CN110596398A - Protein chip for detecting blood coagulation marker and preparation method and application thereof - Google Patents
Protein chip for detecting blood coagulation marker and preparation method and application thereof Download PDFInfo
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- CN110596398A CN110596398A CN201910746725.8A CN201910746725A CN110596398A CN 110596398 A CN110596398 A CN 110596398A CN 201910746725 A CN201910746725 A CN 201910746725A CN 110596398 A CN110596398 A CN 110596398A
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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
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to the technical field of biology, in particular to a protein chip for detecting a blood coagulation marker and a preparation method and application thereof; a protein chip for detecting a blood coagulation marker, wherein the protein chip simultaneously contains four antibody indexes of TAT, PIC, TM and t-PAI-C; the preparation method comprises the following steps: pretreating a black glass slide; spotting an antibody solution; a sealing process is carried out to prepare the protein chip; the protein chip for detecting the blood coagulation marker simultaneously comprises four antibody indexes of TAT, PIC, TM and t-PAI-C, realizes the aim of joint detection, has the advantages of high detection efficiency and low detection cost, and can realize the aim of simultaneously and rapidly detecting 4 indexes by only one patient sample in the actual detection process; furthermore, the like products of the protein core product for detecting the blood coagulation marker and the application thereof are not available in the market at present.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a protein chip for detecting a blood coagulation marker and a preparation method and application thereof.
Background
Thrombus is a disease with hidden onset, sudden onset and high lethality disability rate, can be seen in almost all clinical departments, and the pathological process of the thrombus relates to three major systems of vascular endothelium, blood coagulation and fibrinolysis. Researches have proved that when the organism is in a prothrombotic state, the vascular endothelium, the blood coagulation and the fibrinolysis system are changed, TAT (thrombin-antithrombin complex), PIC (plasmin-antiplasmin complex), TM (thrombomodulin) and t-PAI-C (tissue plasminogen activator-plasminogen activator inhibitor-1 complex) are effective indexes reflecting the early change of the vascular endothelium, the blood coagulation and the fibrinolysis system of the organism, and are suitable for early diagnosis, risk assessment and treatment effect evaluation of thrombus of high risk groups of thrombus in various clinical subjects and thrombus risk screening of healthy people.
These four markers are now widely accepted by hospitals. There are also a variety of kits for individually detecting these four markers. However, the mainstream method adopted at present is an immunoassay method (for example, ELISA or chemiluminescence method). Although these four markers can be measured, there are disadvantages such as a slow detection speed and a high detection cost. And the four indexes are respectively detected, so that the practical problems of inconvenient operation, high error probability and the like exist. Thrombus patients are generally emergent patients, and in order to accelerate detection, a plurality of hospitals have to take multitubular blood samples simultaneously for detecting TAT, PIC, TM and t-PAI-C respectively, so that great waste and burden are caused.
Disclosure of Invention
The purpose of the invention is: the protein chip for detecting the blood coagulation marker has the advantages of high detection efficiency and low detection cost;
another object of the invention is: the preparation method is simple in process, and the capture antibody is fixed on a chip substrate with glass as a carrier, so that the protein chip for detecting the blood coagulation marker is prepared.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a protein chip for detecting coagulation markers, which comprises four antibody indexes including TAT, PIC, TM and t-PAI-C.
A method for preparing a protein chip for detecting a coagulation marker, the method comprising the steps of:
(1) pretreating a black glass slide;
(2) spotting an antibody solution, wherein the antibody solution comprises a TAT monoclonal antibody solution, a PIC monoclonal antibody solution, a TM monoclonal antibody solution and a t-PAI-C monoclonal antibody solution;
(3) and (3) sealing to obtain the protein chip.
Further, the method for pretreating the black glass slide in the step (1) comprises the following steps:
soaking a black glass slide in a glass slide pretreatment solution containing ethanol for 16-24 hours, and then cleaning with purified water for 2-8 times;
secondly, soaking the black glass slide in 1-2% silane water solution for 60-120 min;
thirdly, placing the soaked black glass slide into an oven, and baking for 0.1-0.3 h at the temperature of 200-240 ℃.
Further, the spotting method in the step (2) is machine-automated spotting.
Further, the sealing process in the step (3) is as follows: and immersing the spotted black glass slide into the sealing liquid for 1-24 h, then taking out the black glass slide, and centrifuging to remove residual sealing liquid to obtain the protein chip.
Further, the blocking protein is whey protein; the buffer solution is one or more of PBS buffer solution, Tris buffer solution, HEPS buffer solution and MOPS buffer solution.
The application of the protein chip is used for preparing a kit for detecting the blood coagulation marker.
Further, the kit further comprises a secondary antibody solution labeled with HRP enzyme, and a chemiluminescent substrate sensitive to HRP enzyme.
The technical scheme adopted by the invention has the beneficial effects that:
the protein chip for detecting the blood coagulation marker simultaneously comprises four antibody indexes of TAT, PIC, TM and t-PAI-C, realizes the aim of combined detection, has the advantages of high detection efficiency and low detection cost, and can realize the aim of simultaneously and rapidly detecting 4 indexes by only one patient sample in the actual detection process. Furthermore, the like products of the protein core product for detecting the blood coagulation marker and the application thereof are not available in the market at present.
The preparation method of the protein chip for detecting the blood coagulation marker is simple in process, a chemiluminescent substrate sensitive to HRP enzyme is added in the preparation process of the protein chip kit for chemiluminescence, a CCD camera is used for collecting an optical signal of the chemiluminescent substrate, and an experimenter judges the concentration of the specific marker antigen in a detected sample according to the intensity of the optical signal.
Compared with single detection methods of other domestic manufacturers, the protein chip applied to the kit has the following advantages: (1) because the antibody on the protein chip has high specificity and strong affinity for combining with the antigen and is slightly influenced by impurities, the kit prepared by the protein chip has very low requirement on a biological sample, and the pretreatment process of the sample is simplified;
(2) because the protein chip simultaneously contains four antibodies, the goal of quantitative analysis of protein samples with high flux and parallelization is realized, and the operation method is simple and the detection accuracy is high; most importantly, the patient can quickly obtain a plurality of detection results by only collecting one part of the Asahi Yang;
(3) the amount of reagents and samples required by detection is small, and the use cost is low.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic diagram of a thrombus in the background art of the present invention.
FIG. 2 is a schematic diagram of the layout of the protein chip of the present invention.
FIG. 3 is a schematic diagram showing the correlation between TAT of the present invention and the detection result of the conventional kit (chemiluminescence method).
FIG. 4 is a schematic diagram showing the correlation between the detection results of PIC of the present invention and that of a conventional kit (chemiluminescence method).
FIG. 5 is a graph showing the correlation between the detection results of the TM of the present invention and the conventional kit (chemiluminescence method).
FIG. 6 is a graph showing the correlation between the detection results of t-PAI-C of the present invention and those of a conventional kit (chemiluminescence method).
In the figure: column 1: positive quality control column, columns 2 and 5: blank, column 3: TAT assay column; column 4: PIC assay column, column 6: a TM assay column; column 7: t-PAI-C assay column, column 8: negative quality control.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one preferred embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
A protein chip for detecting a blood coagulation marker is prepared by the following steps:
(1) pretreating a black glass slide;
soaking a black glass slide in a glass slide pretreatment solution containing 75% ethanol for 16 hours, and then cleaning the black glass slide for 2-8 times by using purified water;
soaking the black glass slide in 1% concentration silane solution for 120 min;
thirdly, the soaked black glass slide is placed into an oven and baked for 0.3h at the temperature of 200 ℃.
(2) Spotting an antibody solution;
referring to FIG. 2, TAT monoclonal antibody solution, PIC monoclonal antibody solution, TM monoclonal antibody solution and t-PAI-C monoclonal antibody solution were spotted by machine automatically, the concentration of the antibody solution was 0.1mg/mL, 20nL of spots were spotted per spot, and the distribution of spotting of each antibody was as shown in FIG. 2.
(3) Sealing process;
and immersing the spotted black glass slide into a blocking solution (PBS buffer solution containing 1% whey protein) for 10 hours, taking out the black glass slide, and centrifuging to remove residual blocking solution to obtain the protein chip.
(4) Reagent kit
The protein chip was packaged together with a secondary antibody solution labeled with HRP enzyme (concentration 1ug/ml, wherein the medium was an enzyme-labeled secondary antibody diluent of semelafin, purchased from outsource, pH 6.0), detection solution a (containing 1% hydrogen peroxide), and detection solution B (containing 1% luminol and 2% Tris) into a kit.
Example 2
A protein chip for detecting a blood coagulation marker is prepared by the following steps:
(1) pretreating a black glass slide;
soaking a black glass slide in a glass slide pretreatment solution containing 75% ethanol for 24 hours, and then cleaning the black glass slide for 2-8 times by using purified water;
soaking the black glass slide in 1.5% concentration silane water solution for 90 min;
thirdly, the soaked black glass slide is placed into an oven and baked for 0.2h at the temperature of 220 ℃.
(2) Spotting an antibody solution;
referring to FIG. 2, TAT monoclonal antibody solution, PIC monoclonal antibody solution, TM monoclonal antibody solution and t-PAI-C monoclonal antibody solution were spotted by machine automatically, the concentration of the antibody solution was 0.1mg/mL, 20nL of spots were spotted per spot, and the distribution of spotting of each antibody was as shown in FIG. 2.
(3) Sealing process;
and immersing the spotted black glass slide into a blocking solution (PBS buffer solution containing 2% whey protein) for 24 hours, taking out the black glass slide, and centrifuging to remove residual blocking solution to obtain the protein chip.
(4) Reagent kit
The protein chip was packaged together with a secondary antibody solution labeled with HRP enzyme (concentration 1ug/ml, wherein the medium was an enzyme-labeled secondary antibody diluent of semelafin, purchased from outsource, pH 6.0), detection solution a (containing 1% hydrogen peroxide), and detection solution B (containing 1% luminol and 2% Tris) into a kit.
Example 3
A protein chip for detecting a blood coagulation marker is prepared by the following steps:
(1) pretreating a black glass slide;
soaking a black glass slide in a glass slide pretreatment solution containing 75% ethanol for 20 hours, and then cleaning the black glass slide for 2-8 times by using purified water;
soaking the black glass slide in 2% concentration silane solution for 60 min;
thirdly, the soaked black glass slide is placed into an oven and baked for 0.1 hour at the temperature of 240 ℃.
(2) Spotting an antibody solution;
referring to FIG. 2, TAT monoclonal antibody solution, PIC monoclonal antibody solution, TM monoclonal antibody solution and t-PAI-C monoclonal antibody solution were spotted by machine automatically, the concentration of the antibody solution was 0.1mg/mL, 20nL of spots were spotted per spot, and the distribution of spotting of each antibody was as shown in FIG. 2.
(3) Sealing process;
and immersing the spotted black glass slide into a blocking solution (Tris buffer solution containing 3% whey protein) for 8 hours, then taking out the black glass slide, and centrifuging to remove residual blocking solution to obtain the protein chip.
(4) Reagent kit
The protein chip was packaged together with a secondary antibody solution labeled with HRP enzyme (concentration 1ug/ml, wherein the medium was an enzyme-labeled secondary antibody diluent of semelafin, purchased from outsource, pH 6.0), detection solution a (containing 1% hydrogen peroxide), and detection solution B (containing 1% luminol and 2% Tris) into a kit.
Comparison of test results with the kit
The correlation of the results of the two reagents was verified by regression analysis, using y ═ a + bx and R, in comparison with the commercial Simmetin TAT, PIC, TM, t-PAI-C test kit (chemiluminescence method)2Is given as a fitting equation for regression analysis, wherein: y is the assessment reagent result, x is the reference reagent result, b is the slope of the equation, a is the y-intercept, R2Is the decision coefficient. See fig. 3, 4, 5 and 6, respectively.
Compared with the measured result of a reference reagent, the correlation coefficient R of the product of my department is compared with that of the imported product of the same type2All are more than 0.90, and have higher accuracy consistency.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (8)
1. A protein chip for detection of a coagulation marker, characterized in that: the protein chip comprises four antibody indexes including TAT, PIC, TM and t-PAI-C.
2. A preparation method of a protein chip for detecting a blood coagulation marker is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) pretreating a black glass slide;
(2) spotting an antibody solution, wherein the antibody solution comprises a TAT monoclonal antibody solution, a PIC monoclonal antibody solution, a TM monoclonal antibody solution and a t-PAI-C monoclonal antibody solution;
(3) and (3) sealing to obtain the protein chip.
3. The protein chip for detecting a blood coagulation marker according to claim 2, wherein: the method for pretreating the black glass slide in the step (1) comprises the following steps:
soaking a black glass slide in a glass slide pretreatment solution containing ethanol for 16-24 hours, and then cleaning with purified water for 2-8 times;
secondly, soaking the black glass slide in 1-2% silane water solution for 60-120 min;
thirdly, placing the soaked black glass slide into an oven, and baking for 0.1-0.3 h at the temperature of 200-240 ℃.
4. The protein chip for detecting a blood coagulation marker according to claim 2, wherein: the spotting method in the step (2) is machine-automated spotting.
5. The protein chip for detecting a blood coagulation marker according to claim 2, wherein: the sealing process in the step (3) comprises the following steps: and immersing the spotted black glass slide into the sealing liquid for 1-24 h, then taking out the black glass slide, and centrifuging to remove residual sealing liquid to obtain the protein chip.
6. The protein chip for detecting a blood coagulation marker according to claim 2, wherein: the blocking protein is whey protein; the buffer solution is one or more of PBS buffer solution, Tris buffer solution, HEPS buffer solution and MOPS buffer solution.
7. Use of the protein chip of claim 1, wherein: the protein chip is used for preparing a blood coagulation marker detection kit.
8. Use according to claim 6, characterized in that: the kit also includes a solution of a secondary antibody labeled with HRP enzyme, a chemiluminescent substrate sensitive to HRP enzyme.
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| CN201910746725.8A CN110596398A (en) | 2019-08-14 | 2019-08-14 | Protein chip for detecting blood coagulation marker and preparation method and application thereof |
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Cited By (4)
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| CN111948408A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting TM and application thereof |
| CN111948406A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting thrombin-antithrombin complex and application thereof |
| CN111948407A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluorescence immunoassay kit for detecting PIC and application thereof |
| CN111948390A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting t-PAI-C and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111948408A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting TM and application thereof |
| CN111948406A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting thrombin-antithrombin complex and application thereof |
| CN111948407A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluorescence immunoassay kit for detecting PIC and application thereof |
| CN111948390A (en) * | 2020-08-31 | 2020-11-17 | 浙江盛域医疗技术有限公司 | Time-resolved fluoroimmunoassay kit for detecting t-PAI-C and application thereof |
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