TW201323879A - Soluble CD14 as a biomarker for inspecting coronary artery disease - Google Patents

Abstract

The present invention provides a method for inspecting, preliminary screening or detecting coronary artery disease (CAD) in an individual human body, in which if the level of soluble CD14(sCD14) in an urine sample of the human individual is higher than a standard value, the human individual will be identified as having coronary artery disease.

Description

Soluble CD14 as a biomarker for detecting coronary artery disease

The present invention relates to soluble CD14 (sCD14) in urine as a biomarker for detecting coronary artery disease (CAD). In particular, the present invention provides a method for detecting, initially screening or monitoring a coronary artery disease in a human subject, wherein the human individual has a sCD14 level higher than a standard value in the urine sample of the human individual It is considered to have coronary artery disease.

Coronary artery disease [also known as coronary heart disease (CHD) or atherosclerotic heart disease] is a cardiovascular disease caused by stenosis or obstruction of the coronary arteries. Cardiovascular disease is one of the leading causes of global human death today. Patients with coronary artery disease may have myocardial ischemia, myocardial infarction, angina pectoris, ischemic cardiomyopathy, and congestive heart failure. And symptomatic (symptoms) such as aneurysm formation, which are usually diagnosed after onset of symptoms. Therefore, researchers in the art are looking for a reliable biomarker for diagnosis, treatment, and monitoring of the disease course of coronary artery disease.

Studies have shown that proteins present in serum or plasma, such as lipocalin-type prostaglandin D synthase, monocyte chemoattractant protein-1 (monocyte) Chemoattractant protein-1), Oxidized low-density lipoprotein (oxLDL)/beta(2)-glycoprotein I(beta2GPI) complexes, and eosinophilic globular protein Eosinophil cationic protein), etc., can be used to detect coronary artery disease (Inoue T. et al . (2008), Atherosclerosis , 201: 385-391; Harsimran K. et al . (2009), Diab. Vasc. Dis. Res. , 6: 288-290; Greco TP et al . (2010), Am. J. Clin. Pathol ., 133: 737-743; Niccoli G. et al . (2010), Atherosclerosis, 211: 606-611).

Other studies have shown that proteins present in the urine, such as matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1) And 8-isoprostane, which can be used to diagnose coronary artery disease (PJ Fitzsimmons et al . (2007), Atherosclerosis , 194: 196-203; I. Basarici et al . (2008), Acta. Cardiol ., 63: 415-422).

CD16 is a Fc receptor that binds to the Fc portion of an IgG antibody and is mainly found in natural killer cells, monocytes, and macrophages. ) on the cell surface. CD14 is a glycoprotein associated with the innate immune system, which acts as a co-receptor for detecting lipopolysaccharides (LPS). CD14 mainly exists in the following two forms in the human body:

(1) membrane-bound CD14 (mCD14): it is a glycosylphosphatidylinositol (GPI)-linked protein with a molecular weight of about 55 kDa, mainly Anchored to myeloid cells [such as mononuclear spheres, macrophages, neutrophils, and polymorphonuclear phagocytes] by the GPI tail (GPI tail) On the cell surface;

(2) Soluble CD14 (soluble CD14, sCD14): It has the same amino acid sequence as mCD14, but lacks the GPI tail, which can be mainly expressed by the expression of CD14 gene in mononuclear or hepatocytes. Generated and secreted directly to the extracellular (coat molecular weight of approximately 56 kDa) prior to coupling to the GPI tail; or mCD14 on the surface of mononuclear spheres by phospholipase or protease Digestion is carried out and released from the GPI tail (molecular weight of approximately 48 kDa). Therefore, sCD14 is mainly present in plasma and serum.

CD14 ⁺ CD16 ⁺ monocyte (CD14 ⁺ CD16 ⁺ monocyte) is a proinflammatory monocyte that has an increased ability to produce proinflammatory cytokines [such as tumor necrosis factor- ( (tumor necrosis factor-alpha, TNF-α)]. Studies have shown that patients with coronary artery disease (CAD) have an increased number of CD14 ⁺ CD16 ⁺ mononuclear spheres, while the number of increased CD14 ⁺ CD16 ⁺ mononuclear spheres and the level of TNF-α in serum (level) is related. Therefore, CD14 ⁺ CD16 ⁺ mononuclear spheres are thought to play an important role in disease development of coronary atherosclerosis (A. Schlitt et al. (2004), Thromb. Haemost ., 92:419-24).

WO 2011/083145 A1 discloses that CD14 in exosomes can be used as a predictor of a cardiovascular event [such as a fatal or non-fatal stroke, coronary artery disease, peripheral arterial disease (peripheral). Biomarker of arterial disease or heart failure, etc., wherein the exosomes can be isolated from plasma, serum, blood, urine, and the like. In particular, in the examples of this patent publication, plasma is mainly used as a test sample, and the experimental results show that the biomarker is combined with conventional cardiovascular risk factors [such as cholesterol (cholesterol), After the systolic blood pressure, the history of peripheral arterial disease, and the history of coronary artery disease, etc., the measured area under the ROC curve (AUC) is compared to the use of the ROC curve (AUC) alone. The cardiovascular risk factor has an increased situation.

Studies have shown that sCD14 in serum or plasma can be used as a method for detecting inflammatory diseases [such as sepsis, rheumatoid arthritis, systemic lupus erythematosus). And biomarkers of kawasaki disease (KD) (G. Horneff et al. (1993), Clin. Exp. Immunol ., 91: 207-213; WA Nockher et al . (1994), Clin. Exp. Immunol ., 96: 15-19; S. Takeshita et al . (2000), Clin. Exp. Immunol ., 119: 376-381).

In addition, RG TANG et al. and JW ZHU and CY LIU reported that the level of sCD14 in serum is closely related to the severity of coronary artery disease and the disease process, and thus can be used as a diagnostic tool for coronary artery disease. Biomarkers (RG TANG et al . (2007), Chin J. Crit. Care Med ., 27: 326-328; JW ZHU and CY LIU (2008), Acta Academiae Medical Qingdao Universitatis , 44: 156-157, 161).

Compared with the above literature, it is reported that serum samples are used to detect coronary artery disease. Applicants found that sCD14 in urine is more suitable as a biomarker for detecting coronary artery disease than sCD14 in serum. And has better specificity and sensitivity for the diagnosis of coronary artery disease.

Summary of invention

Thus, in a first aspect, the present invention provides a method for detecting or preliminary screening for coronary artery disease in a human subject, comprising: detecting a sCD14 taken from a urine sample of the human subject Level; and comparing the detected sCD14 level in the urine sample with a standard value, wherein if the sCD14 level detected in the urine sample is higher than the standard value, the human individual is It is considered to have coronary artery disease.

In a second aspect, the present invention provides a method for monitoring coronary artery disease in a human subject, comprising: detecting a sCD14 level periodically taken from a urine sample of the human subject; Having the sCD14 level detected in the urine sample compared to a standard value, wherein if the sCD14 level detected in the urine sample is above the standard value, the human individual is considered to have a coronary shape Arterial disease.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the pre-existing publication forms a common general knowledge in the art. Part of it.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

As used herein, the term "coronary artery disease (CAD)" and the term "coronary heart disease (CHD)" or "atherosclerotic heart disease" may be alternately Use, and means a cardiovascular disease caused by obstruction of the coronary arteries. Clinical signs and symptoms caused by coronary artery disease include, but are not limited to, myocardial hypoxia, myocardial infarction, angina pectoris, ischemic cardiomyopathy (Ischemic cardiomyopathy), congestive heart failure, and aneurysm formation.

Urine samples can be collected in large quantities in a non-invasive manner compared to serum samples collected by using a needle and in an invasive manner, and thus are considered a comparison Good and convenient to test samples. In the present invention, the applicant simultaneously compares morning midstream urine specimens or serum specimens between patients with coronary artery disease (CAD) and normal individuals. sCD14 level. Applicants unexpectedly found that the sCD14 level in the urine of CAD patients was significantly higher than that in normal individuals, but the sCD14 level in serum was not significant between CAD patients and normal individuals. Land difference. Applicants further used the receiver operating characteristics (ROC) curve analysis to find that the cutoff value for urine sCD14 level was 3.51 μg/mL. Based on this cutoff value, the sensitivity and specificity of CAD diagnostics can reach 84% and 70%, respectively.

Accordingly, the present invention provides a method for detecting or preliminary screening for coronary artery disease in a human subject, comprising: detecting a level of sCD14 in a urine sample taken from the human subject; and The sCD14 level detected in the urine sample is compared with a standard value, wherein if the sCD14 level detected in the urine sample is higher than the standard value, the human individual is considered to have coronary artery disease. .

The invention also provides a method for monitoring coronary artery disease in a human subject, comprising: detecting a sCD14 level periodically taken from a urine sample of the human individual; and preparing the urine sample The sCD14 level detected in the comparison is compared with a standard value, wherein if the sCD14 level detected in the urine sample is higher than the standard value, the human individual is considered to have coronary artery disease.

According to the invention, the urine sample can be taken from the human individual at any time. Preferably, the urine sample is a first morning urine sample of the human subject, and more preferably a midstream sample of the first morning urine.

In accordance with the present invention, the sCD14 level in the urine sample can be detected by any means known to those of ordinary skill in the art for quantifying sCD14. Preferably, the sCD14 level in the urine sample can be detected by an immunoassay selected from the group consisting of: multiplex immunoassay, enzyme Combined with immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), fluorescent immunoassay (FIA), chemistry Chemiluminescent immunoassay and immunoglobulinometry.

According to the present invention, quantification of sCD14 is carried out using an antibody-based binding moiety that specifically binds sCD14.

In a preferred embodiment of the invention, the antibody-based binding moiety is an antibody. In another preferred embodiment of the invention, the antibody-based binding moiety is labeled with a detectable label selected from the group consisting of: a radioactive label ( A radioactive label), a half hapten label, a fluorescent label, a chemiluminescent label, an enzymatic label, and an epitope tag.

As used herein, the term "antibody-based binding moiety" or "antibody" includes immunoglobulin molecules and immunologically active determinants of an immunoglobulin molecule [eg, containing a sCD14 specifically binds to the (immune-reactive) antigen-binding site molecule] and is intended to encompass all of the isotypes (eg, IgG, IgA, IgM, IgE, etc.) Antibodies and fragments comprising them that also specifically react with sCD14.

According to the invention, the term "antibody-based binding moiety" or "antibody" includes a capture antibody and a detection antibody.

As used herein, the term "capture antibody" means an antibody (whether an antibody of a single strain, an antibody of a plurality of strains, or an immunoreactive fragment of the antibody) capable of binding an antigen of interest. (antigen), and thus allows identification of the antigen by an antibody that is subsequently administered. The capture antibody can be used in a heterogeneous (solid phase) or homogeneous (solution phase) assay. Preferably, the capture antibody is immobilized on a solid phase.

As used herein, the term "detecting antibody" includes an antibody comprising a detectable label that is specific to the presence of one or more analytes of interest in a sample. Sex [that is, binds, is bound by, or forms a complex with the analyte]. The term also encompasses an antibody that is specific for one or more analytes of interest, wherein the antibody can be combined with another species comprising a detectable label. Examples of detectable labels include, but are not limited to, hapten markers [eg, biotin/streptavidin and digoxigenin (Dig)], nucleic acids [eg, oligonucleic acid] An oligonucleotide] label, a chemiluminescent label, a fluorescent label, an enzyme label, a radioactive label, an epitope tag (eg, T7, c-Myc, HA, VSV-G, HSV, FLAG, V5, and HIS), And their combination.

Antibodies can be fragmented using conventional techniques. Thus, the term "fragment thereof" comprises segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule which are capable of Selectively reacts with a specific protein. Non-limiting examples of such protein hydrolyzed fragments and/or recombinant fragments include Fab, F(ab')2, Fab', Fv, dabs and one that is ligated by a peptide linker. Single-chain antibody (scFv) in the VL and VH domains. The scFv's can be joined covalently or non-covalently to form an antibody having two or more binding sites.

The term "antibody-based binding moiety" encompasses a plurality of strained, single-plant or other purified preparations consisting of antibodies as well as recombinant antibodies. The term "antibody-based binding moiety" is further intended to include humanized antibodies, bi-specific antibodies, and antigen-binding epitopes having at least one molecule derived from an antibody molecule. Chimeric molecules.

In a preferred embodiment of the invention, the antibody-based binding moiety is detectably labeled. As used herein, "labeled antibody" includes antibodies that are attached or conjugated to a detectable label by standard techniques well known to those of ordinary skill in the art, including However, it is not limited to: enzyme-based, radioactively, fluorescently, and chemiluminescently labeled antibodies.

In accordance with the methods of the present invention, when an antibody-based binding moiety is used to detect sCD14, the sCD14 level in a urine sample correlates with the intensity of the signal emitted from the detectably labeled antibody.

In a preferred embodiment of the invention, the antibody-based binding moiety is detectably labeled by binding the antibody to an enzyme. The enzyme, in turn, when exposed to its matrix, reacts with the substrate in a manner that produces a chemical moiety, such as by means of a spectrophotometer. (spectrophotometric mean), fluorometric mean or detected by visual mean. Detectable enzymes suitable for use in the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase ), yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, base Alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, Catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

In another preferred embodiment of the invention, the antibody-based binding moiety is detectably labeled by binding the antibody to a fluorescent compound. When a fluorescently labeled antibody is exposed to light of a suitable wavelength, its presence can be detected by fluorescence. Detectable fluorescent compounds suitable for use in the present invention include, but are not limited to, CYE dyes, fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin ( Phycoerythrin), coriphosphine-O (CPO), phycocyanin (PE), allophycocyanin (APC), o-phthaldehyde, fluorescamine And tandem dyes [such as PE-Cy5 (PC5), PE-Cy7 (PC7) and PE-Texas Red].

According to the invention, the antibody-based binding moiety can also be detected by attaching the antibody to fluorescent emitting metals such as ¹⁵² Eu or other lanthanide series. Ground mark. These metals can be attached to the antibody using metal-chelating groups [such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA)].

In another preferred embodiment of the invention, the antibody-based binding moiety is detectably labeled by coupling the antibody to a chemiluminescent compound. The presence of chemiluminescence-antibodies is then determined by detecting the presence of luminescence that occurs during a chemical reaction. Detectable chemiluminescent compounds suitable for use in the present invention include, but are not limited to, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole (imidazole), acridine salt and oxalate ester.

In still another preferred embodiment of the invention, the antibody-based binding moiety is detectably labeled by labeling the antibody with a radioactive isotope. The radioisotope can be detected by using a gamma counter, a scintillation counter or by autoradiography. Radioisotopes suitable for use in the present invention include, but are not limited to, ³ H, ³¹ P, ³⁵ S, ¹⁴ C, and ¹²⁵ I.

According to the invention, the term "standard value" may mean a normal range, a normal value or a normal cutoff value for the sCD14 level in the urine of a healthy individual (eg by a selection) Method of measurement). A suitable cutoff value for a sCD14 level between a normal individual and a patient with coronary artery disease can be readily determined by standard techniques well known to those of ordinary skill in the art.

As used herein, the terms "healthy individual" and "normal individual" are used interchangeably and mean an individual who does not have symptoms associated with coronary artery disease or who is not at risk of developing coronary artery disease. That is, when the individual is examined by a medical professional, it will be diagnosed as healthy. Preferably, the normal individual has a normal coronary artery and does not have a history of active infectious disease, prior stroke, acute coronary syndrome, and malignancies.

According to the present invention, in addition to the sCD14 level in the urine of a normal individual, in order to evaluate the progression of a pathological condition of coronary artery disease or the effect of treatment for coronary artery disease, The level of sCD14 in the urine detected by a human subject in a previous examination can also be used as a standard value for the human individual. In this case, the sCD14 level detected by the human subject in a subsequent examination (eg, a follow-up examination after a period of several months) may be detected in the previous examination. Compared to the person.

In a preferred embodiment of the invention, the standard value is obtained by using a human sCD14 enzyme linked immunosorbent assay kit (Cat. No. HK320, Hycult Biotechnology, Uden, the Netherlands). The enzyme was measured in combination with immunosorbent assay, whereby a value of 3.51 μg/mL was obtained and used as a cutoff value for the sCD14 level.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example

Experimental individuals:

The experimental individuals participating in the study were recruited by using an operational procedure approved by The medical ethics committee of Kaohsiung Municipal United Hospital. Exclusion criteria included active infection. History of disease, previous stroke, acute coronary syndrome, and malignant disease. Furthermore, the human subjects studied in the present invention have obtained informed consent for their serum samples and donations of urine samples.

A total of 108 human subjects participated in the study, including 73 individuals who were diagnosed with coronary artery disease (CAD) from cardiovascular internal medicine and via angiography. (hereinafter referred to as the CAD group), and 35 individuals with normal coronary arteries (hereinafter referred to as the normal control group). Clinical information about these individuals [such as gender, age, SYNTAX score, and number of diseased vessels] are shown in Table 1 below, where SYNTAX scores Values are evaluated in the manner described in Sianos G. et al. (2005), Eurontervention , 1:219-227 and http://www.syntaxscore.com/, if the measured SYNTAX score is higher High indicates that the complexity and severity of CAD are higher.

General experimental method:

1. Collection of serum samples:

After experiencing at least 8 hours of overnight fasting, blood was collected from the radial artery of each experimental individual by using a needle, and then centrifuged at 3,000 rpm for 10 minutes at 4 ° C. This gave a serum sample.

2. Collection of urine samples:

The first midstream morning urine of each experimental individual was collected and centrifuged at 1,000 g for 5 minutes, and the obtained supernatant was used as a urine sample.

3. Statistical analysis:

In the following examples, each set of experimental data was repeated 3 times, and the experimental data was expressed as mean ± standard deviation (SD). All data were analyzed by Fractional Analysis (ANOVA) followed by Chi-square tests to assess differences between groups. If the statistical analysis obtained is p < 0.05, it represents statistical significance.

Example 1. Analysis of the amount of soluble CD14 (soluble CD14, sCD14) in the urine of normal individuals and patients with CAD

In order to understand the correlation between the amount of expression of sCD14 in urine and the severity of CAD, in the present example, the urine of normal individuals and patients with different SYNTAX scores was compared for comparison.

experimental method:

A. Prepare protein samples from urine:

Applicants selected 2 normal individuals from the normal control group and CAD group (they had SYNTAX scores of 0) and 6 had different CAD complexity and severity (with different complexity and severity of coronary artery) Patients with disease (they had SYNTAX scores of 2, 2, 4, 12, 25, and 34, respectively) to perform the following experiments.

First, the urine samples of the 8 individuals were collected according to the method described in the second item "Collection of urine samples" in the "General Experimental Methods" above, and then 7 mL of each of the obtained urine samples were taken and separately Add 7 mL of 10% trichloroacetic acid (TCA) solution [containing 6 mM dithiothreitol (DTT)], and then carry out the reaction on ice for 30 minutes, thereby making protein precipitate (protein pellets) were precipitated. Thereafter, centrifugation was carried out at 13,000 rpm for 30 minutes at 4 ° C, and then the protein precipitate was collected and washed twice with ice-cold 100% acetone (containing 6 mM DTT), and then centrifuged at 13,000 rpm. . The resulting protein precipitate was suspended in 0.3 mL of rehydration buffer [containing 9.8 M urea, 0.5% Triton X-100, 65 mM DTT, and 0.5% ampholytes (ampholytes)] Thereby, a protein sample is obtained.

The resulting protein samples were quantified using a 2-D Quant Kit (Amersham Biosciences, Piscataway, NJ) and then subjected to the analysis of item B below.

B. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis:

Take 10 μg of the protein sample prepared in item A above and add 5 volumes of sample loading buffer [containing 10% SDS, 0.3125 M Tris-HCl (pH 6.8), 10%) Glycerin, 0.5 M DTT, and 0.01% bromphenol blue] were then subjected to a denaturation reaction at 95 ° C for 5 minutes.

Next, SDS-PAGE analysis was performed using a vertical electrophoresis tank (Hoefer SE260, Amersham Biosciences, Buckinghamshire, UK). First, a polypropylene decyl gel gel sheet was prepared, which was divided into two layers, which were 12.5% SDS-PAGE separating gel and 5% SDS-PAGE accumulation gel (SDS-PAGE). Stacking gel). Thereafter, the obtained denatured protein sample was loaded into a sample well of the gel sheet and subjected to electrophoresis at 80 ° C for 30 minutes at 8 ° C, followed by 120 V. The voltage is electrophoresed and the electrophoresis is terminated when the tracking dye reaches about 0.1 to 0.2 cm from the bottom of the gel.

After the end of the electrophoresis, the protein separated on the gel sheet was transferred to polydifluorocarbon using a transfusion electrophoresis tank (TE 22 Mini Tank Transfer Unit, Amersham Biosciences, Buckinghamshire, UK) at a fixed current of 40 mA. A 12-hour period was carried out on a polyvinylidene difluoride membrane (PVDF) membrane (Millipore, Bed-ford, MA, USA). Next, the transferred PVDF membrane was taken out and washed with 5% skimmed milk at room temperature [in Tris-buffered saline/Tween-20 (TBS-T)). The blocking treatment was carried out for 1 hour, followed by washing 3 times with TBS-T for 10 minutes each time. Thereafter, a rabbit anti-CD14 polyclonal antibody (Cat. No. GTX101342, Genetex, San Antonio, TX, USA) was added as a primary antibody (1000 times diluted with TBS-T). After being reacted at 4 ° C overnight, it was washed 3 times with TBS-T for 10 minutes each time. Next, a goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Cat. No. L3032, Signalway Antibody, Pearland, TX, USA) was added as a secondary antibody. (secondary antibody) (diluted 5000 times with TBS-T) and allowed to react at room temperature for 1 hour, then washed 3 times with TBS-T for 10 minutes each time.

Subsequently, an enhanced chemiluminescence by mass (enhanced chemiluminescence substrate) (Pierce, Rockford, IL, USA) treated PVDF membrane, and then using a ChemiDoc ^TM XRS ⁺ system ^{(ChemiDoc TM XRS + System) (} Bio-Rad, Hercules, CA, USA) to take pictures. The obtained photograph was subjected to densitometric analysis using scanning densitometry software (Multi Gauge V3.0), whereby the signal intensity of sCD14 in the urine of each experimental individual was obtained. A corresponding value can be quantitatively calculated from the ribbons of the photographs. In addition, the non-ribbons of the photographs were used as a background and the same optical density analysis was performed.

Thereafter, the quantitative value of the obtained sCD14 signal intensity is substituted into the following formula (1) to calculate the sCD14 expression amount (%) in the urine of each experimental individual:

Formula (1): A=(B-C/C)×100

Of which: A = sCD14 performance (%)

Quantitative value of B=sCD14 signal intensity

C = quantitative value of background signal strength

result:

Figure 1 shows the amount of sCD14 expression in the urine of normal control individuals and CAD patients. It can be seen from Figure 1 that patients with different SYNTAX scores have a significant increase in sCD14 performance and are more likely to increase with the increase in SYNTAX scores compared with individuals in the normal control group. Obviously [trend P value ( p for trend) <0.001]. The results of this experiment show that the level of sCD14 in urine has a significant positive correlation with the severity of CAD, and thus can be used as a potential biomarker for detecting CAD.

Example 2. Analysis of the amount of soluble CD14 (sCD14) in urine and serum of normal individuals and patients with CAD

In the present example, serum and urine of normal individuals and patients with different numbers of diseased vessels were taken for enzyme-linked immunosorbent assay (ELISA) of sCD14 to evaluate serum and The correlation between the amount of sCD14 in urine and the degree of disease in CAD.

experimental method:

First, collect 73 patients with CAD (they have single or multiple vascular diseases) and 35 normal according to the method described in item 1 "Collection of Serum Samples" in the "General Experimental Methods" above. Serum samples from individual controls. Further, urine samples of these individuals were collected in accordance with the method described in the second item "Collection of urine samples" of the "General Experimental Method" above.

Thereafter, the human sCD14 enzyme linked immunosorbent assay kit (Cat. No. HK320, Hycult Biotechnology, Uden, the Netherlands) was used and the obtained serum was measured according to the manufacturer's instructions. Sample and sCD14 concentration in urine samples.

In addition, SPSS 16.00 software (SPSS, Chicago, IL) was used to analyze the sCD14 concentration measured in the urine samples of each experimental individual, thereby obtaining a receiver operating characteristic (ROC) curve. The area of the urine sCD14 (urinary sCD14) below the ROC curve [ie, the area under the ROC curve (AUC)] was calculated and used as a method for evaluating urine sCD14. Whether it has a good diagnostic ability can distinguish the index of the CAD group from the normal control group.

result:

Table 2 below shows the serum and urine sCD14 concentrations in the normal control group as well as in the CAD group. As can be seen from Table 2, there was no significant difference between the normal control group and the CAD group in terms of the sCD14 concentration in the serum. Conversely, in terms of sCD14 concentration in urine, the sCD14 level of patients with a single vascular disease or multiple vascular diseases was significantly increased compared with the normal control group. Patients with vascular disease have an increased sCD14 level compared to CAD patients with a single vascular disease.

In addition, it was found by subject operating characteristic (ROC) curve analysis that the area under the ROC curve (AUC) was 0.846, and the cutoff value for the sCD14 level of urine was 3.51 μg/mL, which indicates When a human individual has a sCD14 position of 3.51 μg/mL in the urine, the human individual may have coronary artery disease (CAD). Based on the cutoff value of the sCD14 level, the sensitivity and specificity of CAD diagnosis can reach 84% and 70%, respectively.

Based on the above experimental results, sCD14 in urine is more suitable as a biomarker for detecting CAD than sCD14 in serum, and has better specificity and sensitivity for CAD diagnosis. Therefore, Applicants believe that sCD14 in urine has the potential to be a diagnostic marker and thus can be used to rapidly detect or initially screen coronary artery diseases in a human subject.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Figure 1 shows the amount of sCD14 expression in the urine of normal control individuals and CAD patients.

Claims (14)

A method for detecting or initially screening for coronary artery disease in a human subject, comprising: detecting a level of sCD14 in a urine sample taken from the human subject; and preparing the urine sample The detected sCD14 level is compared to a standard value, wherein if the sCD14 level detected in the urine sample is above the standard value, the human individual is considered to have coronary artery disease. The method of claim 1, wherein the sCD14 level is detected by quantifying sCD14 using any of the following methodologies: multiplex immunoassay, enzyme-bound immunosorbent assay, radioimmunoassay Method, immunoradiometric assay, fluorescent immunoassay, chemiluminescence immunoassay, and immunoturbidimetric assay. The method of claim 2, wherein the quantification of sCD14 is carried out using an antibody-based binding moiety that specifically binds to sCD14. The method of claim 3, wherein the antibody-based binding moiety is an antibody. The method of claim 3, wherein the antibody-based binding moiety is labeled with a detectable label. The method of claim 5, wherein the detectable label is selected from the group consisting of: a radioactive label, a half antigen label, a fluorescent label, a chemiluminescent label, an enzyme label, and An epitope tag. The method of claim 1, wherein detecting the sCD14 level in the urine sample is performed by an enzyme-binding immunosorbent assay, and the sCD14 level detected in the urine sample is The cutoff value was determined to be 3.51 μg/mL. A method for monitoring coronary artery disease in a human subject, comprising: detecting a sCD14 level periodically taken from a urine sample of the human individual; and detecting the urine sample The sCD14 level is compared to a standard value, wherein if the sCD14 level detected in the urine sample is above the standard value, the human individual is considered to have coronary artery disease. The method of claim 8, wherein the sCD14 level is detected by quantifying sCD14 using any of the following methodologies: multiplex immunoassay, enzyme-binding immunosorbent assay, radioimmunoassay Method, immunoradiometric assay, fluorescent immunoassay, chemiluminescence immunoassay, and immunoturbidimetric assay. The method of claim 9, wherein the quantification of sCD14 is carried out using an antibody-based binding moiety that specifically binds to sCD14. The method of claim 10, wherein the antibody-based binding moiety is an antibody. The method of claim 10, wherein the antibody-based binding moiety is labeled with a detectable label. The method of claim 12, wherein the detectable label is selected from the group consisting of: a radioactive label, a half antigen label, a fluorescent label, a chemiluminescent label, an enzyme label, and An epitope tag. The method of claim 8, wherein detecting the sCD14 level in the urine sample is performed by an enzyme-binding immunosorbent assay, and the sCD14 level detected in the urine sample is The cutoff value was determined to be 3.51 μg/mL.