Application of protein marker in preparation of AAA diagnosis product and kit

Abstract

The invention discloses application of a protein marker in preparation of a product for diagnosing AAA, a kit and a use method of the kit. The kit is high in sensitivity and specificity and has a good diagnosis effect. According to the application of the protein marker in preparation of a product for diagnosing AAA, the protein marker is any one of the following proteins or a combination of the following proteins: a fibrinogen alpha chain, a fibrinogen gamma chain, histone H3.1 and S100-P protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedicine, and particularly to application of a protein marker in preparation of a product for diagnosing AAA, and a kit for diagnosing AAA. BACKGROUND

[0002] AAA (abdominal aortic aneurysm) is defined as a local expansion of the abdominal aorta with a maximum diameter of more than 3 cm or 50% of the normal aortic diameter. AAA is a common aortic lesion in the elderly population, and its incidence increases with age. The incidence of AAA in the population aged 65-75 is about 2.98%. The main complication of AAA is aneurysm rupture, and its mortality rate can be as high as 90%. With the rapid progress of population aging, AAA will seriously threaten people's life and health.

[0003] Most of the AAA in clinical practice is insidious and has no obvious clinical symptoms, making it difficult to identify early. Most AAA patients in China are accidentally discovered during unrelated imaging examinations or even aneurysm rupture. There is no effective treatment drug for AAA in clinical practice. Surgical treatment is more traumatic and expensive, which brings heavy economic burden to patients and society, and there are still risks of aortic expansion or in-stent leakage after surgery; patients who do not meet the surgical indications need long-term follow-up monitoring, and the aneurysm may rupture at any time, which brings heavy psychological burden to patients. Therefore, it is of great significance to develop a product and kit for diagnosing AAA. SUMMARY

[0004] To overcome the defects of the prior art, the technical problem to be solved by the present application is to provide application of a protein marker in preparation of a product for diagnosing AAA, which has high sensitivity, strong specificity and good diagnostic effect.

[0005] The technical solution of the present application is: the application of the protein marker in preparation of a product for diagnosing AAA, wherein the protein marker is any one of the following proteins or a combination thereof: fibrinogen alpha chain, fibrinogen gamma chain, histone H3.1 and S100-P protein.

[0006] The protein marker for the application in preparation of a product for diagnosing AAA has high sensitivity, strong specificity and good diagnostic effect.

[0007] A kit for diagnosing AAA is also provided, which comprises the above protein marker, and the diagnostic reagent of the kit is selected from mass spectrometry identification reagents, antibodies or antigen-binding fragments thereof.

[0008] A method for using the kit for diagnosing AAA is also provided, which comprises the following steps: (1) Collect biological samples from the subjects; (2) Blood exosomes were separated using exosome separation reagents; (3) Use the protein lysis reagent in the kit to lyse the blood exosomes isolated and extracted in step (2) to obtain blood exosome proteins; (4) Compare the expression levels of the protein in the subjects with the expression levels of the protein in the controls; (5) Determine whether the subject has an abdominal aortic aneurysm or assess the risk of having an abdominal aortic aneurysm. Attached Figure Description

[0009] Figure 1 The results of electron microscopy, particle size analysis, and immunoblotting identification of blood exosomes are shown.

[0010] Figure 2 The receiver operating characteristic (ROC) curves for four blood exosome protein expression levels are shown.

[0011] Figure 3 The ROC curves are shown when using a combination of fibrinogen α chain, fibrinogen γ chain, histone H3.1 and S100-P protein. Detailed Implementation

[0012] Unless otherwise stated, the technical terms used in this application have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0013] To more clearly and in detail illustrate the present invention, the technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples, and should not be used to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the experimental materials used are all available from commercial companies.

[0014] In this invention, the term "subject" refers to an individual (preferably a person) selected in the process of examining the diagnostic efficacy of exosomal protein markers for abdominal aortic aneurysms, who has or is suspected of having a certain disease, including subjects with abdominal aortic aneurysms and healthy subjects. This term may also be used interchangeably with "patient," "subject of testing," "subject of treatment," etc.

[0015] In this invention, the term "marker" refers to an indicator that can serve as an objective measure and evaluation of the normal biological process, pathogenic process, or pharmacological response to a therapeutic intervention, and can be used to determine the presence of a specific disease or the severity of a disease. This term may also be used interchangeably with "biomarker."

[0016] In this invention, the term "diagnosis" refers to the process of determining whether a subject has a certain disease by detecting the nature and condition of the disease. In this invention, the diagnosis of abdominal aortic aneurysm refers to the determination of whether a subject has an abdominal aortic aneurysm.

[0017] In this invention, the term "exosome" refers to nanovesicles secreted by different types of cells. These vesicles act as carriers for various biomolecules (such as proteins, nucleic acids, and lipids) from one cell to another and are present in all biological fluids under normal and pathophysiological conditions. In this invention, "exosome" refers to exosomes found in biological samples.

[0018] The detection of the expression level of the biomarker protein in the method of the present invention includes both quantitative and qualitative detection of the biomarker.

[0019] The biological samples are collected from the subject's fluids or tissues, including blood, serum, plasma, saliva, sputum, lymph, cerebrospinal fluid, pleural effusion, and urine.

[0020] The method of this invention can be used to evaluate the therapeutic efficacy of existing treatments for abdominal aortic aneurysms or the pharmacological effects of drugs for treating abdominal aortic aneurysms. For example, biological samples are obtained from the subject before, during, or after treatment, and the expression levels of the biomarkers are detected using the protein biomarker detection method described in this invention. The treatment status of the subject is then identified and analyzed based on the detection results, thereby determining the therapeutic efficacy of the treatment method and the pharmacological effects of the therapeutic drug.

[0021] The method of the present invention can also be used to identify the recurrence status of aneurysm in a subject after surgery. During the postoperative period, the subject's biological samples are obtained, and the expression level of the biomarker is detected according to the protein biomarker detection method of the present invention. The recurrence status of the abdominal aortic aneurysm in the subject is determined by the detection results.

[0022] This invention also protects a method for detecting protein biomarker expression levels using the kit, comprising the following steps: biological sample collection, exosome extraction and separation, exosome protein extraction, and ELISA detection of biomarker expression levels.

[0023] The application of this protein biomarker in the preparation of diagnostic AAA products, wherein the protein biomarker is any one of the following proteins or a combination thereof: fibrinogen α chain, fibrinogen γ chain, histone H3.1, S100-P protein.

[0024] The beneficial effects of this invention are: this protein biomarker, when used in the preparation of diagnostic AAA products, has high sensitivity, strong specificity, and good diagnostic effect.

[0025] Preferably, fibrinogen α chain, fibrinogen γ chain, histone H3.1, and S100-P protein are used as biomarkers, and the abdominal aortic aneurysm is identified by detecting the difference in expression levels of protein biomarkers in biological samples of abdominal aortic aneurysm and control group.

[0026] Preferably, compared with the control group, a decrease in the expression level of the following protein indicates that the subject has an abdominal aortic aneurysm, said protein being selected from any one or a combination of the following: histone H3.1, S100-P protein; Compared to the control group, increased expression levels of the following proteins indicate that the subject has an abdominal aortic aneurysm, and the proteins are selected from any one or a combination of the following: fibrinogen α chain and fibrinogen γ chain; The control group refers to individuals who do not have abdominal aortic aneurysm.

[0027] Preferably, the protein is a protein from serum exosomes.

[0028] Preferably, the biological sample is the subject's blood, serum, plasma, saliva, sputum, lymph, cerebrospinal fluid, pleural fluid, or urine.

[0029] A kit for diagnosing AAA is also provided, which includes the above protein biomarkers. The diagnostic reagents in the kit are selected from mass spectrometry identification reagents, antibodies or their antigen-binding fragments.

[0030] Preferably, the antibody is a monoclonal antibody.

[0031] Preferably, the antigen-binding fragment includes: Fv, ScFv, Fab, Fab', (Fab')2, bispecific antibody, trispecific antibody, tetraspecific antibody, bi-scFv, and mimi antibody.

[0032] Instructions for using the kit for diagnosing AAA are also provided, including the following steps: (1) Collect biological samples from the subjects; (2) Blood exosomes were separated using exosome separation reagents; (3) Use the protein lysis reagent in the kit to lyse the blood exosomes isolated and extracted in step (2) to obtain blood exosome proteins; (4) Compare the expression levels of the protein in the subjects with the expression levels of the protein in the controls; (5) Determine whether the subject has an abdominal aortic aneurysm or assess the risk of having an abdominal aortic aneurysm.

[0033] Preferably, in step (4), the protein biomarker expression level detection method is selected from one or more of the following methods: protein chip, mass spectrometry, ELISA, chemiluminescence, and Western spectroscopy.

[0034] The embodiments of the present invention will be described in detail below.

[0035] Serum exosome samples were collected from 17 patients diagnosed with abdominal aortic aneurysm and 17 healthy controls in the cohort. A total of 3038 proteins were identified at a protein identification FDR < 1%. Compared with the control samples, differentially expressed proteins with a fold change of more than 2-fold and an independent samples t-test p < 0.05 were selected. Based on whether the protein function was related to the pathogenesis of abdominal aortic aneurysm, four exosome proteins were selected as candidate diagnostic biomarkers for AAA (Table 1).

[0036] Table 1. Candidate biomarkers of serum exosomal proteins that differentiate between AAA patients and healthy controls .

[0037] To further confirm the sensitivity and specificity of candidate exosomal protein biomarkers in the diagnosis of AAA, PRM-targeted proteomics was used to validate the candidates in a new clinical cohort for the proteins listed in Table 1, and ROC curves were plotted to distinguish AAA patients from subjects. The specific steps were as follows: Serum samples were collected from 45 newly identified AAA patients and 30 healthy controls in the validation cohort, and exosomes were extracted. A blinded method was used to test the expression levels of candidate proteins in the samples using PRM-targeted proteomics quantification. The results showed that compared with healthy controls, the fold change in fibrinogen α-chain in the exosomes of AAA patients was 13.0 (p < 0.001); the fold change in fibrinogen γ-chain was 10.29 (p < 0.001); the fold change in histone H3.1 was 0.22 (p < 0.001); and the fold change in S100-P protein was 0.67 (p = 0.012). The expression levels of these proteins, alone or in combination, could distinguish AAA patients from subjects.

[0038] For example, such as Figure 1 As shown, A represents the morphology of isolated blood exosomes under an electron microscope (scale bar: 200 nm); B represents the particle size concentration analysis of blood exosomes; and C represents the immunoblotting detection using exosome markers CD9, Tsg101, CD63, and Calnexin. This invention demonstrates the successful isolation of blood exosomes through electron microscopy, particle size analysis, and immunoblotting.

[0039] For example, such as Figure 2As shown, the AUC value of fibrinogen α chain for the diagnosis of AAA was 0.929, with a sensitivity of 87.5% and a specificity of 100%; the AUC value of fibrinogen γ chain was 0.920, with a sensitivity of 87.5% and a specificity of 97.1%; the AUC value of histone H3.1 was 0.754, with a sensitivity of 75% and a specificity of 65.7%; and the AUC value of S100-P protein was 0.665, with a sensitivity of 90% and a specificity of 40%.

[0040] For example, such as Figure 3 As shown, the combination of fibrinogen α chain, fibrinogen γ chain, histone H3.1 and S100-P protein has a good diagnostic effect on AAA, with an area under the ROC curve (AUC) of 0.974, a sensitivity of 90% and a specificity of 100%.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. The application of protein biomarkers in the preparation of diagnostic AAA products, characterized by: The protein marker is any one of the following proteins or a combination thereof: fibrinogen α chain, fibrinogen γ chain, histone H3.1, or S100-P protein.

2. The application of the protein biomarker according to claim 1 in the preparation of diagnostic AAA products, characterized in that: Using fibrinogen α chain, fibrinogen γ chain, histone H3.1, and S100-P protein as biomarkers, the abdominal aortic aneurysm was identified by detecting the difference in expression levels of these protein biomarkers in biological samples from the abdominal aortic aneurysm and the control group.

3. The application of the protein biomarker according to claim 2 in the preparation of diagnostic AAA products, characterized in that: Compared to the control group, a decrease in the expression level of the following proteins indicates that the subject has an abdominal aortic aneurysm, and the proteins are selected from any one or a combination of the following: histone H3.1 and S100-P protein; Compared to the control group, increased expression levels of the following proteins indicate that the subject has an abdominal aortic aneurysm, and the proteins are selected from any one or a combination of the following: fibrinogen α chain and fibrinogen γ chain; The control group refers to individuals who do not have abdominal aortic aneurysm.

4. The application of the protein biomarker according to claim 1 in the preparation of diagnostic AAA products, characterized in that: The protein in question is a protein found in serum exosomes.

5. The application of the protein biomarker according to claim 1 in the preparation of diagnostic AAA products, characterized in that: Biological samples include the subject's blood, serum, plasma, saliva, sputum, lymph, cerebrospinal fluid, pleural fluid, or urine.

6. A reagent kit for diagnosing AAA, characterized in that: It includes the protein biomarker according to claim 1, and the diagnostic reagents in the kit are selected from mass spectrometry identification reagents, antibodies or their antigen-binding fragments.

7. The kit for diagnosing AAA according to claim 6, characterized in that: The antibody is a monoclonal antibody.

8. The reagent kit for diagnosing AAA according to claim 6, characterized in that: The antigen-binding fragments include: Fv, ScFv, Fab, Fab', (Fab')2, bispecific antibody, trispecific antibody, tetraspecific antibody, bi-scFv, and mimi antibody.

9. The method of using the reagent kit for diagnosing AAA according to claim 6, characterized in that: It includes the following steps: (1) Collect biological samples from the subjects; (2) Blood exosomes were separated using exosome separation reagents; (3) Use the protein lysis reagent in the kit to lyse the blood exosomes isolated and extracted in step (2) to obtain blood exosome proteins; (4) Compare the expression levels of the protein in the subjects with the expression levels of the protein in the controls; (5) Determine whether the subject has an abdominal aortic aneurysm or assess the risk of having an abdominal aortic aneurysm.

10. The method of using the reagent kit for diagnosing AAA according to claim 9, characterized in that: In step (4), the protein biomarker expression level detection method is selected from one or more of the following methods: protein chip, mass spectrometry, ELISA, chemiluminescence, and Western spectroscopy.

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