CN116183586A - Method and kit for detecting thrombomodulin - Google Patents
Method and kit for detecting thrombomodulin Download PDFInfo
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- CN116183586A CN116183586A CN202211654564.8A CN202211654564A CN116183586A CN 116183586 A CN116183586 A CN 116183586A CN 202211654564 A CN202211654564 A CN 202211654564A CN 116183586 A CN116183586 A CN 116183586A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N2021/0106—General arrangement of respective parts
- G01N2021/0112—Apparatus in one mechanical, optical or electronic block
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention aims to provide a method and a kit for detecting thrombomodulin, which have the advantages of high detection speed, high sensitivity, good specificity, small variation coefficient and good stability of each reagent component, and can meet the existing detection requirements, and are characterized in that: the detection method comprises the following steps: step 1, adding 30 mu L of a sample to be detected and 100 mu L of biotin-labeled thrombomodulin antibody into a coating plate, uniformly mixing and reacting; washing with 300. Mu.L of washing solution 5 times after the reaction; step 2, adding 100 mu L of horseradish peroxidase-labeled thrombomodulin antibody into a coating plate, uniformly mixing and reacting; washing with 300. Mu.L of washing solution 5 times after the reaction; step 3, sequentially adding 50ul of substrate A and 50ul of substrate B into the coating plate, uniformly mixing, and measuring the luminous intensity in a chemiluminescence analyzer; and 4, drawing a luminous intensity standard curve by using the calibrator with known concentration, and calculating the content of thrombomodulin in the sample to be detected according to the luminous intensity obtained in the step 3 by comparing with the luminous intensity standard curve.
Description
Technical Field
The invention belongs to the technical field of thrombomodulin detection, and particularly relates to a thrombomodulin detection method and a kit.
Background
The change of thrombomodulin content on the membrane surface and in plasma is caused by abnormal expression and secretion of thrombomodulin and release into blood when normal endothelial cells of human body are diseased or damaged. Therefore, the content of thrombomodulin can reflect the endothelial cell state of the organism and is the most reliable index for judging whether the endothelial cells in the organism are damaged or not.
The existing thrombomodulin detection kit has the problems of low detection speed, low sensitivity, poor specificity and the like. Therefore, there is a need to design a novel method and kit for detecting thrombomodulin to meet the needs
Disclosure of Invention
The invention aims to provide a method and a kit for detecting thrombomodulin, which have the advantages of high detection speed, high sensitivity, good specificity, small variation coefficient and good stability of each reagent component and can meet the existing detection requirements.
In order to achieve the above purpose, the present invention provides the following technical solutions.
A method for detecting thrombomodulin, characterized by: the detection method comprises the following steps:
step 2, adding 100 mu L of horseradish peroxidase-labeled thrombomodulin antibody into a coating plate, uniformly mixing and reacting; washing with 300. Mu.L of washing solution 5 times after the reaction;
step 3, sequentially adding 50ul of substrate A and 50ul of substrate B into the coating plate, uniformly mixing, and measuring the luminous intensity in a chemiluminescence analyzer;
and 4, drawing a luminous intensity standard curve by using the calibrator with known concentration, and calculating the content of thrombomodulin in the sample to be detected according to the luminous intensity obtained in the step 3 by comparing with the luminous intensity standard curve.
Further, the solution concentration of the biotin-labeled thrombomodulin antibody was 2.2. Mu.g/mL.
Further, the concentration of the solution of the horseradish peroxidase-labeled thrombomodulin antibody was 0.16. Mu.g/mL.
Further, the coated plate is a streptavidin coated chemiluminescent microplate.
Further, the washing solution was Tris-HCl solution containing 1% Tween-20, 0.1% Proclin300 and 0.05M.
Further, the substrate A is an aqueous solution containing 0.4% luminol, pH9.0, and the substrate B is an aqueous solution containing 0.06% Na2B4O7, pH 5.0.
Further, the calibrator comprises thrombomodulin antigen solution at a concentration of 0TU/mL, 5TU/mL, 40TU/mL, 100TU/mL, 200 TU/mL.
Further, the reaction conditions in the step 1 and the step 2 are respectively 37 ℃ for 10 minutes.
Based on the detection method, the invention also provides a detection kit of thrombomodulin, which is characterized in that: the kit comprises a coating plate, a biotin-labeled thrombomodulin antibody, a horseradish peroxidase-labeled thrombomodulin antibody, a calibrator, a substrate A, a substrate B and a washing solution.
Further, the substrate A, the substrate B and the washing liquid are all independently packaged.
Compared with the prior art, the invention has the following beneficial effects:
the kit for detecting thrombomodulin has the advantages of high detection speed, high sensitivity, good specificity, small variation coefficient, good stability of each reagent component, stability of more than one year at 2-8 ℃, low detection cost and capability of meeting the existing detection requirements.
Drawings
FIG. 1 is a linear fit curve for the calculation of example reagents.
Detailed Description
Examples: based on the detection method of the present invention, a kit was prepared to detect various performance indexes as follows. The kit is used as an experimental group, and a commercial kit is used as a control group for experiments.
the measurement was repeated 20 times using a zero-concentration dilution as a sample, and the mean value (M) and Standard Deviation (SD) of the RLU values (relative luminescence values) were calculated to obtain M+2SD. The concentration value corresponding to (m+2sd) was calculated as the lowest limit of detection using the dose-response curve.
TABLE 1 minimum limit of detection results
The detection data show that other various components added into the kit can not influence the minimum detection limit, and the kit still keeps a good minimum detection limit.
Performance index 2, linear range and correlation coefficient:
diluting high-value samples near the upper limit of the linear range to 5 concentrations in a certain proportion, wherein the low-value samples near the lower limit of the linear range are repeatedly detected for 3 times, calculating the average value of the samples, performing linear fitting on the average value and the dilution proportion of the result by using a least square method, and calculating a linear correlation coefficient R 2 ，R 2 Not less than 0.99.
As shown in FIG. 1, the experimental results show that the R of the linear result of the kit 2 And the detection result is higher than 0.99, and the accurate detection result is proved in the linear range of the reagent.
Performance index 3, accuracy:
the comparison experiments were performed using TM commercial quality control 1 and quality control 2 as samples, using TM detection kits approved in the market as control groups, and each detection was repeated 3 times, and each detection result was designated as (Mi), and the relative deviation (Bi) of each detection result was calculated.
TABLE 3 accuracy test results
As can be seen from the detection data in Table 3, the relative deviation between the detection kit and the target value of the quality control substance is within 10%, which accords with the technical requirements formulated by enterprises, and the test result is better than the test result within 15% of the control kit, which shows that the accuracy performance of the kit is better than that of the TM detection kit approved in the market, and the other various components added by the kit are proved to improve the accuracy.
Performance index 4, repeatability:
the TM commercial quality control 1 and quality control 2 were used as samples, and a comparison experiment was performed using a TM detection kit approved in the market as a control group, and each of the samples was repeatedly tested 10 times, and the average value M and standard deviation SD of the measured concentration results of 10 times for each reference were calculated, and the coefficient of variation was calculated according to the formula cv=sd/m×100%.
TABLE 4 repeatability test results
The experimental results show that the variation coefficient of the detection results of the reagent and the reagent of the comparison example is within 10%, and the detection results of the embodiment are superior to the results of the comparison example, thus proving that the reagent is superior to the commercial reagent results.
Performance index 5, stability:
the kits of the three examples were all placed in 37℃for 7 days, and measured three times at 0 day, 1 day, 3 days, 5 days, and 7 days, respectively, and the average was compared with the detection results of the fresh comparative example 1 reagent.
TABLE 5 stability test results
The results of comparative examples 1 to 3 show that the degradation rates of the reagents of examples 1, 2 and 3 stored for 7 days in a light-resistant environment are respectively 7.07%, 3.53% and 5.17%, and the test results of the reagents of examples 1, 2 and 3 stored for 7 days in a light-resistant environment at 37 ℃ are respectively reduced by 2.80%, 1.81% and 2.21%, and the reduction amplitude is smaller than that of the reagents of comparative examples, so that the stability of the reagents of the invention can meet the requirements of products.
Claims (10)
1. A method for detecting thrombomodulin, characterized by: the detection method comprises the following steps:
step 1, adding a sample to be tested and a biotin-labeled thrombomodulin antibody into a coating plate, uniformly mixing and reacting; washing with washing liquid after the reaction;
step 2, adding the horseradish peroxidase-labeled thrombomodulin antibody into a coating plate, uniformly mixing and reacting; washing with washing liquid after the reaction;
step 3, sequentially adding a substrate A and a substrate B into the coating plate, uniformly mixing, and measuring the luminous intensity in a chemiluminescence analyzer;
and 4, drawing a luminous intensity standard curve by using the calibrator with known concentration, and calculating the content of thrombomodulin in the sample to be detected according to the luminous intensity obtained in the step 3 by comparing with the luminous intensity standard curve.
2. The method for detecting thrombomodulin according to claim 1, characterized in that: the solution concentration of the biotin-labeled thrombomodulin antibody was 2.2. Mu.g/mL.
3. The method for detecting thrombomodulin according to claim 1, characterized in that: the concentration of the solution of the horseradish peroxidase-labeled thrombomodulin antibody was 0.16. Mu.g/mL.
4. The method for detecting thrombomodulin according to claim 1, characterized in that: the coating plate is a streptavidin-coated chemiluminescent microplate.
5. The method for detecting thrombomodulin according to claim 1, characterized in that: the washing solution is Tris-HCl solution containing 1% Tween-20, 0.1% Proclin300 and 0.05M.
6. The method for detecting thrombomodulin according to claim 1, characterized in that: the substrate A is an aqueous solution containing 0.4% luminol, pH9.0, and the substrate B is an aqueous solution containing 0.06% Na2B4O7, pH 5.0.
7. The method for detecting thrombomodulin according to claim 1, characterized in that: the calibrator comprises thrombomodulin antigen solution at a concentration of 0TU/mL, 5TU/mL, 40TU/mL, 100TU/mL, 200 TU/mL.
8. The method for detecting thrombomodulin according to claim 1, characterized in that: the reaction conditions in the step 1 and the step 2 are respectively 37 ℃ for 10 minutes.
9. A thrombomodulin assay kit based on the assay method according to any one of claims 1-8, characterized in that: the kit comprises a coating plate, a biotin-labeled thrombomodulin antibody, a horseradish peroxidase-labeled thrombomodulin antibody, a calibrator, a substrate A, a substrate B and a washing solution.
10. The kit for detecting thrombomodulin of claim 9 wherein: further, the substrate A, the substrate B and the washing liquid are all independently packaged.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211654564.8A CN116183586A (en) | 2022-12-22 | 2022-12-22 | Method and kit for detecting thrombomodulin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211654564.8A CN116183586A (en) | 2022-12-22 | 2022-12-22 | Method and kit for detecting thrombomodulin |
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| Publication Number | Publication Date |
|---|---|
| CN116183586A true CN116183586A (en) | 2023-05-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211654564.8A Pending CN116183586A (en) | 2022-12-22 | 2022-12-22 | Method and kit for detecting thrombomodulin |
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| CN (1) | CN116183586A (en) |
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2022
- 2022-12-22 CN CN202211654564.8A patent/CN116183586A/en active Pending
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