Galectin-3 antigen epitope peptide, Galectin-3 antigen, Galectin-3 antibody and chemiluminescence kit

Abstract

The invention provides a Galectin-3 antigen epitope peptide, a Galectin-3 antigen, a Galectin-3 antibody and a chemiluminescence kit, and relates to the technical field of biological detection, the Galectin-3 antigen epitope peptide is amino acid of a Galectin-3 antigen epitope sequence 196-225, and the amino acid sequence is shown as SEQ ID NO: 1. Based on the antigen epitope peptide, the invention also provides a corresponding antigen and an antibody, and provides an application of disaster resistance in preparation of a Galectin-3 detection kit. Compared with a common kit, the chemiluminescence kit is high in sensitivity, can detect target substances with extremely low concentration, is suitable for trace analysis, and ensures the accuracy of a detection result. The reaction system is simple, the operation is convenient, the luminescence reaction can be completed only by adding a proper amount of reagent, and the detection efficiency is greatly improved. The chemiluminescence kit is good in specificity, interference substances can be effectively eliminated, and the accuracy of a detection result is ensured. Finally, the chemiluminescence kit is high in portability, field detection can be conveniently carried out in different places, and an accurate result can be rapidly obtained.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to Galectin-3 antigen epitope peptide, antigen, antibody and chemiluminescence kit. Background Art

[0002] The detection of Galectin-3 (Gal-3 for short) has shown broad application prospects and important clinical significance in the medical field. As a protein that plays a key role in a variety of pathological processes, the detection of Gal-3 is of great value in the diagnosis and treatment of cardiovascular diseases, kidney diseases, tumors and nervous system diseases.

[0003] In terms of cardiovascular diseases, especially heart failure, Gal-3 as a biomarker can accurately predict the risk of hospitalization and death of patients, providing a strong basis for clinical decision-making. At the same time, in kidney diseases, the detection of Gal-3 levels can help diagnose diseases such as idiopathic membranous nephropathy and lupus nephritis, and can determine the degree of kidney damage, providing an important reference for early intervention and treatment of kidney diseases.

[0004] In addition, the overexpression of Gal-3 in a variety of malignant tumors makes it a potential tool for tumor diagnosis, staging and prognosis assessment. In neurological diseases such as Alzheimer's disease, the detection of Gal-3 also helps in disease diagnosis and treatment monitoring.

[0005] The detection of Galectin-3 can achieve early diagnosis of the disease and buy time for timely intervention and treatment. At the same time, the level of Gal-3 is closely related to the prognosis of many diseases. Regular testing can evaluate the prognosis of patients and provide a basis for the development of personalized treatment plans. During the treatment process, monitoring the changes in Gal-3 levels can also reflect the effectiveness of the treatment, and timely adjust the treatment plan to improve the treatment effect. Therefore, the detection of Galectin-3 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] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses an antigenic epitope peptide of Galectin-3, wherein the antigenic epitope peptide is the antigenic epitope sequence 196-225 amino acids of Galectin-3, and the amino acid sequence is shown in SEQ ID NO:1.

[0008] Preferably, the steps of the method for synthesizing the antigen epitope peptide of Galectin3 are as follows: S1: Deprotection: The resin was used as a solid phase carrier, and the resin was swollen in dimethylformamide (DMF), and the first amino acid (i.e., C-terminal amino acid H) in the antigen epitope sequence 196-225 of Galectin-3 was coupled to the solid phase carrier through a linker, diisopropylcarbonyldiimidazole; S2: Cleaning: Use DMF containing 20% ​​piperidine as a solution protecting agent to remove the amino protecting group, which is the Fmoc protecting group; use DMF 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 have a protecting group (such as Fmoc); S3: Amino Acid Coupling coupling the amino acid to the deprotected amino acid using a coupling reagent and repeatedly washing to remove unreacted reagents and byproducts; Repeat S2 and S3 to couple all amino acids in sequence until the entire polypeptide sequence is synthesized.

[0009] S4: Cleavage and deprotection: Use a cleavage solution to cleave the synthesized peptide chain from the solid support and remove all remaining protecting groups; The cleavage solution used is a mixture of trifluoroacetic acid (TFA) and other reagents. S5: Isolation and purification: The peptides were isolated by cold ether precipitation and further purified by reverse phase high performance liquid chromatography (RP-HPLC) to ensure the desired purity.

[0010] Preferably, the coupling agent is N,N'-diisopropylcarbodiamide DIC.

[0011] Preferably, the formula of the cutting fluid is TFA: water: TIPS: phenol = 95: 2.5: 2.5: 1.

[0012] The present application also provides a Galectin-3 antigen, which is obtained by coupling the Galectin-3 antigen epitope peptide described above with a carrier protein.

[0013] Preferably, the carrier protein is hemocyanin (KLH); and during the coupling process, a cross-linking reagent glutaraldehyde is used.

[0014] The present application also provides an antibody to Galectin-3, and the method for preparing the antibody is as follows: Mouse immunization: Four immunizations were performed. For the first immunization, 100 μg of the immunogen was mixed with Freund's complete adjuvant in a ratio of 1:1 and injected into 6-8 week old female BALB / C mice at multiple points on the back. From the second to the fourth immunization, 50 μg of immunogen was injected intraperitoneally into mice.

[0015] The immunogen is the Galectin-3 antigen described above; Prepare antibodies: After the fourth immunization, BALB / c mice with serum titers reaching the standard were subjected to immunization shock. The serum of the immunized mice was collected and the antibodies were purified by protein A / G column.

[0016] Preferably, there is a two-week interval between each immunization from the first immunization to the fourth immunization.

[0017] The present application also provides an application of a Galectin-3 antibody in the preparation of a Galectin-3 detection kit, wherein the Galectin-3 antibody is the Galectin-3 antibody described above, and the detection kit is a chemiluminescence detection kit.

[0018] The above-described kit for detecting the content of Galectin-3 using chemiluminescence method, compared with ordinary kits, has high sensitivity of chemiluminescence kit, can detect extremely low concentration of target substances, is suitable for trace analysis, and ensures the accuracy of test results. Secondly, its reaction system is simple and easy to operate, and only needs to add appropriate amount of reagents to complete the luminescence reaction, which greatly improves the detection efficiency. In addition, the specificity of the chemiluminescence kit is good, and interfering substances can be effectively excluded to ensure the accuracy of the test results. Furthermore, the kit is stable and reliable, with good repeatability, can maintain high sensitivity for a long time, and is suitable for screening of large batches of samples. Finally, the chemiluminescence kit is highly portable, convenient for on-site detection in different places, and accurate results can be obtained quickly. In summary, the chemiluminescence detection kit is superior to ordinary kits in terms of sensitivity, ease of operation, specificity, stability and portability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 For screening the antigenic epitope 196-225 amino acids of Galectin-3 in one embodiment of the present invention; Figure 2 The figure is a comparison of the correlation between the Galectin-3 chemiluminescence detection kit in one embodiment of the present invention and the product kit of Roche, a reference manufacturer, on clinical specimens. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments.

[0021] An antigenic epitope peptide of Galectin-3, see Figure 1 The antigen epitope peptide is the antigen epitope sequence 196-225 amino acids of Galectin-3, and the amino acid sequence is shown in SEQ ID NO: 1.

[0022] The amino acid sequence of the antigen epitope peptide is: KPFKIQVLVEPDHFKVAVNDAHLLQYNHRV. The present application provides a method for synthesizing an antigen epitope peptide, the steps of which are as follows: S1: Deprotection: Specifically, the resin is used as a solid phase carrier, the resin is swollen in dimethylformamide (DMF), and the first amino acid (i.e., the C-terminal amino acid H) in the antigen epitope sequence 196-225 of Galectin-3 is coupled to the solid phase carrier through a linker diisopropylcarbonyldiimidazole.

[0023] S2: Cleaning: In one embodiment, DMF containing 20% ​​piperidine is used as a solution protecting agent to remove the amino protecting group, which 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 provided with a protecting group (such as Fmoc).

[0024] S3: Amino Acid Coupling coupling the amino acid to the deprotected amino acid using a coupling reagent and repeatedly washing to remove unreacted reagents and byproducts; The coupling reagent is N,N'-diisopropylcarbodiamide DIC.

[0025] Repeat S2 and S3 to couple all amino acids in sequence until the entire polypeptide sequence is synthesized.

[0026] S4: Cleavage and deprotection: Use a cleavage solution to cleave the synthesized peptide chain from the solid support and remove all remaining protecting groups; In one embodiment, the cleavage solution used is a mixture of trifluoroacetic acid (TFA) and other reagents (such as water, triisopropylsilane (TIPS) and phenol), and the preferred formula is TFA: water: TIPS: phenol = 95:2.5:2.5:1.

[0027] S5: Isolation and purification: The peptides were isolated by cold ether precipitation and further purified by reverse phase high performance liquid chromatography (RP-HPLC) to ensure the desired purity.

[0028] A Galectin-3 antigen is obtained by coupling the Galectin-3 antigen epitope peptide mentioned above with a carrier protein.

[0029] In one embodiment, the carrier protein is hemocyanin (KLH); and during the coupling process, a cross-linking reagent glutaraldehyde is used.

[0030] Based on the above-mentioned antigen, the present application also provides an antibody against Galectin-3, and the method for preparing the antibody is as follows: Immunization of mice: In one embodiment, four immunizations are performed. For the first immunization, 100 μg of the immunogen is mixed with Freund's complete adjuvant in a ratio of 1:1 and injected into female BALB / C mice of 6 to 8 weeks old at multiple points on the back. From the second to the fourth immunization, 50 μg of immunogen was injected intraperitoneally into mice.

[0031] From the first immunization to the fourth immunization, each immunization was separated by two weeks. The immunogen and the antigen described above.

[0032] Prepare antibodies: After the fourth immunization, BALB / c mice with serum titers reaching the standard were subjected to immunization shock. The serum of the immunized mice was collected and the antibodies were purified by protein A / G column.

[0033] The present application also provides the use of the above-mentioned antibody in the preparation of a Galectin-3 detection kit, wherein the Galectin-3 detection kit is a chemiluminescence kit.

[0034] The present application also provides a chemiluminescent kit, which includes R1 reagent, R2 reagent, instructions and radio frequency card, wherein the R1 reagent includes Galectin-3 antibody (3 μg / mL) coated with magnetic microparticles, 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% Proclin300; The R2 includes an acridinium ester-labeled Galectin-3 antibody (2 μg / mL), an aqueous solution containing 0.5% bovine serum albumin, 1% trehalose, 0.93% sodium dihydrogen phosphate, 0.32% sodium dihydrogen phosphate, 0.88% sodium chloride, 0.1% Tween 20, and 0.05% Proclin 300.

[0035] Based on the kit described above, the present application also provides a method for using and a principle of the chemiluminescent kit. The chemiluminescent kit described in the present application adopts a competitive method for detection, and uses a specific antibody labeled with a luminescent agent and a solid phase carrier coated with the antibody to react with the sample to form two forms of complexes, namely "solid phase carrier-antibody-antigen" and "solid phase carrier-antibody-antigen-luminescent agent". The solid phase carrier is adsorbed by a magnetic field, and the unbound sample and reagent are washed and removed. Finally, a substrate is added to allow the complex labeled with the luminescent agent to undergo a luminescent reaction, and its luminescence intensity is measured. The measured specific antigen concentration will be inversely proportional to the luminescence intensity, that is, the lower the specific antigen concentration in the sample, the higher its luminescence intensity.

[0036] Specifically, the method of use comprises the following steps: S1: Incubation The successfully prepared Galectin-3 antibody labeled with acridinium ester, the Galectin-3 antibody coated with magnetic microparticles and the sample to be tested. Galectin-3 in the sample to be tested combines with the Galectin-3 antibody labeled with acridinium ester and the Galectin-3 antibody coated with magnetic microparticles to form an immune complex.

[0037] S2: Cleaning After the incubation is completed, the immune complex is adsorbed to the wall of the reaction cup under the action of the magnetic field, and the unbound substances are washed away.

[0038] S3: Provocation and calculation After cleaning, add pre-excitation solution and excitation solution to the reaction mixture. 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.

[0039] The Galectin-3 chemiluminescent detection kit in the example was compared with the product kit of Roche, a reference manufacturer, on clinical specimens. The data are as follows: Figure 2 shown.

[0040] The Galectin-3 used in the present application is Homo sapiens galectin-3, and its protein sequence is shown in SEQ ID NO:2.

[0041] Final determination, correlation coefficient R 2 The result showed that the kit of the present invention can accurately detect the content of Galectin-3, provide a basis for clinical diagnosis, and fully meet the requirements of clinical in vitro diagnostic testing (see Figure 2 ).

[0042] In summary, the present application provides a kit for detecting the content of Galectin-3 using chemiluminescence. Compared with ordinary kits, chemiluminescence kits have high sensitivity, can detect extremely low concentrations of target substances, are suitable for trace analysis, and ensure the accuracy of test results. Secondly, its reaction system is simple and easy to operate. It only needs to add an appropriate amount of reagents to complete the luminescent reaction, which greatly improves the detection efficiency. In addition, the chemiluminescence kit has good specificity, can effectively exclude interfering substances, and ensure the accuracy of the test results. Furthermore, the kit is stable and reliable, has good repeatability, can maintain high sensitivity for a long time, and is suitable for screening of large quantities of samples. Finally, the chemiluminescence kit is highly portable, convenient for on-site detection in different places, and accurate results are quickly obtained. In summary, the chemiluminescence detection kit is superior to ordinary 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 antigen epitope peptide is the antigen epitope sequence 196-225 amino acids of Galectin-3, and the amino acid sequence is shown in SEQ ID NO:

1.

2. The antigenic epitope peptide of Galectin-3 according to claim 1, characterized in that: The steps of the synthesis method of the Galectin-3 antigen epitope peptide are as follows: S1: Deprotection: The resin was used as a solid phase carrier, and the resin was swollen in dimethylformamide (DMF), and the first amino acid (i.e., C-terminal amino acid H) in the antigen epitope sequence 196-225 of Galectin-3 was coupled to the solid phase carrier through a linker, diisopropylcarbonyldiimidazole; S2: Cleaning: Use DMF containing 20% ​​piperidine as a solution protecting agent to remove the amino protecting group, which is the Fmoc protecting group; use DMF 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 have a protecting group (such as Fmoc); S3: Amino Acid Coupling coupling the amino acid to the deprotected amino acid using a coupling reagent and repeatedly washing to remove unreacted reagents and byproducts; Repeat S2 and S3 to sequentially couple all amino acids until the entire polypeptide sequence is synthesized; S4: Cleavage and deprotection: Use a cleavage solution to cleave the synthesized peptide chain from the solid support and remove all remaining protecting groups; The cleavage solution used is a mixture of trifluoroacetic acid (TFA) and other reagents. S5: Isolation and purification: The peptides were isolated by cold ether precipitation and further purified by reverse phase high performance liquid chromatography (RP-HPLC) to ensure the desired purity.

3. The antigen epitope peptide of Galectin-3 according to claim 2, characterized in that: The coupling reagent is N,N'-diisopropylcarbodiamide DIC.

4. The Galectin-3 antigen epitope peptide according to claim 2, characterized in that: The formula of the cutting solution is TFA: water: TIPS: phenol = 95: 2.5: 2.5:

1.

5. A Galectin3 antigen, characterized in that: The method is obtained by coupling the Galectin-3 antigen epitope peptide described in claims 1 to 4 with a carrier protein.

6. The Galectin-3 antigen according to claim 5, characterized in that: The carrier protein is hemocyanin (KLH); and in the coupling process, a cross-linking reagent glutaraldehyde is used.

7. An antibody against Galectin-3, characterized in that: The antibody preparation method is as follows: Mouse immunization: Four immunizations were performed. For the first immunization, 100 μg of the immunogen was mixed with Freund's complete adjuvant in a ratio of 1:1 and injected into 6-8 week old female BALB / C mice at multiple points on the back. From the second to the fourth immunization, 50 μg of immunogen was injected intraperitoneally into mice; The immunogen is the Galectin-3 antigen according to claim 5; Prepare antibodies: After the fourth immunization, BALB / c mice with serum titers reaching the standard were subjected to immunization shock; the serum of the immunized mice was collected and the antibodies were purified using a protein A / G column.

8. The Galectin-3 antibody according to claim 7, characterized in that: From the first to the fourth immunization, there is a two-week interval between each immunization.

9. Use of an antibody against Galectin-3 in the preparation of a Galectin-3 detection kit, characterized in that: The Galectin 3 antibody is the Galectin-3 antibody according to claim 7, and the detection kit is a chemiluminescence detection kit.

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