Creld2 as a biomarker for atherosclerosis

Abstract

The application relates to the field of biological medicine, and particularly discloses Creld2 as a biomarker of atherosclerosis. For the first time, the Creld2 level of mouse models of obesity, diabetes, hyperlipidemia and atherosclerosis is detected, and it is found that the serum Creld2 level is greatly increased only when atherosclerosis occurs. The increase of the Creld2 level is related to the development process of atherosclerosis, and with the aggravation of atherosclerotic plaques, the serum Creld2 level is further increased. The serum Creld2 level of the atherosclerosis model group is greatly increased compared with the normal control group, and the Creld2 level ranges of the two groups do not overlap, so that the Creld2 is taken as the biomarker of atherosclerosis and has high diagnostic efficiency, can be used as an early blood diagnostic marker of atherosclerosis, is beneficial to low-cost, large-scale screening and monitoring, can be used for early diagnosis and treatment of atherosclerosis, and improves the life quality of patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to Creld2 as a biomarker for atherosclerosis. Background Technology

[0002] CRELD2 is a cysteine-rich epidermal growth factor-like domain, belonging to the epidermal growth factor (EGF) superfamily and a novel secreted protein homologous to CRELD1. The CRELD2 gene was first recognized for its role in regulating endoplasmic reticulum stress, specifically in its involvement in endoplasmic reticulum stress signaling and unfolded protein responses; it also participates in the intracellular transport of acetylcholine receptor α4 and β2 subunits. In bone marrow mesenchymal stem cells, CRELD2 enhances osteogenic and matrix mineralization induced by bone morphogenetic protein-9, and binds to cartilage matrix protein-3, playing a fundamental role in cartilage development. Furthermore, CRELD2 is a paracrine factor of tumor-associated fibroblasts in the PERK-ROCK pathway, participating in the formation of the tumor microenvironment; studies have also shown that CRELD2 plays an important role in maintaining and regulating hepatic metabolic homeostasis. In addition, CRELD2 has the potential to serve as a biomarker for diagnosing artificial joint infections and for early prediction of postoperative acute kidney injury.

[0003] The inventors' research group previously conducted studies on the expression level of CRELD2 in mouse tissues. Using real-time quantitative polymerase chain reaction and Western blotting, they determined the expression levels in various mouse tissues. The results showed that CRELD2 is ubiquitous in all tissues, but the relative expression levels among tissues were not entirely consistent. Subsequently, a 2022 patent document disclosed that CRELD2 can counteract intestinal damage caused by noxious stimuli. Furthermore, a 2019 patent document disclosed that CRELD2 can serve as a pathogenic and / or susceptibility gene for congenital heart disease. Currently, there are very few reports on the function of the CRELD2 protein and gene, and its relationship with cardiovascular diseases is rarely reported.

[0004] Atherosclerosis is the main pathological basis of cardiovascular disease, leading to serious complications such as heart disease and stroke. Early diagnosis helps in timely intervention, delaying or stopping disease progression, thereby reducing morbidity and mortality. Currently, clinical diagnosis of atherosclerosis mainly relies on imaging examinations, such as vascular ultrasound, CTA, or angiography, while blood tests are relatively non-invasive, low-cost, and more suitable for large-scale screening and monitoring. Therefore, the development of high-efficiency blood diagnostic biomarkers for early atherosclerosis is of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing Creld2 as a biomarker for atherosclerosis. By detecting the Creld2 protein level in the individual being tested, it can be used to diagnose whether or not atherosclerosis has been diagnosed.

[0006] In a first aspect, the present invention provides the application of Creld2 protein as a diagnostic marker in the preparation of diagnostic reagents or kits for atherosclerosis.

[0007] Secondly, the present invention provides the use of reagents for detecting Creld2 protein levels, for the preparation of diagnostic reagents or kits for atherosclerosis.

[0008] As another preferred embodiment of the present invention, the reagent for detecting Creld2 protein levels is a specific antibody against Creld2 protein, including monoclonal antibodies or polyclonal antibodies.

[0009] As another preferred embodiment of the present invention, the atherosclerosis diagnostic reagent or kit further includes an enzyme-linked immunosorbent assay (ELISA) reagent or related reagent, which is other reagent or system required for performing routine ELISA reactions.

[0010] As another preferred embodiment of the present invention, the diagnostic sample of the atherosclerosis diagnostic reagent or kit is blood, plasma or serum.

[0011] Thirdly, the present invention provides a kit for diagnosing atherosclerosis, the kit comprising reagents for detecting Creld2 protein levels.

[0012] As another preferred embodiment of the present invention, the reagent for detecting Creld2 protein levels is a specific antibody against Creld2 protein, including monoclonal antibodies or polyclonal antibodies.

[0013] As another preferred embodiment of the present invention, the atherosclerosis diagnostic reagent or kit further includes an enzyme-linked immunosorbent assay (ELISA) reagent or related reagent, which is other reagent or system required for performing routine ELISA reactions.

[0014] As another preferred embodiment of the present invention, the diagnostic sample of the atherosclerosis diagnostic reagent or kit is blood, plasma or serum.

[0015] The advantages of this invention are:

[0016] This invention is the first to detect Creld2 levels in mouse models of obesity, diabetes, hyperlipidemia, and atherosclerosis. It found that only the atherosclerosis model showed a significant increase in serum Creld2 levels. The elevated Creld2 level is related to the development and progression of atherosclerosis. In 8-week-old model mice, no obvious atherosclerosis was detected, and the serum Creld2 levels were not significantly different from the control group. As age increased, atherosclerosis gradually worsened, and the Creld2 levels in 16-week-old model mice were significantly different from the control group. Furthermore, as the atherosclerotic plaques worsened, the serum Creld2 levels increased further. In this invention, the serum Creld2 level in the atherosclerosis model group was significantly higher than that in the normal control group. The Creld2 level ranges in the atherosclerosis model group and the normal control group did not overlap, and the serum Creld2 level in the atherosclerosis model animals was also much higher than that in obese, diabetic, and hyperlipidemic model animals. Therefore, using the Creld2 gene or protein as a biomarker for atherosclerosis has high diagnostic efficacy and can serve as an early blood diagnostic marker for atherosclerosis. This facilitates low-cost, large-scale screening and monitoring, enabling early diagnosis and treatment of atherosclerosis and improving the quality of life for patients. Attached Figure Description

[0017] Figure 1 Serum CRELD2 levels in obese and diabetic mice. (A) Body weight, (B) tissue weight of periepididymal adipose tissue (PAT), liver, muscle, and heart, and tissue weight to body weight ratios (PAT / body weight, liver / body weight, muscle / body weight, heart / body weight), (C) Blood glucose, (D) Serum lipid concentrations (triglycerides: TG, total cholesterol: TC, low-density lipoprotein cholesterol: LDL-C, high-density lipoprotein cholesterol: HDL-C), and (E) Serum CRELD2 levels in obese and diabetic mice. n = 5 animals per group. ***P < 0.001, **P < 0.01, *P < 0.05; NS, no significant difference. All data are mean ± standard error.

[0018] Figure 2 Blood CRELD2 levels in hyperlipidemic and atherosclerotic mice. (A) ApoE - / - Oil Red O staining of mouse aorta. C57BL / 6J mice, high-fat fed mice (C57+HFD), and ApoE mice. - / -(B) Blood glucose, (C) Serum lipid concentrations (triglycerides: TG, total cholesterol: TC, low-density lipoprotein cholesterol: LDL-C, high-density lipoprotein cholesterol: HDL-C), and (D) Serum CRELD2 levels in mice. n = 9 animals per group. ***P < 0.001, **P < 0.01, *P < 0.05 vs C57; NS, no significant difference. All data are mean ± standard error.

[0019] Figure 3 Blood Creld2 levels in mice with atherosclerosis and ischemic stroke. A. ApoE - / - Preoperative body weight of mice and C57 mice. n = 7-8 per group. B. ApoE - / - Representative images of TTC staining in brain slices from mice and C57 mice 24 hours post-surgery. (C. ApoE) - / - A representative image of atherosclerosis in the aortic arch and branches, and bilateral carotid arteries and branches in mice. (D.ApoE) - / - Serum Creld2 levels in mice (Sham and MCAO) and C57 mice (Sham) 24 hours post-surgery. n = 7–8 per group. **P < 0.01; ns, no statistical significance. All data are mean ± standard error.

[0020] Figure 4 A. Body weight, blood lipids, blood Creld2 levels, and atherosclerotic plaque formation in mice. A. ApoE levels at different ages. - / - Body weight of mice and C57 mice. BE. ApoE at different ages. - / - Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mice and C57 mice. F. ApoE at different ages. - / - Serum Creld2 levels in mice and C57 mice. G. ApoE levels in different age groups. - / - Representative images of Oil Red O staining in the aortic root of mice and C57 mice, scale bar at 500 μm (left) and 200 μm (right). H. Statistical analysis of Oil Red O (ORO) plaque area in the aortic root. 8-week-old animals, n=9 per group; 18-month-old animals, n=7 per group; other groups, n=5 per group. *P<0.05, ***P<0.001 vs C57 8 weeks; # P<0.05, ### P<0.001 vs ApoE - / - 8 weeks; && P<0.01 vs ApoE - / - 16 weeks; $ P<0.05, $$P<0.01, $$$ P < 0.001. All data are mean ± standard error. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0022] Creld2 protein

[0023] The Creld2 protein used in this invention can be naturally occurring, for example, it can be isolated and purified from mammals. Alternatively, the Creld2 protein can be artificially prepared, for example, using conventional genetic engineering techniques. Any suitable Creld2 protein is applicable to this invention. The Creld2 protein includes the full-length Creld2 protein or its biologically active fragment. As a specific embodiment, the amino acid sequence of the Creld2 is identical or substantially identical to the amino acid sequence disclosed in the patent specification with application number CN2022106123809.

[0024] Furthermore, the amino acid sequence of the Creld2 protein, formed by the substitution, deletion, or addition of one or more amino acid residues, is also included in this invention. The Creld2 protein or its bioactive fragment includes a sequence of substitutions for a portion of conserved amino acids, the substitutions of which do not affect its activity or retain a portion of its activity. Appropriate amino acid substitutions are well-known techniques in the art, which can be readily implemented and ensure that the biological activity of known molecules is not altered. These techniques have made it clear to those skilled in the art that, generally, changing a single amino acid in a non-essential amino acid region of a polypeptide does not change its biological activity.

[0025] Any bioactive fragment of the Creld2 protein can be used in this invention. Here, a bioactive fragment of the Creld2 protein means a polypeptide that retains all or part of the function of the full-length Creld2 protein. Typically, the bioactive fragment retains at least 50%, 60%, and up to 99% or 100% of the activity of the full-length Creld2 protein.

[0026] This invention can also employ a modified or altered Creld2 protein, comprising all or some of its amino acids. For example, a Creld2 protein modified or altered to improve half-life, efficacy, metabolism, and / or protein potency. The modified or altered Creld2 protein can be a conjugate of a Creld2 protein, or a substitute or artificial amino acid thereof. The modified or altered Creld2 protein or gene may differ from the natural Creld2 protein or gene to some extent, but still possess the functions described in this invention without causing other adverse reactions or toxicity. In other words, any variation that does not affect the biological activity of the Creld2 protein or the biological function of the gene can be used in this invention.

[0027] Example 1: Blood Creld2 levels in patients with obesity, diabetes, hyperlipidemia, and atherosclerosis

[0028] I. Experimental Methods

[0029] (1) Animal source

[0030] Obese and diabetic mouse models were created using leptin-deficient ob / ob mice (also known as Leptin-deficient mice). ob / Lep ob Mice). Male ob / ob mice and their control C57BL / 6J mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0031] The hyperlipidemic C57BL / 6 mouse model was established by feeding mice with a high-fat diet (HFD) containing 42% fat for energy for 5 consecutive weeks. Male C57BL / 6 mice were purchased from Shanghai Bikaico Biotechnology Co., Ltd.

[0032] The mouse model of atherosclerosis is the apolipoprotein E (ApoE) gene knockout (ApoE) model. - / - Male mice aged 13 months and older. ApoE - / - The mice were constructed using CRISPR / Cas9 technology and purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.

[0033] (2) Blood glucose measurement

[0034] Before blood glucose testing, the experimental mice were fasted but allowed to drink water. The fasting period was from 8:00 PM to 8:00 AM the next day (12 hours of fasting). The following morning, the mice were placed individually in a quiet, undisturbed environment for 40 minutes, allowing them to move freely. At 8:00 AM, the blood glucose of the mice was measured by cutting off 1-2 mm from the end of the mouse's tail and dropping a drop of blood from the tail end onto a test strip of a GA-3 blood glucose meter.

[0035] (3) Animal sampling and weight determination

[0036] On the day of sampling, mice were weighed and then anesthetized by intraperitoneal injection of 1% sodium pentobarbital (100 mg / kg). The heart was dissected and exposed, and blood was collected from the inferior vena cava at the junction of the heart using a 1 mL syringe. After being stored at room temperature for 2 hours, the blood was centrifuged at 4°C for 3000 g and 15 min to obtain serum samples. After perfusion of the heart with 1xPBS, the perididymal adipose tissue (PAT), liver, heart, and gastrocnemius muscle were dissected and the tissue weights were obtained.

[0037] (4) Serum lipid profile test

[0038] The four lipid parameters in mouse serum were measured by Wuhan Saiweier Biotechnology Co., Ltd. A fully automated biochemical analyzer (Shenzhen Raydu Life Science Co., Ltd.) and reagent kits (S03027, S03042, S03025, S03029, Shenzhen Raydu Life Science Co., Ltd.) were used to detect triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum.

[0039] (5) Serum CRELD2 level measurement

[0040] Serum CRELD2 concentration was detected using enzyme-linked immunosorbent assay (ELISA). The sample loading volume of serum was 50 μL. The assay method and procedure were based on the Mouse CRELD2 ELISA Kit (EK1984, Wuhan Boster Biological Engineering Co., Ltd.). Serum CRELD2 concentration was obtained by conversion from the standard curve.

[0041] II. Experimental Results

[0042] like Figure 1 As shown in A-1D, compared with control C57BL / 6J wild-type mice, ob / ob mice showed significantly increased body weight, tissue weight (PAT, liver), tissue weight to body weight ratio (PAT / body weight, liver / body weight), blood glucose, and serum TC, and significantly decreased tissue weight (muscle), tissue weight to body weight ratio (muscle / body weight, heart / body weight), and serum LDL-C, demonstrating that ob / ob mice are a model of obesity and type II diabetes. Further analysis of serum CRELD2 levels in ob / ob mice and their control C57BL / 6J wild-type mice revealed that although the mean serum CRELD2 concentration in ob / ob mice was higher than that in normal control mice, there was no statistically significant difference between the two groups. Figure 1 E). This result suggests that obesity and type 2 diabetes did not significantly affect blood CRELD2 levels.

[0043] In a hyperlipidemic mouse model, compared with standard-diet C57BL / 6J mice, high-fat-fed mice (C57+HFD) had higher blood glucose and TC, LDL-C, and HDL-C concentrations, and lower TG concentrations. Figure 2 (B-2C). Serum CRELD2 measurements showed a trend of higher serum CRELD2 concentrations in C57+HFD mice, but no statistically significant difference compared to C57BL / 6J mice. These results suggest that high-fat diet-induced hyperlipidemia did not significantly affect serum CRELD2 levels.

[0044] ApoE for atherosclerosis was also detected. - / - Mouse model. For example... Figure 2 As shown in Figure A, the 9 ApoEs used in this experiment - / - Obvious atherosclerotic plaques were observed in the aorta and its branches of the mice, consistent with a disease model. Compared with C57BL / 6J normal control mice, ApoE... - / - There was no significant difference in blood glucose levels in mice, but TG, TC, and LDL-C were significantly increased, while HDL-C was significantly decreased. Figure 2 B-2C). ApoE - / - The serum CRELD2 levels of mice and C57BL / 6J normal control mice are shown in the table below. (ApoE) - / - Serum CRELD2 concentrations in mice were significantly higher than in C57BL / 6J wild-type mice. Figure 2 D). This result suggests that blood CRELD2 levels are significantly elevated in atherosclerosis.

[0045] Table 1. ApoE in Atherosclerosis - / - Serum Creld2 levels in mice and normal control C57BL / 6 mice

[0046]

[0047] Example 2: Blood Creld2 Levels in Atherosclerosis and Ischemic Stroke

[0048] I. Experimental Methods

[0049] Apolipoprotein E (ApoE) gene knockout (ApoE) used as a model of atherosclerosis - / - Mice were constructed using CRISPR / Cas9 technology and purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. C57BL / 6 mice were purchased from Shanghai Bicoyte Biotechnology Co., Ltd. Mice were fed a standard diet; male C57BL / 6 mice were 9 months old, and male ApoE mice... - / -Mice were used in the experiment at 13 months of age. Mice underwent middle cerebral artery occlusion (MCAO) via electrocoagulation under intraperitoneal anesthesia with sodium pentobarbital (70 mg / kg) to establish a permanent ischemic stroke model. Control mice underwent sham surgery. Twenty-four hours post-surgery, mice were euthanized after blood was collected from the inferior vena cava at the junction of the heart and administered an overdose of sodium pentobarbital (100 mg / kg) intraperitoneal injection. Brain tissue was collected and stained with 2,3,5-triphenyltetrazolium chloride (TTC) to confirm ischemic stroke in all MCAO animals. The aortic arch and branches, and bilateral carotid arteries and branches were dissected to confirm ApoE. - / - All mice exhibited obvious atherosclerotic plaques. After blood samples were allowed to stand at room temperature for 2 hours, serum was separated at 3000g and 4℃ for 15 minutes, placed into sterile tubes, and stored at -80℃ until detection. Serum Creld2 levels were measured according to the instructions of the mouse Creld2 ELISA kit (EK1984, Wuhan Boster Biological Engineering Co., Ltd.), with a sample loading volume of 50 μL.

[0050] II. Experimental Results

[0051] like Figure 3 As shown. There was no significant difference in preoperative body weight among the three groups of mice. Figure 3 A). TTC staining showed significant cerebral infarction in animals 24 hours after MCAO, and all ApoEs were present. - / - Obvious atherosclerotic plaques were observed in the aorta, bilateral carotid arteries, and their branches of the mice, demonstrating that the disease model conforms to (…). Figure 3 B and C). Compared with normal control C57BL / 6 mice undergoing sham surgery, atherosclerotic ApoE... - / - Serum Creld2 levels were significantly elevated in mice after sham surgery (4.3 times the normal level), indicating atherosclerosis (ApoE). - / - Serum Creld2 levels in mice were also significantly elevated 24 hours after MCAO treatment (4.6 times the normal level). However, ApoE... - / - There was no significant difference in serum Creld2 levels between the sham-operated and MCAO groups in mice. Figure 3 D). These results suggest that blood Creld2 levels are significantly elevated in atherosclerosis, while ischemic stroke may slightly increase blood Creld2 levels, but this increase is not statistically significant.

[0052] Example 3: Changes in blood Creld2 levels during the development and progression of atherosclerosis

[0053] I. Experimental Methods

[0054] Male apolipoprotein E (ApoE) gene knockout (ApoE) - / - Mice were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. (introduced from Jackson Laboratory, USA, animal number 002052). Male C57BL / 6 mice were purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. Mice were fed a standard diet. C57BL / 6 mice were ApoE at 8, 16, and 24 weeks of age. - / - Mice were used in experiments at 8, 16, 24 weeks, and 18 months of age. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg / kg), and blood was collected from the inferior vena cava at the junction of the heart and aortic arch. The blood samples were allowed to stand at room temperature for 2 hours, then serum was separated at 3000g and 4℃ for 15 minutes, placed in sterile tubes, and frozen at -80℃ until testing. Four lipid indicators were measured using an automated biochemical analyzer and kits (Shenzhen Raydu Life Sciences Co., Ltd.) by Wuhan Saiwei Biotechnology Co., Ltd. Serum Creld2 levels were measured according to the instructions of the mouse Creld2 ELISA kit, with a sample loading volume of 100 μL. The aortic arch and heart were fixed in paraformaldehyde for 24 hours, and the aortic root was frozen sectioned. The sections were hydrated in 1x PBS, stained with Oil Red O for 15 minutes, observed and photographed under a microscope, and the Oil Red O staining area was measured as an indicator of atherosclerosis.

[0055] II. Experimental Results

[0056] like Figure 4 As shown. Under the same age conditions, C57 and ApoE... - / - There was no significant difference in mouse body weight. Weight gradually increased with age; while the body weight of 18-month-old mice was higher than that of 8-week-old mice, it was lower than that of 16- and 24-week-old mice. Figure 4 A). Compared to C57 of the same age, ApoE - / - Total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were significantly elevated in the blood of mice, and ApoE levels were significantly increased in 18-month-old mice. - / - The TC and LDL-C levels in mice were also significantly higher compared to C57 mice; triglycerides (TG) were only elevated at 8 and 16 weeks of age in ApoE. - / - Mice showed significantly elevated ApoE levels in high-density lipoprotein cholesterol (HDL-C) across all age groups. - / - There was no significant difference between the mice and the C57 mice. Figure 4 BE). ApoE - / -Atherosclerosis was not detectable in the thoracic and abdominal aortas of 8-week-old mice; therefore, the aortic root (a more sensitive site for atherosclerosis detection) was used for observation. In C57 mice aged 8-24 weeks, Oil Red O staining of the aortic root was not obvious, and ApoE staining was... - / - Oil Red O staining of the aortic root in 8-week-old mice showed no significant difference compared to C57 mice, indicating no obvious atherosclerosis. However, with increasing age, ApoE staining... - / - The presence of Oil Red O staining in the aortic root of mice at 16, 24, and 18 weeks of age gradually increased, indicating that atherosclerosis gradually worsens with age. Figure 4 GH). Meanwhile, serum Creld2 levels also significantly increase with the occurrence and aggravation of atherosclerosis. Figure 4 F), while at 8 weeks of age, blood Creld2 levels were at ApoE - / - There was no significant difference between ApoE mice and C57 mice. These results suggest that ApoE... - / - At 8 weeks of age, mice showed significant changes in blood lipids, but no obvious atherosclerosis or changes in blood Creld2 levels. However, after 16 weeks of age, obvious atherosclerotic plaques appeared at the aortic root, and serum Creld2 levels increased significantly. As the plaques worsened, serum Creld2 levels increased further. This suggests that blood Creld2 levels can serve as a biomarker for atherosclerosis.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Application of Creld2 protein as a diagnostic biomarker in the preparation of diagnostic reagents or kits for atherosclerosis.

2. The purpose of the reagent for detecting Creld2 protein levels, characterized in that, Used to prepare diagnostic reagents or kits for atherosclerosis.

3. The use according to claim 2, characterized in that, The reagent used to detect Creld2 protein levels is a specific antibody against Creld2 protein.

4. The use according to claim 3, characterized in that, The diagnostic reagents or kits for atherosclerosis also include enzyme-linked immunosorbent assay (ELISA) reagents.

5. The use according to claim 4, characterized in that, The test refers to a blood, plasma, or serum test.

6. A reagent kit for diagnosing atherosclerosis, characterized in that, This includes reagents for detecting Creld2 protein levels.

7. The reagent kit according to claim 6, characterized in that, The reagent used to detect Creld2 protein levels is a specific antibody against Creld2 protein.

8. The reagent kit according to claim 7, characterized in that, The atherosclerosis diagnostic kit also includes an enzyme-linked immunosorbent assay (ELISA) reagent.

9. The reagent kit according to claim 8, characterized in that, The test refers to a blood, plasma, or serum test.

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