectin-3 is obtained by conjugating the antigenic epitope peptide of Galectin-3 described above with a carrier protein.
[0057] In one embodiment, the carrier protein is hemocyanin (KLH); and during the coupling process, the cross-linking agent glutaraldehyde is used.
[0058] Based on the antigen described above, this application also provides an antibody against Galectin-3, the preparation method of which is as follows:
[0059] Immunizing mice:
[0060] In one embodiment, four immunizations are performed. For the first immunization, 100 μg of immunogen is mixed with Freund's complete adjuvant at a 1:1 ratio and the immunogen is injected into 6-8 week old female BALB / C mice using a multi-point injection method on the back.
[0061] From the second to the fourth immunization, 50 μg of immunogen was injected intraperitoneally into mice.
[0062] The initial to fourth immunizations were administered two weeks apart. The immunogens and antigens described above were also mentioned.
[0063] Antibody preparation:
[0064] BALB / c mice that achieved the target serum titer were subjected to an immunization shock after the fourth immunization. Serum from the immunized mice was collected, and antibodies were purified using a protein A / G column.
[0065] This application also provides the application of the antibody described above in the preparation of a Galectin-3 detection kit, wherein the Galectin-3 detection kit is a chemiluminescent assay kit.
[0066] This application also provides a chemiluminescence reagent kit, which includes reagent R1, reagent R2, instructions, and an RFID card. The reagent R1 includes magnetically microparticle-coated Galectin-3 antibody (3 μg / mL) and 0.05 mol / L Tris hydrochloric acid buffer (pH 7.4±0.5) containing 0.5% bovine serum albumin, 1% sucrose, 0.5% trehalose, 0.2% Tween 20, and 0.05% Proclin 300.
[0067] R2 comprises an acridinium ester-labeled Galectin-3 antibody (2 μg / mL) in an aqueous solution containing 0.5% bovine serum albumin, 1% trehalose, 0.93% sodium dihydrogen phosphate, 0.32% disodium hydrogen phosphate, 0.88% sodium chloride, 0.1% Tween 20, and 0.05% Proclin 300.
[0068] Based on the kit described above, this application also provides the method and principle of using the chemiluminescence kit. The chemiluminescence kit described in this application uses a competitive method for detection. It uses a specific antibody labeled with a luminescent agent and a solid-phase carrier coated with the antibody to react with the sample, forming two types of complexes: "solid-phase carrier-antibody-antigen" and "solid-phase carrier-antibody-antigen-luminescent agent". The solid-phase carrier is adsorbed by a magnetic field, and unbound sample and reagents are removed by washing. Finally, a substrate is added to make the complex containing the luminescent agent luminescent reaction. The luminescence intensity is measured. The measured specific antigen concentration is inversely proportional to the luminescence intensity, that is, the lower the specific antigen concentration in the sample, the higher the luminescence intensity.
[0069] Specifically, the method of use includes the following steps:
[0070] S1: Incubation
[0071] Acridinium ester-labeled Galectin-3 antibody, magnetic microparticle-coated Galectin-3 antibody, and the test sample were prepared. Galectin-3 in the test sample binds to both the acridinium ester-labeled Galectin-3 antibody and the magnetic microparticle-coated Galectin-3 antibody, forming immune complexes.
[0072] S2: Cleaning
[0073] After incubation, the immune complexes are adsorbed onto the reaction vessel wall under the influence of the magnetic field, and the unbound substances are washed away.
[0074] S3: Excitation and Computation
[0075] After washing, add the pre-activation solution and activation solution to the reaction mixture.
[0076] The Galectin-3 content in the sample is proportional to the relative luminescence intensity (RLU) detected by the optical system, and the Galectin-3 concentration in the sample is calculated from the calibration curve.
[0077] The Galectin-3 chemiluminescence detection kit used in this example was compared with the kit from Roche, a control manufacturer, on clinical specimens. The data are as follows: Figure 2 As shown.
[0078] The Galectin-3 used in this application is Homo sapiens galectin-3, whose protein sequence is shown in SEQ ID NO: 2.
[0079] Finally, the correlation coefficient R was determined. 2 The value was 0.9907. This indicates that the kit of the present invention can accurately detect Galectin-3 levels, providing a basis for clinical diagnosis and fully meeting the requirements of clinical in vitro diagnostic testing (see...). Figure 2 ).
[0080] In summary, this application provides a kit for detecting Galectin-3 using chemiluminescence immunoassay. Compared to conventional kits, the chemiluminescence kit offers higher sensitivity, capable of detecting extremely low concentrations of the target substance, making it suitable for trace analysis and ensuring accurate results. Secondly, its reaction system is simple and easy to operate; only the addition of appropriate reagents is needed to complete the luminescence reaction, significantly improving detection efficiency. Furthermore, the chemiluminescence kit exhibits good specificity, effectively eliminating interfering substances and ensuring accurate results. Moreover, the kit is stable, reliable, and reproducible, maintaining high sensitivity for extended periods, making it suitable for screening large batches of samples. Finally, the chemiluminescence kit is highly portable, facilitating on-site testing in various locations and enabling rapid acquisition of accurate results. In conclusion, the chemiluminescence detection kit outperforms conventional kits in terms of sensitivity, ease of operation, specificity, stability, and portability.
Claims
1. An antigenic epitope peptide of Galectin-3, characterized in that: The antigenic epitope peptide is the 196-225 amino acid sequence of Galectin-3, and the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO:
1.
2. The Galectin-3 antigenic epitope peptide according to claim 1, characterized in that: The steps for synthesizing the Galectin-3 antigenic epitope peptide are as follows: S1: Deprotection: Using resin as a solid support, the resin was swelled in dimethylformamide (DMF), and the first amino acid (i.e., C-terminal amino acid H) in the antigenic epitope sequence 196-225 of Galectin-3 was coupled to the solid support by the linker diisopropylcarbazolium. S2: Cleaning: DMF containing 20% piperidine was used as a solution protectant to remove the amino protecting group, wherein the amino protecting group is an Fmoc protecting group; DMF was used to wash the resin to ensure that all deprotecting agents were removed; the next amino acid must first have a protecting group, wherein the next amino acid refers to the next monomer at the N-terminus, and the protecting group is Fmoc; S3: Amino acid coupling The amino acid is coupled to the deprotected amino acid using a coupling agent, and repeated washing is performed to remove unreacted reagents and byproducts. Repeat steps S2 and S3 to sequentially couple all amino acids until the entire polypeptide sequence is synthesized. S4: Cutting and removing protection: The synthesized peptide chains are cut from the solid support using a cleavage solution, and all remaining protecting groups are removed. The cutting solution used was a mixture of trifluoroacetic acid (TFA) and other reagents. S5: Separation and purification: The peptides were separated by cold ether precipitation; the peptides were further purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to ensure that they met the required purity requirements.
3. The Galectin-3 antigenic epitope peptide according to claim 2, characterized in that: The coupling reagent is N,N'-diisopropylcarbodiamide (DIC).
4. The Galectin-3 antigenic epitope peptide according to claim 2, characterized in that: The cutting solution is formulated as follows: TFA:water:TIPS:phenol = 95:2.5:2.5:
1.
5. An antigen of Galectin3, characterized in that: It is obtained by conjugating the antigenic epitope peptide of Galectin-3 as described in any one of claims 1-4 with a carrier protein.
6. The Galectin-3 antigen according to claim 5, characterized in that: The carrier protein is hemocyanin (KLH); and glutaraldehyde, a cross-linking agent, is used during the coupling process.