Serum diagnostic markers of coronary artery spasm and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a serum diagnosis marker and application thereof, in particular to application of serum selenium binding protein SELENBP1 in preparation of a clinical medicine and forensic medicine coronary artery spasm (CAS) precise diagnosis product. The application provides a corresponding kit, which contains an anti-human SELENBP1 serum protein specific antibody, a chromogenic agent, a detergent, a standard product, biotin and the like. Experiments prove that the content of serum SELENBP1 has good reference value for the diagnosis of CAS, and is superior to the currently commonly used CK-MB and cTn-I indexes for diagnosing myocardial injury. The application provides a highly specific and sensitive serum biochemical index of coronary artery spasm and a diagnosis threshold thereof, and can realize rapid and precise cause diagnosis of CAS.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a serum diagnostic marker for coronary artery spasm (CAS) and application thereof, in particular to serum selenoprotein SELENBP1 as a diagnostic marker and application of content change of the serum selenoprotein SELENBP1 in clinical medical etiological diagnosis and forensic cause-of-death diagnosis of coronary artery spasm. BACKGROUND

[0002] The prior art discloses that coronary artery spasm (CAS) is a strong vasoconstriction causing total or subtotal occlusion of the coronary artery, and plays an important role in myocardial ischemic syndromes including stable and unstable angina pectoris, acute myocardial infarction and sudden cardiac death. Studies have shown that CAS plays an important role in the pathogenesis of myocardial infarction regardless of coronary artery stenosis. In ischemic heart disease caused by spasm, even if the onset is painless, arrhythmia such as ventricular fibrillation, tachycardia and complete atrioventricular block can be repeatedly observed. In dynamic electrocardiogram monitoring, transient sympathetic vagal imbalance can be observed, that is, significant reduction of heart rate variability in a period before ST segment change is considered to be a triggering factor for ischemic sudden death.

[0003] In clinical practice, coronary angiography plus drug provocation test is the only precise method for diagnosing CAS. At present, different CAS diagnostic guidelines all take the reduction of coronary artery diameter as the main diagnostic standard, and the symptoms of myocardial ischemia such as angina pectoris and / or ECG ST segment changes during drug provocation test as the secondary diagnostic standard. However, different guidelines have different requirements for the degree of coronary artery diameter narrowing during drug-induced provocation. Some guidelines require coronary artery diameter narrowing >50%, while some guidelines require coronary artery diameter narrowing >75% or >90%. Therefore, although 'coronary angiography plus drug provocation test' is the most specific and sensitive 'gold standard' for diagnosing CAS, there is still a lack of internationally unified and consensus-based guideline standards. In addition, ergonovine and acetylcholine are the most commonly used drugs in provocation tests. Through intracoronary administration of methyl ergonovine, ECG changes and CAS clinical manifestations appear, which can be diagnosed. The test process needs to strictly follow the safety procedures, stop using vasodilators (calcium antagonists, nitrates) for more than 48 hours before the test, and be prepared with nitroglycerin solution, atropine and other emergency drugs, with a dose of 5-10 μg. Early provocation tests are mainly intravenous administration, but patients receiving high doses of ergonovine are prone to cause severe angina pectoris and death, leading to the abandonment of intravenous provocation tests and the replacement of intracoronary administration. Although studies have shown that intracoronary administration is safer than intravenous administration in inducing CAS, there are still great safety risks, and complications include angina pectoris, various arrhythmias, hypotension, dyspnea, and even death. And the invasive test method has limitations in clinical application. Provocation tests are rarely used in Europe and the United States, and only Japan in Asia. Our country is very cautious in drug provocation tests. In addition, coronary angiography of some multi-vessel onset CAS patients can show positive. It can be seen that the only gold standard for diagnosing CAS in current clinical practice has the risk of causing severe angina pectoris and death, and is not suitable for clinical promotion.

[0004] In forensic practice, sudden cardiac death is an important category of problems. Among them, a considerable number of cases of sudden cardiac death can only see slight lesions in the coronary artery after systematic autopsy, and the degree of lumen stenosis is lower than grade II, which is not enough to determine that it is caused by coronary atherosclerotic heart disease. For this part of the case, some scholars suggest that if the deceased has obvious inducement before death (such as arguing with others, cold stimulation, etc.), it is highly suspected that the death is caused by coronary artery spasm before death. However, the sudden death caused by CAS often does not cause significant morphological changes in the heart, and there is a lack of effective detection means in forensic pathology identification, so it is difficult to accurately diagnose, and generally only a "compliance" diagnosis opinion can be issued, which is easy to cause controversy. Unlike clinical medicine, forensic cause of death diagnosis is often limited by postmortem natural changes, and it is difficult to diagnose CAS and specific spasm arterial segments before death through coronary angiography. Some data suggest that after death caused by CAS, the smooth muscle in the middle layer of the coronary artery can appear contraction band. The longitudinal shortening of the smooth muscle cells in the middle layer of the coronary artery, nuclear folding, twisting and other morphological changes are considered to be the changes after death caused by CAS. The elastic membrane and intimal folds in the coronary artery are also proved to be morphological changes caused by CAS. However, these morphological changes of the coronary artery are subjective, and their specificity needs to be studied. So far, there is no clear marker for objective diagnosis of CAS in forensic medicine. It can be seen that from the perspective of clinical medical etiological diagnosis and forensic medical cause of death diagnosis, accurate, non-invasive and objective diagnostic indicators for identifying the onset of CAS are urgently needed.

[0005] Selenium is an essential element for the human body and has strong anti-cancer properties. Some studies have shown that a lack of selenium can lead to certain nervous system diseases. The role of selenium in preventing cancer and nervous system diseases may be mediated by selenium-binding protein 1 (SELENBP1). It is known that reduced expression of SELENBP1 protein is associated with various types of cancer. However, the association of SELENBP1 with cardiovascular disease remains to be studied.

[0006] Based on the current status of the prior art, the inventors of the present application propose a serum diagnostic marker for coronary artery spasm and its application. The present application has been identified through experiments that serum SELENBP1 is closely related to CAS; by detecting the content of serum SELENBP1, the clinical diagnosis of CAS and the forensic accurate diagnosis of death caused by CAS before life can be realized. SUMMARY

[0007] The purpose of the present application is to provide a diagnostic marker for coronary artery spasm (CAS) based on the current status of the prior art; specifically, it relates to the application of changes in the serum concentration of SELENBP1 in clinical and forensic diagnostic products for CAS.

[0008] The present application is based on the fact that coronary artery spasm (CAS) refers to a pathophysiological state in which the coronary artery is partially or completely blocked due to abnormal vasomotor function, which can cause myocardial ischemia, myocardial infarction, arrhythmia, syncope, and even sudden cardiac death. However, the symptoms of CAS are similar to other coronary artery diseases, making it difficult to diagnose and easily leading to misdiagnosis and missed diagnosis. In forensic medicine, CAS death is often caused by a short onset time (usually less than 6 hours), and specific ischemic pathological changes have not yet appeared on the heart, so it is difficult to make an accurate diagnosis through postmortem examination of heart tissue. Currently, forensic doctors can only make a presumptive diagnosis, which is prone to disputes and doubts. The present application provides a highly specific and sensitive serum biochemical marker for coronary artery spasm and its diagnostic threshold, which can achieve rapid and accurate cause diagnosis of CAS. The present application has been proven by experiments that the content of serum SELENBP1 has good diagnostic value for CAS and is superior to the commonly used CK-MB and cTn-I markers for diagnosing myocardial injury.

[0009] The present application also provides a corresponding kit containing anti-human SELENBP1 serum protein specific antibodies, color developing agents, detergents, standards, biotin, etc. Preferably, the kit for diagnosing coronary artery spasm of the present application contains reagents for quantitatively detecting the content of human serum SELENBP1 and anti-human SELENBP1 antibodies. The kit is particularly suitable for diagnosing coronary artery spasm in clinical medicine and forensic medicine.

[0010] In the embodiments of the present application, the above-mentioned human serum SELENBP1 was quantitatively detected in the serum of 21 healthy subjects and 25 CAS patients by ELISA. The results showed that the concentration of SELENBP1 in the serum of CAS patients was significantly lower than that of healthy subjects (p<0.001). Based on the quantitative results, ROC curve analysis of the SELENBP1 protein was further performed, which showed that the area under the curve (AUC) generated by the SELENBP1 protein was 0.9810 (p value <0.0001), and when the threshold was set to 186.5 ng / L, the diagnostic specificity was 100.0% and the sensitivity was as high as 96.00%. At the same time, the commonly used cTNT for diagnosing CAS had an area under the curve (AUC) of 0.7652 (p value = 0.0065), and when the threshold was set to 0.0275 ng / mL, the diagnostic specificity was 80.95% and the sensitivity was as high as 80.00%. The commonly used CK-MB for diagnosing CAS had an area under the curve (AUC) of 0.5306 (p value = 0.7508), and when the threshold was set to 17.40 U / L, the diagnostic specificity was 79.17% and the sensitivity was as high as 46.67%. The results showed that the serum SELENBP1 protein of the present application as a diagnostic marker can achieve more sensitive and specific clinical diagnostic effect.

[0011] In another embodiment of the present application, ELISA was used to quantitatively detect the serum SELENBP1 and two traditional indicators (CK-MB and cTnI) of 23 cases of dead human samples (11 cases of non-cardiac sudden death group and 12 cases of CAS death group). The results showed that the expression of CK-MB in the CAS group was significantly increased (p value was 0.0372), the content of SELENBP1 was significantly decreased (p value was <0.0001), and the expression difference of cTnI was not significant; based on the quantitative results, ROC curve analysis was carried out on the SELENBP1 protein, and the area under the curve (AUC) generated by the SELENBP1 protein was 0.9091 (p value was 0.0009), and when the threshold value was set to 127.7 ng / L, the diagnostic specificity was 72.73%, and the sensitivity was as high as 100%.

[0012] Compared with the area under the curve generated by the traditional myocardial injury indicator CK-MB, which was 0.7311 (p value was 0.0605), and when the threshold value was 187.7 ng / ml, the diagnostic sensitivity was 83.33%, and the specificity was 63.64%; and the area under the curve generated by cTnI was 0.5455 (p value was 0.7119), and when the threshold value was 1176 ng / L, the diagnostic sensitivity was only 25%, and the specificity was 100%. The results of the analysis of the human death cases in the present application showed that the use of SELENBP1 alone for diagnosing CAS death could obtain a sensitivity of 100% and a specificity of 72.73%, and in comprehensive consideration, the diagnostic performance of SELENBP1 in the death blood sample was better than that of the traditional myocardial damage indicator.

[0013] The present application provides a diagnostic marker for coronary artery spasm (CAS); in particular, the serum concentration change of SELENBP1 as a marker can be used in the preparation of clinical medicine and forensic diagnosis products for CAS. The detection method of the present application is non-invasive, the results are objective, and has the characteristics of more safety, and is suitable for clinical medicine and forensic application. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 (A) The concentration difference box plot of SELENBP1 in 21 cases of healthy human serum and 25 cases of CAS patient serum; (B) ROC curve analysis of the area under the curve, sensitivity and specificity of clinical serum SELENBP1 in diagnosing CAS, and *** p<0.001.

[0015] Figure 2The specificities and sensitivities of traditional biochemical markers such as CK-MB, cTnI and SELENBP1 serum protein in diagnosing CAS were displayed in the serum samples of dead bodies, wherein, (A, B) the content difference of CK-MB in the control group (ctrl) and the experimental group (coronary artery spasm CAS group) and the ROC curve for diagnosing CAS death; (C, D) the content difference of cTnI in the control group (ctrl) and the experimental group (coronary artery spasm CAS group) and the ROC curve for diagnosing CAS death; (E, F) the content difference of SELENBP1 in the control group (ctrl) and the experimental group (coronary artery spasm CAS group) and the ROC curve for diagnosing CAS death; NS, not significant (no significant statistical difference); *, p<0.05. ****, p<0.0001. DETAILED DESCRIPTION

[0016] The specific experimental materials, methods and steps in the embodiments of the application are as follows:

[0017] 1. Reagents

[0018] Pituitrin (5.1 U / kg) was purchased from Shanghai First Pharmaceutical Co., Ltd. Physiological saline (0.9% sodium chloride solution) was prepared in the laboratory.

[0019] The ELISA kit includes: anti-human CK-MB, cTnI, SELENBP1 detection kit, purchased from Jiangsu Zhenmian Biological Co., Ltd.

[0020] All the primary antibodies used in this experiment were verified by the manufacturer.

[0021] 2. Animal experiments

[0022] All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised in 1996). The protocol for animal experiments was approved by the Experimental Animal Ethics Committee of Fudan University School of Basic Medical Sciences (No. 20170223-004). Every effort was made to minimize animal suffering during the experiment.

[0023] Six male rabbits weighing about 220 g were purchased from the Experimental Animal Center of Fudan University and used for in vivo experiments in a 20-22℃, 12-hour light / dark cycle environment with free access to food and water. All experimental rabbits were acclimated to the environment for 1 week before any treatment. All experimental rabbits received pentobarbital sodium (50 mg / kg, i.p.) anesthesia. To construct a coronary artery spasm model, the internal carotid artery of the 6 experimental rabbits was opened after anesthesia, and a catheter was placed into the left anterior descending branch of the coronary artery, and a contrast agent was injected. Three rabbits were randomly selected for intracardiac injection of pituitrin (5.1 U / kg) to induce coronary artery spasm (CAS group), and the remaining 3 rabbits were injected with an equal amount of normal saline as a control group (Ctrl group). When the imaging showed that coronary artery spasm caused significant blood perfusion obstruction, the rabbits died, and immediately through the pre-placed internal carotid artery, 1 ml of arterial blood was drawn, centrifuged at 4℃, 12,000 rpm for 15 minutes, and the supernatant was stored at -80℃.

[0024] 3. Serum protein extraction and enzymolysis

[0025] The present application uses SDT (4% (w / v) SDS, 100 mM Tris / HCl, pH 7.6, 0.1 M DTT) lysis method to extract proteins from 6 experimental rabbit serum samples, and then uses BCA protein determination kit (Bio-Rad, USA) to determine the protein amount. An appropriate amount of protein is taken from each sample and subjected to trypsin enzymolysis using the Filter aided proteome preparation (FASP) method, desalted using C18 Cartridge, and the peptide segments are freeze-dried and resuspended in 40 μL of 0.1% formic acid solution; peptide segment quantification is performed under OD 280 conditions.

[0026] 4. SDS-PAGE separation

[0027] 20 μg of protein was taken from each of the 6 serum samples, mixed with 5x loading buffer, and boiled for 5 minutes; all samples were separated by 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel (constant current 14 mA, 90 minutes). Coomassie blue R-250 staining was used to visualize the protein bands.

[0028] 5. Label-free proteome analysis

[0029] Label-free technology has become an important mass spectrometry quantitative method in recent years. In the present invention, MS1 is used as the basis for calculating the integral of each peptide signal on the LCMS chromatogram for quantification. The advantage of Label-free technology is that it does not require complex labeling or processing of samples to achieve protein quantification and significant difference analysis of multiple groups of samples. The present process is divided into two parts: preliminary experiment and formal experiment. The preliminary experiment includes protein extraction, protein quantification, SDS-PAGE, protein digestion, LC-MS / MS analysis, database query, quality control, and preparation of preliminary experiment report. The formal experiment is based on the preliminary experiment. The samples that pass the quality control in the preliminary experiment are used to obtain mass spectrometry raw data by using a high-resolution mass spectrometer for formal experiment. Then, MaxQuant software (version number 1.5.3.17) is used for database search, and LFQ (Label Free Quantitation) algorithm is used for quantitative analysis.

[0030] 6. LC-MS / MS data acquisition

[0031] The samples of the CAS group and the Ctrl group are separated by a nanoliter flow rate HPLC liquid phase system Easy nLC. Buffer A is 0.1% formic acid aqueous solution, and buffer B is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%). The chromatographic column is equilibrated with 95% A liquid, and the sample is loaded onto the loading column (Thermo Scientific Acclaim PepMap100, 100μm*2cm, nanoViper C18) by the automatic sampler, separated by the analysis column (Thermo scientific EASY column, 10cm, ID75μm, 3μm, C18-A2), and the flow rate is 300nL / min.

[0032] After chromatographic separation, the samples were analyzed by mass spectrometry using a Q-Exactive mass spectrometer. The detection mode was positive ion, the parent ion scan range was 300-1800 m / z, the first mass spectrometry resolution was 70,000 at 200 m / z, the AGC (Automatic gain control) target was 1e6, the Maximum IT was 50 ms, and the dynamic exclusion time was 60.0 s. The mass-to-charge ratio of polypeptides and polypeptide fragments was collected according to the following method: 20 fragment maps (MS2 scan) were collected after each full scan, the MS2 Activation Type was HCD, the Isolation window was 2 m / z, the secondary mass spectrometry resolution was 17,500 at 200 m / z, the Normalized Collision Energy was 30 eV, and the Underfill was 0.1%.

[0033] 7. Statistical and bioinformatic analysis

[0034] The present application adopts a data-dependent acquisition (DDA) method to collect proteomics raw data, uses MaxQuant software (version number 1.5.3.17) to perform database search, and then uses an LFQ (Label Free Quantitation) algorithm to perform quantitative analysis, with Peptide FDR≤0.01 and Protein FDR≤0.01 as screening standards to detect high-quality data. The basic information of the source of the human sample, such as the age, CK-MB and cTnI, is expressed as an average value ± standard deviation (SD). The R 4.0.3 tool is used to perform protein double clustering and PCA analysis on the data. GraphPad Prism 8.2.0 statistical software (GraphPad, La Jolla, CA, USA) is used to perform Venn diagram, volcano plot and ROC curve analysis. Blast2GO is used to perform GO annotation on the target protein set. KAAS (KEGG Automatic Annotation Server) software is used to perform KEGG pathway annotation on the target protein set. MedCalc (v19.2.6) is used to perform two-factor ROC curve statistical analysis. The present application experiment uses unsupervised principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models of proteomic data. The r3.6.0 tool is used to perform PCA and PLS-DA analysis on the proteomic data, and double clustering and volcano plot analysis are performed on the DAPs. Blast2GO (version 3.3.5) is searched in the process of gene ontology mapping and annotation. On the basis of Fisher's exact test, GO annotation enrichment analysis of three modules of biological processes (GO-BP), molecular functions (GO-MF) and cellular components (GO-CC) is applied, and p<0.05 is considered as a statistically significant functional category.

[0035] 8. Multiple reaction monitoring (PRM) validation

[0036] The present application further performs LC-PRM analysis on 40 selected proteins from the same sample to test the protein abundance obtained previously by label-free. After preparing the peptides, non-labeled analysis is performed according to the protocol, and TOMHAQ is used for verification (Shanghai Applied Protein Technology Co., Ltd., Shanghai, China); the raw data is analyzed using the bioinformatics tool Skyline (University of Washington, Seattle, WA, USA, MacCoss Lab); the signal intensity of the peptide sequence of the significantly changed protein is quantified relative to each sample, and is standardized to a standard reference.

[0037] 9. Human sample verification and ethical statement

[0038] The clinical samples in the present application come from Zhongshan Hospital Affiliated to Fudan University, a total of 46 cases of serum of patients, of which 21 cases are healthy physical examination, and 25 cases are CAS cases. The healthy physical examination is a group of people who come to Zhongshan Hospital Affiliated to Fudan University for routine annual examination. The CAS group is the patient admitted by the department of cardiology, which is clearly a CAS patient through symptoms, predisposing factors, characteristics of onset, coronary angiography and calcium ion antagonist treatment effective. The use of clinical serum samples is approved by the Human Ethics Review Committee of Zhongshan Hospital Affiliated to Fudan University.

[0039] The forensic samples in the present application come from the Department of Forensic Medicine of the Basic Medical School of Fudan University, a total of 23 cases of serum samples of death cases, of which 11 cases of control serum samples include 4 cases of death due to respiratory depression caused by alcoholism, 7 cases of death due to hemorrhagic shock caused by mechanical trauma, and 12 cases of death due to acute cardiac ischemia caused by coronary artery spasm. The 12 cases of coronary artery spasm cases were subjected to systematic postmortem autopsy, and obvious heart disease, drug poisoning was excluded, combined with the symptoms before death, the morphological changes of coronary artery, and the judgment was made comprehensively; the heart blood of each case was collected. The written informed consent of the family members of each deceased patient for the use of these samples for research purposes was obtained. The samples were collected in accordance with the Declaration of Helsinki. The use of human samples is approved by the Human Ethics Review Committee of the Basic Medical School of Fudan University. In animal experiments, this program is approved by the Experimental Animal Ethics Committee of the Basic Medical School of Fudan University.

[0040] 10. Blood sample collection and processing

[0041] The postmortem heart blood of the forensic human cases was collected within 4 hours after death, and centrifuged at 4℃, 12,000 rpm for 15 minutes. The supernatant of each sample was collected after centrifugation, and stored at -80℃ until ELISA experiment.

[0042] 11. ELISA bioquantification

[0043] The present application determines the content level of 46 cases of SELENBP1 in clinical medicine, and the content level of CK-MB, cTnI and SELENBP1 in 23 cases of human serum samples in forensic medicine, which is determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.

[0044] 12. Statistical analysis

[0045] The data is shown as mean ± standard deviation. Non-paired t test is used for comparison between 2 groups. The receiver operating characteristic curve analysis is performed by graphpad8.0.

[0046] Example 1 Proteomics and PRM verification screen 15 candidate proteins for diagnosing coronary artery spasm

[0047] Through coronary angiography, it was found that the coronary blood perfusion was unobstructed at different times when normal saline was perfused into the coronary artery. However, when an equal volume of pituitrin was injected, the coronary blood perfusion of the rabbits was significantly slowed down, and the diameter of some coronary arteries became narrower, that is, coronary artery spasm of the rabbits was successfully induced. Then, label-free proteomics analysis was performed on 6 serum samples from the CAS group and the Ctrl group to preliminarily screen out candidate proteins. Among the 377 high-abundance protein data detected, 310 proteins were identified in both groups, with 45 specific proteins in the Ctrl group and 22 specific proteins in the CAS group. Next, the expression difference fold change (CAS / Ctrl) ≧ 2 (i.e., log2 (CAS / Ctrl) ≧ 1) and p-value < 0.05 (i.e., -log 10 Using the two factors (p-value)≧1.3) as the criteria, volcano plot analysis and heat map analysis were performed on 325 proteins that could be detected in both groups. The heat map showed that the proteomics data had good consistency within the group and had obvious discrimination for CAS samples. PRM targeted validation was further performed on the 67 proteins screened by label-free proteomics. Using p < 0.05 and fold change ≧ 2 times as the screening criteria, a total of 28 peptides from 15 proteins were identified, of which 11 proteins had the same trend as the initial proteomics screening. Subsequently, principal component analysis (PCA) was used to evaluate the discriminatory power of this data for the CAS group. The top 10 proteins with the highest fold difference were taken as parameters, and the first two principal components of PCA accounted for 68.1% of the total variability. The first part accounted for 42.5%, which was positively correlated with proteins such as G1TM88 (SERPINA3), and negatively correlated with proteins such as G1TT06 (SELENBP1), G1SJ56 (VCL), G1SGW4 (Proteasome subunit beta), and P29751 (ACTB); the second part accounted for 25.6%, which was positively correlated with G1TT06 (SELENBP1) protein, and negatively correlated with the remaining proteins.

[0048] Example 2 SELENBP1 serum protein can be used as a new marker to effectively distinguish the CAS group from the Ctrl group in clinical serum samples

[0049] Based on the experimental example 1, this embodiment collects the serum samples of the experimental animals with coronary artery spasm for proteomics analysis, and then performs PRM targeted analysis to confirm that there are differences in serum proteomics between the CAS group and the Ctrl group, which can be distinguished by dozens of proteins. The highest difference ratio of SELENBP1 protein is particularly noteworthy. 46 clinical serum samples were collected for detection, including 21 healthy subjects and 25 CAS patients. The results showed that compared with healthy subjects, the concentration of SELENBP1 in the serum of CAS patients was significantly reduced by 42.2% (average 271.1 ng / L in healthy subjects vs. average 156.7 ng / L in CAS group, p<0.001, Figure 1 A). Based on the quantitative results, further ROC curve analysis of SELENBP1 protein was performed, and the results showed that the area under the curve (AUC) generated by SELENBP1 protein was 0.9810 (p value <0.0001), and when the threshold was set to 186.5 ng / L, the diagnostic specificity reached 100.0%, and the sensitivity was as high as 96.00% (B). Figure 1 B).

[0050] cTNT and CK-MB are commonly used myocardial enzymes for clinical detection of myocardial injury. Experiments show that in the diagnosis of CAS, the area under the curve (AUC) generated by cTNT is 0.7652 (p value = 0.0065), and when the threshold is set to 0.0275 ng / mL, the diagnostic specificity reaches 80.95%, and the sensitivity is as high as 80.00%. The area under the curve (AUC) of the commonly used CK-MB for the diagnosis of CAS is 0.5306 (p value = 0.7508), and when the threshold is set to 17.40 U / L, the diagnostic specificity reaches 79.17%, and the sensitivity is as high as 46.67%. The results show that the serum SELENBP1 protein can obtain more sensitive and specific clinical diagnostic effect.

[0051] Example 3 SELENBP1 serum protein as a new indicator can more effectively distinguish CAS group and Ctrl group in forensic samples.

[0052] Further, the more serious situation of coronary artery spasm (causing death) was investigated. In this experiment, human samples caused by acute ischemic death after coronary artery spasm were used. Through screening, 12 CAS samples died of sudden acute myocardial infarction and had no obvious coronary heart disease history, and 11 human serum samples died of trauma such as car accident, falling, assassination, and alcoholism were used as controls.

[0053] Based on the fact that the most common traditional biological indicators that can be used in clinical practice to assist in the diagnosis of coronary artery spasm are CK-MB and cTnI, combined with the candidate protein SELENBP1 screened out in this study, this experiment performed ELISA quantitative detection on the three serum indicators. The results showed that the expression of CK-MB was significantly increased in the CAS group (p value was 0.0372, Figure 2 A), while the difference in cTnI expression was not significant ( Figure 2 C), the content of SELENBP1 in the CAS lethal group was significantly decreased (p value < 0.0001, Figure 2 E). Based on the quantitative results, ROC curve analysis of the three proteins showed that CK-MB performed well among the traditional indicators. When the threshold value was 187.7 ng / mL, the diagnostic sensitivity reached 83.33% and the specificity reached 63.64%. However, the area under the curve was 0.7311 (p value 0.0605, Figure 2 B); while the area under the curve of cTnI was 0.5455 (p value was 0.7119), with a threshold of 1176 ng / L, the specificity was 100%, but the sensitivity was only 25% ( Figure 2 D). The area under the curve (AUC) of SELENBP1 protein was 0.9091 (p value was 0.0009). When the threshold was set at 127.7 ng / L, its specificity reached 72.73% and its sensitivity was as high as 100% ( Figure 2 F). The results showed that the diagnostic sensitivity of serum SELENBP1 for diagnosing CAS-related mortality was significantly higher than that of traditional indicators, while its specificity was similar to that of traditional indicators.

Claims

1. Application of serum selenoprotein SELENBP1 as a marker in the preparation of a product for diagnosing coronary artery spasm in clinical medicine.

2. Use according to claim 1, wherein The application is to determine the serum SELENBP1 protein concentration from a clinical patient suspected of having coronary artery spasm.

3. Use according to claim 2, wherein the compound is ###0002### In the application, a serum SELENBP1 protein concentration level below the corresponding threshold indicates that the patient has developed coronary artery spasm.

4. Use according to claim 3, wherein the compound is ###0002### The SELENBP1 threshold is 186.5 ng / L.

5. Application of serum selenoprotein SELENBP1 as a marker in the preparation of a product for diagnosing coronary artery spasm in forensic medicine.

6. Use according to claim 5, wherein The application is to determine the serum SELENBP1 protein concentration from a deceased person suspected of having coronary artery spasm.

7. Use according to claim 6, wherein In the application, a serum SELENBP1 protein concentration level below the corresponding threshold indicates that the coronary artery spasm caused sudden cardiac death.

8. Use according to claim 7, wherein the compound is ###0002### The SELENBP1 threshold is 127.7 ng / mL.

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