CN113252806A - Application of S-adenosyl homocysteine in preparation of product for diagnosing or treating kawasaki disease - Google Patents
Application of S-adenosyl homocysteine in preparation of product for diagnosing or treating kawasaki disease Download PDFInfo
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Abstract
The application discloses application of S-adenosyl homocysteine in preparation of products for diagnosing or treating Kawasaki disease, the application screens, identifies and finally confirms that S-adenosyl homocysteine in serum can be used as a biomarker for early diagnosis of the Kawasaki disease for the first time through metabonomics, and a diagnosis product for early diagnosis of the Kawasaki disease is developed based on the biomarker, has high sensitivity and high specificity, and can solve the problems of difficulty in early diagnosis and easiness in misdiagnosis of the Kawasaki disease. Meanwhile, the kit has the characteristics of low detection cost and good repeatability, and the stability and reliability of S-adenosyl homocysteine in serum for diagnosing the Kawasaki disease are verified in a discovery and verification mode, so that the kit has important clinical development and application values.
Description
Technical Field
The invention relates to the technical field of medical biological detection, in particular to application of S-adenosyl homocysteine as a biomarker or a drug target in preparation of products for diagnosing or treating Kawasaki disease.
Background
Kawasaki disease is an acute self-limiting systemic vasculitis which occurs well in children under 5 years old, the main complication is coronary artery injury, and 20-25% of kawasaki patients can form coronary aneurysm and even myocardial infarction without being treated in time. In recent years, the incidence of Kawasaki disease is gradually increased year by year, and the Kawasaki disease exceeds rheumatic fever at present, and becomes one of important factors causing the acquired heart disease of children in developed countries and developing countries. The pathogenesis and pathogenesis of kawasaki disease are not clear, and the diagnosis mode is mainly based on some clinical manifestations (such as fever lasting for more than 5 days, waxberry tongue, hand and foot molting, rash and the like). However, since the main symptoms of kawasaki disease are not specific and often do not occur simultaneously, many febrile diseases such as respiratory tract infection, drug eruption, scarlet fever, EB virus infection, measles, infantile idiopathic arthritis, etc. have similar clinical manifestations with kawasaki disease, which causes difficulties in diagnosis and often leads to misdiagnosis or missed diagnosis. Therefore, in order to improve the sensitivity and specificity of early clinical diagnosis of kawasaki disease, development of new diagnostic products is imperative.
Disclosure of Invention
The application finds a biological marker S-adenosyl homocysteine for early diagnosis of the Kawasaki disease, develops a diagnosis product for early diagnosis of the Kawasaki disease based on the biological marker, and can solve the problems of difficult early diagnosis and easy misdiagnosis of the Kawasaki disease.
Based on the discovery that S-adenosyl homocysteine can be used as a biomarker for early diagnosis of Kawasaki disease:
the application provides application of S-adenosyl homocysteine as a diagnosis marker of Kawasaki disease in preparation of a Kawasaki disease diagnosis product.
Optionally, the test sample of the diagnostic product is serum of the subject.
Optionally, the diagnostic product is a diagnostic kit, and the diagnostic kit contains a detection reagent for specifically detecting S-adenosyl homocysteine in a biological sample.
The application also provides an application of the reagent for in vitro detection of S-adenosyl homocysteine in serum in preparation of Kawasaki disease diagnostic products. Optionally, the diagnostic product is a diagnostic kit or a detection probe.
The application also provides application of the S-adenosyl homocysteine serving as a drug target of the Kawasaki disease in preparing a drug for treating the Kawasaki disease.
The application also provides a Kawasaki disease diagnostic kit based on human serum S-adenosyl homocysteine detection, which comprises a detection reagent for specifically detecting the S-adenosyl homocysteine in the serum of a subject.
Optionally, the kawasaki disease diagnostic kit comprises:
(1) (ii) an S-adenosine homocysteine standard;
(2) diluting the solution;
(3) a derivatizing agent;
(4) an internal standard solution;
(5) eluting the solution;
(6) a protein solution that mimics a serum environment.
Optionally, the diluent is an acetonitrile solution containing formic acid; the derivatization reagent is triethylamine, phenyl isothiocyanate or o-phthalaldehyde; the internal standard solution is an acetonitrile solution of DL-2 amino pimelic acid; the mobile phase A of the eluent is an ammonium formate solution containing formic acid, and the mobile phase B of the eluent is acetonitrile; the protein solution is bovine serum albumin solution.
Further, the mass percent of formic acid in the acetonitrile solution containing formic acid is 0.05-0.15%, and acetonitrile is acetonitrile solution with mass fraction of 90%; in the acetonitrile solution of the DL-2 aminopimelic acid, the concentration of the DL-2 aminopimelic acid is 55.0 mu g/mL, and the acetonitrile is an acetonitrile solution with the mass fraction of 80.0 percent; in the ammonium formate solution containing formic acid, the mass percentage of the formic acid is 0.005-0.02%; the concentration of the bovine serum albumin in the bovine serum albumin solution is 45.0 mg/mL-50.0 mg/mL.
Most preferably, the diluent is a 90% acetonitrile solution containing 0.1% formic acid; the derivatization reagent is triethylamine, phenyl isothiocyanate or o-phthalaldehyde; the internal standard solution is an 80.0% acetonitrile solution of DL-2 aminopimelic acid, wherein the concentration of DL-2 aminopimelic acid is 55.0 mu g/mL; the mobile phase A of the eluent is ammonium formate solution containing 0.01 percent formic acid, and the mobile phase B is acetonitrile (pure acetonitrile); the protein solution is bovine serum albumin solution with the concentration of 50.0 mg/mL.
The present application also provides a system for detecting S-adenosyl homocysteine in serum, comprising:
the standard curve drawing module is used for drawing a quantitative standard curve by taking the ratio of the peak areas of S-adenosyl homocysteine and DL-2 aminopimelic acid as a vertical axis and the concentration of S-adenosyl homocysteine as a horizontal axis on the basis of taking S-adenosyl homocysteine as a standard substance and DL-2 aminopimelic acid as an internal standard;
the separation module is used for extracting and eluting S-adenosyl homocysteine in a serum sample from a subject and recording the peak area of the S-adenosyl homocysteine;
and the calculation module is used for calculating the concentration of the S-adenosine homocysteine in the serum sample of the subject based on the standard curve drawn by the standard curve drawing module and the peak area recorded by the separation module.
Optionally, the separation module comprises a HILIC chromatographic column and an ultra-high performance liquid chromatograph tandem mass spectrometer.
Compared with the prior art, the method has the following effects:
the application screens, identifies and finally confirms that the S-adenosyl homocysteine in the serum can be used as a biomarker for early diagnosis of the Kawasaki disease through metabonomics for the first time, is used for diagnosis of the Kawasaki disease, has high sensitivity and high specificity, has the characteristics of low detection cost and good repeatability, verifies the stability and reliability of the S-adenosyl homocysteine in the serum for diagnosis of the Kawasaki disease through a discovery and verification mode, and has important clinical development and application values.
Drawings
FIG. 1 is a graph showing the content change (mean. + -. standard error) of S-adenosyl homocysteine in serum samples of Kawasaki disease patients, Kawasaki disease patients complicated with coronary artery injury, non-Kawasaki disease febrile patients, healthy subjects and cured Kawasaki disease patients (KD-nonCALS-Kawasaki disease, KD-CALS-Kawasaki disease complicated with coronary artery injury, FC-febrile patients, HC-healthy control subjects and CKD-cured Kawasaki disease patients).
FIG. 2 is a ROC plot of S-adenosyl homocysteine for patients with Kawasaki disease and for patients with non-Kawasaki disease fever; wherein A shows ROC curves of S-adenosyl homocysteine in experimental groups for patients with Kawasaki disease and patients with non-Kawasaki disease fever; and B shows an ROC curve for verifying that the S-adenosyl homocysteine in the group is used for patients with Kawasaki disease and patients with non-Kawasaki disease fever.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Metabonomics is an emerging discipline behind phylogenomics, proteomics and transcriptomics, and is a scientific field with the aim of researching small-molecule metabolites. Since the presence of small molecule metabolites in body fluids is relatively stable, the analysis of some endogenous small molecule metabolites through metabolomics has been widely used for early diagnosis of diseases. The method detects the metabolites in the serum by a metabonomics method of ultra-high performance liquid chromatography-mass spectrometry, and screens out early diagnosis biomarkers suitable for Kawasaki disease through bioinformatics analysis. The method has the advantages of fast detection, good repeatability, high sensitivity and low cost. The potential diagnosis marker is screened by adopting a strategy from discovery to verification, the early diagnosis marker of the Kawasaki disease based on the human serum S-adenosylhomocysteine is successfully screened, the diagnosis sensitivity and the specificity of the diagnosis marker are good, and a kit of the diagnosis marker is not reported.
The application finds that the process of taking S-adenosyl homocysteine as an early diagnosis biomarker of Kawasaki disease is as follows:
(1) the method comprises the steps of carrying out metabonomic fingerprint analysis on blood serums of Kawasaki disease patients, non-Kawasaki disease fever patients and healthy control subjects by using a metabonomics technology of an ultra-high performance liquid chromatograph tandem mass spectrometer, finding that choline metabolism and amino acid metabolism paths in the blood serums of the Kawasaki disease patients are changed, and finding that 17 metabolites have significant difference between the blood serums of the Kawasaki disease patients and the non-Kawasaki disease fever patients.
(2) The 17 metabolites were analyzed by ROC (receptor Operating spectroscopic dark) using the data statistics software SPSS, and the metabolites with the best diagnostic performance for kawasaki disease were selected and evaluated for sensitivity and specificity. The results are shown in fig. 2 a, which shows that S-adenosylhomocysteine in the experimental group is used for identifying and diagnosing ROC curves of kawasaki disease patients and non-kawasaki disease febrile patients, and finally determines that S-adenosylhomocysteine has the best performance for diagnosing kawasaki disease, wherein AUROC of the S-adenosylhomocysteine is 0.922, and when the cutoff value is 13.98ng/mL, the sensitivity and specificity of the S-adenosylhomocysteine are 96.40% and 79.10% respectively, namely when the content of S-adenosylhomocysteine in serum of children with febrile diseases is lower than 13.98ng/mL, the children with febrile diseases can be diagnosed with kawasaki disease.
(3) And (3) verifying the diagnosis markers found in the step (2) by using a batch of independent double-blind experimental samples (including Kawasaki disease patients and non-Kawasaki disease febrile patients), wherein the result is shown as B in fig. 2, and the results show that the S-adenosyl homocysteine in the verification group is used for identifying and diagnosing ROC curves of the Kawasaki disease patients and the non-Kawasaki disease febrile patients, and the S-adenosyl homocysteine can be used as a diagnosis marker of the Kawasaki disease.
(4) And (3) verifying the diagnosis marker found in the step (2) again by using a batch of blood serum of the patient with the Kawasaki disease which is treated and rehabilitated, and finding that the content of S-adenosylhomocysteine in the blood serum of the patient with the Kawasaki disease is reduced along with the aggravation of the condition of the patient, but the blood serum returns to a normal level after treatment, and further determining the level of the S-adenosylhomocysteine in the blood serum, wherein the level of the S-adenosylhomocysteine can be used for diagnosing the Kawasaki disease and possibly for judging the prognosis of.
Based on this finding:
the present application provides: an application of S-adenosyl homocysteine as a diagnosis marker of Kawasaki disease in preparing a Kawasaki disease diagnosis product; an application of a reagent for in vitro detection of S-adenosyl homocysteine in serum in preparation of Kawasaki disease diagnostic products; an application of S-adenosyl homocysteine as drug target of Kawasaki disease in preparing drugs for treating Kawasaki disease; a Kawasaki disease diagnostic kit; and a detection system for S-adenosyl homocysteine in serum.
The diagnostic product can be a reagent, test paper, a kit and the like, and optionally, the diagnostic product is a diagnostic kit which contains a detection reagent for specifically detecting S-adenosyl homocysteine in a biological sample. The test sample of the diagnostic product is the serum of the subject.
As one embodiment of the kawasaki disease diagnostic kit, the kawasaki disease diagnostic kit comprises:
(1) (ii) an S-adenosine homocysteine standard;
(2) diluting the solution;
(3) a derivatizing agent;
(4) an internal standard solution;
(5) eluting the solution;
(6) a protein solution that mimics a serum environment.
The S-adenosine homocysteine standard is used for qualitative analysis of S-adenosine homocysteine in serum and drawing of a marker curve. The mass numbers of the parent ion and the main fragment ion of the S-adenosylhomocysteine in the mass spectrum MRM mode are 385.08 and 136.055 respectively.
Dilutions were used to pre-condition extracts and standards from serum samples from subjects, optionally in 90% acetonitrile containing 0.1% formic acid.
The derivatization reagent comprises triethylamine, phenyl isothiocyanate or o-phthalaldehyde solution and the like.
The internal standard solution can be 80.0% acetonitrile solution of DL-2 amino pimelic acid, the concentration of DL-2 amino pimelic acid is 55.0 mug/mL, the quantitative analysis is carried out under the mass spectrum SRM mode or MRM mode, and the mass numbers of parent ions and main fragment ions thereof under the mass spectrum MRM mode are 176.10 and 112.10 respectively.
The eluent is used to elute the column, and optionally, mobile phase a may be selected from ammonium formate solution containing 0.01% formic acid, and mobile phase B may be selected from acetonitrile. The chromatographic column can be C18 or HILIC chromatographic column.
The protein solution simulating serum environment may be selected from bovine serum albumin solution with concentration of 50.0 mg/mL.
The kit can be applied to an ultra-high performance liquid chromatograph tandem mass spectrometer, can detect more than 20 amino acids or one-carbon metabolism related metabolites simultaneously, and can analyze the metabolic abnormality of Kawasaki disease patients more comprehensively. Serum samples were used to test the efficacy of the invention. Serum samples of patients with Kawasaki disease and patients without fever caused by Kawasaki disease are adopted, and serum S-adenosyl homocysteine is used as a diagnostic standard, so that the two groups of patients can be well distinguished.
Specifically, one method for detecting S-adenosyl homocysteine in a serum sample of a subject using the kit of the present application comprises the steps of:
(1) taking S-adenosylhomocysteine as a standard substance, adding an internal standard DL-2 aminopimelic acid, firstly performing derivatization or direct sample injection analysis, and drawing a corresponding quantitative standard curve;
(2) serum sample pretreatment from subjects: unfreezing a serum sample at 4 ℃, adding 400.0 mu L of serum into 90.0% acetonitrile solution containing 0.1% formic acid and 50.0 mu L of internal standard solution, violently whirling and uniformly mixing for 15.0S, then centrifuging for 10.0min at 14000g, taking 200.0 mu L of supernatant to be used for detection in an automatic sample injection bottle, and recording the ratio of the elution peak area and the internal standard peak area of S-adenosylhomocysteine in the serum sample;
(3) and (3) calculating the content of the S-adenosyl homocysteine in the serum sample of the subject according to the ratio of the quantitative standard curve in the step (1) to the peak area in the step (2).
The diagnostic kit detects the content of S-adenosyl homocysteine in the serum of patients with fever (including patients with Kawasaki disease and patients with non-Kawasaki disease), takes 13.98ng/mL as a cut-off value, and diagnoses the Kawasaki disease when the content of S-adenosyl homocysteine in the serum of the patients is lower than 13.98 ng/mL.
One embodiment of the system for detecting S-adenosyl homocysteine in serum, comprises:
the standard curve drawing module is used for drawing a quantitative standard curve by taking the ratio of the peak areas of S-adenosyl homocysteine and DL-2 aminopimelic acid as a vertical axis and the concentration of S-adenosyl homocysteine as a horizontal axis on the basis of taking S-adenosyl homocysteine as a standard substance and DL-2 aminopimelic acid as an internal standard;
the separation module is used for extracting and eluting S-adenosyl homocysteine in a serum sample from a subject and recording the peak area of the S-adenosyl homocysteine;
and the calculation module is used for calculating the concentration of the S-adenosine homocysteine in the serum sample of the subject based on the standard curve drawn by the standard curve drawing module and the peak area recorded by the separation module.
The diagnostic system comprises means for: the chromatographic column is HILIC chromatographic column (2.1 × 150mm,3.5 μm, Merck, US), the detecting instrument is ultra-high performance liquid chromatography tandem mass spectrometer, the metabolite is detected under multiple reaction monitoring mode (MRM), and the electrospray ion source adopts positive ions.
One embodiment of the kawasaki disease diagnostic kit comprises:
(1) (ii) an S-adenosine homocysteine standard;
(2) diluting liquid: 90% acetonitrile solution containing 0.1% formic acid;
(3) derivatization reagent: the derivatization reagent comprises triethylamine, phenyl isothiocyanate or o-phthalaldehyde solution and the like.
(4) Internal standard solution: a solution of DL-2 aminopimelic acid in 80.0% acetonitrile at a concentration of 55.0. mu.g/mL;
(5) eluent: the mobile phase A is an ammonium formate solution containing 0.01 percent of formic acid, and the mobile phase B is acetonitrile;
(6) protein solution mimicking serum environment: bovine serum albumin solution at a concentration of 50.0 mg/mL.
The following examples are given for the detection using the kit, and the concentrations in the following examples are given by percentage unless otherwise specified.
Example 1:
1. serum sample collection
Prior to collection, informed consent was obtained from all subjects enrolled in the study.
All subjects fasted for more than 8 hours in 50 febrile patients (including 30 patients with Kawasaki disease and 20 patients with non-Kawasaki disease) and were bled by venipuncture the next morning. After whole blood was collected, it was left to stand for 1 hour, centrifuged at 2000g for 10 minutes, and the supernatant serum was stored in a refrigerator at-80 ℃.
2. Analytical method
2.1 drawing of Standard Curve
10.0mg of S-adenosyl homocysteine standard is precisely weighed, and diluted into different concentration gradients by using 90% acetonitrile solution containing 0.1% formic acid as a solvent. 50.0 mu L of protein solution (bovine serum albumin solution with the concentration of 50.0 mg/mL) is taken, 50.0 mu L of internal standard solution, 50.0 mu L of standard solution and 350.0 mu L of 90% acetonitrile solution containing 0.1% formic acid are sequentially added, the mixture is violently vortexed and uniformly mixed for 15.0s, then 14000g of the mixture is centrifuged for 10.0min, and 200.0 mu L of supernatant is taken and put into an automatic sample injection bottle for drawing a standard curve.
2.2 serum sample pretreatment
Unfreezing a serum sample at 4 ℃, adding 400.0 mu L of serum into 90.0% acetonitrile solution containing 0.1% formic acid and 50.0 mu L of internal standard solution, violently vortexing and uniformly mixing for 15.0s, then centrifuging for 10.0min at 14000g, taking 200.0 mu L of supernatant, derivatizing with o-phthalaldehyde solution, and using the derivatized o-phthalaldehyde solution in an automatic sampling bottle for detection.
2.3 ultra high performance liquid chromatograph tandem mass spectrometer analysis
(1) Liquid phase conditions: the chromatograph is Waters ultra-performance liquid chromatography; the chromatographic column is HILIC chromatographic column; the mobile phase A is an ammonium formate solution containing 0.01 percent of formic acid, and the mobile phase B is acetonitrile; the flow rate is 0.5 mL/min; the sample injection volume is 5.0 mu L; the separation time is 12.0 min; the following gradient elution was used: 0-2.5min, 20% A; 2.5-5.0min, 20% -80% A; 5.0-8.0min, 80% A; 8-8.5min, 80% -20% A; 8.5-12.0min, 20% A. The column temperature was set at 45 ℃ and the autosampler temperature at 8 ℃.
(2) Mass spectrum conditions: the mass spectrometer is Xevo QTof (Waters, US), the metabolite is detected in a multiple reaction monitoring mode (MRM), and the electrospray ion source employs positive ion detection; the voltage of the electrospray capillary was set to 3.0kV, nitrogen was used as the drying gas for solvent evaporation, and the flow rate was 50L/h; the ion source temperature was 100 ℃.
2.4 serum assay results
Taking the concentration of the standard substance as a horizontal coordinate, taking the ratio of the peak area of the standard substance to the peak area of the internal standard substance as a vertical coordinate, and drawing a standard curve; and substituting the detection result of the S-adenosyl homocysteine in the serum sample into a standard curve to finally obtain the content of the S-adenosyl homocysteine in each sample.
The detection results are shown in fig. 1, and the results show that the content of S-adenosyl homocysteine in the serum of patients with Kawasaki disease is greatly reduced compared with the content of S-adenosyl homocysteine in the serum of patients with non-Kawasaki disease, the accuracy of distinguishing two groups of patients is 86.67% when 13.98ng/mL is taken as a cutoff value, and the false positive rate is 10.00%.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
- Application of S-adenosyl homocysteine as a diagnosis marker of Kawasaki disease in preparation of a Kawasaki disease diagnosis product.
- 2. The use according to claim 1, wherein the test sample of the diagnostic product is the serum of a subject.
- 3. The use according to claim 1, wherein the diagnostic product is a diagnostic kit comprising a detection reagent for specifically detecting S-adenosylhomocysteine in a biological sample.
- 4. Application of a reagent for in vitro detection of S-adenosyl homocysteine in serum in preparation of Kawasaki disease diagnostic products.
- 5. The use according to claim 4, wherein the diagnostic product is a diagnostic kit or a detection probe.
- Application of S-adenosyl homocysteine as a drug target of Kawasaki disease in preparing drugs for treating Kawasaki disease.
- 7. The Kawasaki disease diagnostic kit is characterized by comprising a detection reagent for specifically detecting S-adenosyl homocysteine in serum of a subject.
- 8. The Kawasaki disease diagnostic kit of claim 7, wherein the Kawasaki disease diagnostic kit comprises:(1) (ii) an S-adenosine homocysteine standard;(2) diluting liquid: acetonitrile solution containing formic acid;(3) derivatization reagent: triethylamine, phenyl isothiocyanate or o-phthalaldehyde;(4) internal standard solution: acetonitrile solution of DL-2 amino pimelic acid;(5) eluent: the mobile phase A is an ammonium formate solution containing formic acid, and the mobile phase B is acetonitrile;(6) a protein solution that mimics a serum environment.
- 9. The Kawasaki disease diagnostic kit according to claim 8, wherein the formic acid-containing acetonitrile solution contains 0.05-0.15% by mass of formic acid, and the acetonitrile is an acetonitrile solution with a mass fraction of 90%; in the acetonitrile solution of the DL-2 aminopimelic acid, the concentration of the DL-2 aminopimelic acid is 55.0 mu g/mL, and the acetonitrile is an acetonitrile solution with the mass fraction of 80.0 percent; in the ammonium formate solution containing formic acid, the mass percentage of the formic acid is 0.005-0.02%; the protein solution is bovine serum albumin solution; the concentration of the bovine serum albumin in the bovine serum albumin solution is 45.0 mg/mL-55.0 mg/mL.
- 10. A system for detecting S-adenosyl homocysteine in serum, which is characterized by comprising:the standard curve drawing module is used for drawing a quantitative standard curve by taking the ratio of the peak areas of S-adenosyl homocysteine and DL-2 aminopimelic acid as a vertical axis and the concentration of S-adenosyl homocysteine as a horizontal axis on the basis of taking S-adenosyl homocysteine as a standard substance and DL-2 aminopimelic acid as an internal standard;the separation module is used for extracting and eluting S-adenosyl homocysteine in a serum sample from a subject and recording the peak area of the S-adenosyl homocysteine;and the calculation module is used for calculating the concentration of the S-adenosine homocysteine in the serum sample of the subject based on the standard curve drawn by the standard curve drawing module and the peak area recorded by the separation module.
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Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6066467A (en) * | 1997-07-24 | 2000-05-23 | Anticancer, Inc. | High specificity homocysteine assays for biological samples |
| US20060246529A1 (en) * | 1999-07-06 | 2006-11-02 | Chong-Sheng Yuan | Methods and compositions for assaying homocysteine |
| JP2007510892A (en) * | 2003-11-07 | 2007-04-26 | エフ．ホフマン−ラ ロシュ アーゲー | Base markers for arterial thrombosis and inflammation to stratify the risk of coronary artery disease |
| CN101089604A (en) * | 2006-06-12 | 2007-12-19 | 苏州艾杰生物科技有限公司 | Homotype semicystinol diagnostic kit and homotype semicystinol concentration investigating method |
| US20090186370A1 (en) * | 2008-01-18 | 2009-07-23 | Ogle Matthew F | Diagnostic biomarkers for vascular aneurysm |
| US20090191575A1 (en) * | 2006-06-05 | 2009-07-30 | Makoto Watanabe | Tumor marker and method for determination of the occurrence of cancerous disease |
| WO2010025393A2 (en) * | 2008-08-28 | 2010-03-04 | The Regents Of The University Of California | Protein biomarkers and methods for diagnosing kawasaki disease |
| WO2012110253A2 (en) * | 2011-02-18 | 2012-08-23 | Cavadis B.V. | Exosomal biomarkers for cardiovascular events |
| US20120264629A1 (en) * | 2009-08-07 | 2012-10-18 | Joseph Anderberg | Methods and compositions for diagnosis and prognosis of renal injury and renal failure |
| US9216209B1 (en) * | 2011-06-06 | 2015-12-22 | Kilmer S. McCully | Compositions and method for utilization of thioretinamide in therapy of degenerative diseases of aging |
| WO2016077912A1 (en) * | 2014-11-17 | 2016-05-26 | The Hospital For Sick Children | Method for diagnosing an inflammatory disorder |
| CN107132349A (en) * | 2017-04-28 | 2017-09-05 | 苏州博源医疗科技有限公司 | A kind of homocysteine automatic detection reagent, preparation method and detection method |
| CN107502664A (en) * | 2017-09-18 | 2017-12-22 | 苏州贝斯派生物科技有限公司 | A kind of biomarker and kit and purposes for being used to diagnose Kawasaki disease |
| WO2018089764A1 (en) * | 2016-11-11 | 2018-05-17 | Ascendant Dx, Llc | Compositions and methods for diagnosing and differentiating systemic juvenile idiopathic arthritis and kawasaki disease |
| CN110430873A (en) * | 2017-04-11 | 2019-11-08 | 雀巢产品有限公司 | Omega-3 fatty acid, homocysteine and the vitamin D level of cognition aging are identified and weakened in individual |
| CN110726846A (en) * | 2019-12-04 | 2020-01-24 | 南京市儿童医院 | Application of HBP protein as diagnostic marker of Kawasaki disease |
| CN111393519A (en) * | 2019-11-21 | 2020-07-10 | 中国药科大学 | As KRASG12CNovel stapled peptides of/SOS 1 inhibitor and uses thereof |
| US20210102261A1 (en) * | 2019-08-26 | 2021-04-08 | Liquid Lung Dx | Biomarkers for the Diagnosis of Lung Cancers |
-
2021
- 2021-03-25 CN CN202110321840.8A patent/CN113252806B/en active Active
Patent Citations (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6066467A (en) * | 1997-07-24 | 2000-05-23 | Anticancer, Inc. | High specificity homocysteine assays for biological samples |
| US20060246529A1 (en) * | 1999-07-06 | 2006-11-02 | Chong-Sheng Yuan | Methods and compositions for assaying homocysteine |
| JP2007510892A (en) * | 2003-11-07 | 2007-04-26 | エフ．ホフマン−ラ ロシュ アーゲー | Base markers for arterial thrombosis and inflammation to stratify the risk of coronary artery disease |
| US20090191575A1 (en) * | 2006-06-05 | 2009-07-30 | Makoto Watanabe | Tumor marker and method for determination of the occurrence of cancerous disease |
| CN101089604A (en) * | 2006-06-12 | 2007-12-19 | 苏州艾杰生物科技有限公司 | Homotype semicystinol diagnostic kit and homotype semicystinol concentration investigating method |
| US20090186370A1 (en) * | 2008-01-18 | 2009-07-23 | Ogle Matthew F | Diagnostic biomarkers for vascular aneurysm |
| WO2010025393A2 (en) * | 2008-08-28 | 2010-03-04 | The Regents Of The University Of California | Protein biomarkers and methods for diagnosing kawasaki disease |
| US20110189698A1 (en) * | 2008-08-28 | 2011-08-04 | The Regents Of The University Of California | Protein Biomarkers and Methods for Diagnosing Kawasaki Disease |
| US20120264629A1 (en) * | 2009-08-07 | 2012-10-18 | Joseph Anderberg | Methods and compositions for diagnosis and prognosis of renal injury and renal failure |
| WO2012110253A2 (en) * | 2011-02-18 | 2012-08-23 | Cavadis B.V. | Exosomal biomarkers for cardiovascular events |
| US9216209B1 (en) * | 2011-06-06 | 2015-12-22 | Kilmer S. McCully | Compositions and method for utilization of thioretinamide in therapy of degenerative diseases of aging |
| WO2016077912A1 (en) * | 2014-11-17 | 2016-05-26 | The Hospital For Sick Children | Method for diagnosing an inflammatory disorder |
| WO2018089764A1 (en) * | 2016-11-11 | 2018-05-17 | Ascendant Dx, Llc | Compositions and methods for diagnosing and differentiating systemic juvenile idiopathic arthritis and kawasaki disease |
| CN110430873A (en) * | 2017-04-11 | 2019-11-08 | 雀巢产品有限公司 | Omega-3 fatty acid, homocysteine and the vitamin D level of cognition aging are identified and weakened in individual |
| CN107132349A (en) * | 2017-04-28 | 2017-09-05 | 苏州博源医疗科技有限公司 | A kind of homocysteine automatic detection reagent, preparation method and detection method |
| CN107502664A (en) * | 2017-09-18 | 2017-12-22 | 苏州贝斯派生物科技有限公司 | A kind of biomarker and kit and purposes for being used to diagnose Kawasaki disease |
| US20210102261A1 (en) * | 2019-08-26 | 2021-04-08 | Liquid Lung Dx | Biomarkers for the Diagnosis of Lung Cancers |
| CN111393519A (en) * | 2019-11-21 | 2020-07-10 | 中国药科大学 | As KRASG12CNovel stapled peptides of/SOS 1 inhibitor and uses thereof |
| CN110726846A (en) * | 2019-12-04 | 2020-01-24 | 南京市儿童医院 | Application of HBP protein as diagnostic marker of Kawasaki disease |
Non-Patent Citations (13)
| Title |
|---|
| AL BAKER ET AL.: "Diagnosis，Treatment，and Long-term Management of Kawasaki Disease：A Scientific Statement for Health Professionals from the American Heart Association", CIRCULATION, vol. 17, no. 135 * |
| 司万军;: "同型半胱氨酸联合脂蛋白小a对糖尿病早期微血管病变的临床诊断价值", 临床医药文献电子杂志, no. 18 * |
| 姜毅;李温慈;徐海滨;邵山鹰;: "川崎病患儿血清胱抑素C的变化及其与冠状动脉病变的关系", 新医学, no. 10 * |
| 杨一芬;王天菊;卿之驹;: "同型半胱氨酸转甲基酶的制备及纯化", 广东医学, no. 19 * |
| 杨蕊华;李亚蕊;崔涛;赵孟佳;刘丽华;景迎春;: "川崎病患儿同型半胱氨酸C反应蛋白纤维蛋白原水平与冠状动脉损伤的关系及临床意义", 中国药物与临床, no. 15 * |
| 林洁英, 林郁生, 仇烨, 林吉进, 郭继忠: "川崎病患儿血浆同型半胱氨酸的检测及其临床意义", 中国妇幼保健, no. 16 * |
| 段婉莹;刘丽萍;杨中华;温淼;杨波;濮月华;刘大成;刘婧伊;: "动脉瘤性蛛网膜下腔出血后迟发性脑缺血的早期凝血及纤溶生物标志物变化", 中国脑血管病杂志, no. 01 * |
| 王晓霞;: "婴儿不典型川崎病早期误诊分析", 当代医学, no. 32 * |
| 禇茂平 等: "99Tcm心肌灌注显像和血浆同型半胱氨酸水平对判断川崎病心肌缺血的价值", 温州医学院学报, no. 03 * |
| 章国忠,潘继豹: "川崎病患儿血浆同型半胱氨酸水平检测及其意义", 浙江医学, no. 07 * |
| 许海棠;荣星;何跃娥;仇慧仙;张松跃;任跃;褚茂平;吴蓉洲;: "血清8-羟基脱氧鸟苷水平对川崎病冠状动脉损伤的预测价值", 浙江医学, no. 18 * |
| 邵东;陈洁;: "川崎病患儿血清中25-(OH)D3、NT-proBNP 和IL-6表达水平及诊断价值", 中国地方病防治杂志, no. 06 * |
| 陈玲: "基于代谢组学的慢加急性肝衰竭疾病预后标志物的发现和确证", 中国优秀硕士学位论文全文数据库 医药卫生科技辑, no. 08, pages 19 - 24 * |
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