CN114354929A - Kit for monitoring fibrinolytic state of human body and application - Google Patents
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Abstract
The invention relates to a kit for monitoring the fibrinolytic state of a human body, which comprises 6 fluorescent coding microspheres which are respectively coupled with six different monoclonal antibodies of human fibrinogen PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer and mixed in a certain amount, and also comprises a pairing detection antibody marked by biotin. The kit provided by the invention provides a group of 6-color antibody combination schemes by utilizing 6 antibodies, single-tube detection of a single sample is realized by utilizing the kit provided by the invention on a multi-color flow cytometer platform, deep analysis can be carried out on fibrinolysis related factors by bioinformatics analysis, and the fibrinolysis state of a physical examiner can be effectively monitored.
Description
Technical Field
The invention relates to the field of cytobiology and biomedical detection, in particular to a kit for monitoring a human fibrinolytic state and application thereof.
Background
The occurrence and development of blood coagulation comprise multiple links of coagulation, fibrinolysis system, anticoagulation system, endothelial system, inflammation system and the like. Wherein the fibrinolysis-related molecular markers include PLG, PAI, alpha 2-AP, PIC, FIB, and D-Dimer. PLG, plasminogen, is a plasmin precursor and proteolytic enzymes are capable of solubilizing part of the fibrinolytic system fibrinogen/fibrin. Plasminogen can be converted to plasmin by endogenous activators, such as urokinase or tissue-type plasminogen activator, or by external activators such as streptokinase, among others. Plasminogen levels are elevated in patients with tumors and diabetes. Patients with liver disease, Disseminated Intravascular Coagulation (DIC), and therapeutic fibrinolysis, among others, have reduced levels of plasminogen due to decreased synthesis or increased consumption. PAI, a plasminogen activator inhibitor, which specifically inhibits t-PA and inactivates it by forming a 1:1 complex with t-PA, is also known as a t-PA fast inhibitor. PAIs are mainly classified into four categories, namely vascular endothelial cell type (PAI1), placental type (PAI2), PAI3 and protein ligase (PN). Normally, only PAI1 is detected in plasma, and the PAI1 exists mainly in vascular endothelial cells and belongs to serine protease inhibitor. t-PA and PAI are normally in arterial balance, and the imbalance of the pair of physiologically active substances is closely related to the occurrence and development of a plurality of hemorrhagic diseases and thrombotic diseases. Alpha 2-antiplasmin alpha 2-AP is the most important inhibitor of plasmin, which is an enzyme for activating fibrinolysis, and can rapidly form an irreversible and inactive compound. The reduced alpha 2-antiplasmin activity is seen in Disseminated Intravascular Coagulation (DIC) complications, or hyperfibrinolysis caused by surgery on organs containing large amounts of plasminogen activator. Alpha 2-antiplasmin deficiency is seen in obstructive diseases (e.g. severe hepatocellular damage). Alpha 2-antiplasmin can also be used to assist in the assessment of problematic issues arising in fibrinolytic therapy. The activation of the fibrinolytic system is mainly marked by Plasmin-alpha 2-antiplasmin Complex (PIC), which is a Complex of Plasmin and its representative Inhibitor alpha 2 antiplasmin 1:1 and can reflect the degree of activation of fibrinolysis. The liver produces plasmin inhibitor, α 2plasmin inhibitor (α 2PI), α 2PI is expressed in a ratio of 1:1 rapidly associate to form PIC which inhibits fibrinolysis. Therefore, PIC faithfully reflects the molecular marker of plasmin at the final stage of the fibrinolytic system, and is also one of the important indexes of thrombolytic therapy and prevention of re-thrombosis. D-Dimer, the D-Dimer, is one of the specific markers of fibrin degradation by plasmin after crosslinking. Is a good index for distinguishing primary fibrinolysis and secondary fibrinolysis. The fibrinolysis related protein can provide important prompt for the fibrinolysis state of an organism and diagnose certain bleeding and thrombotic diseases, and related detection indexes can play a role in monitoring the state of illness and curative effect in the treatment process, so that the fibrinolysis related protein has important clinical significance.
Disclosure of Invention
The invention aims to provide a kit for detecting the human body fibrinolysis state, and also provides a preparation method of the kit and application of the kit in detecting the human body fibrinolysis state.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a kit for monitoring fibrinolysis state of human body comprises 6 fluorescent coding microspheres which are respectively coupled with six different monoclonal antibodies of human fibrinogen PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer and mixed in a certain amount, and also comprises a pairing detection antibody marked by biotin.
The preparation process comprises two steps, wherein the first step is to couple the capture antibody to the fluorescent coding microspheres, uniformly mix the microspheres after the coupling is finished, and store the microspheres in a sealed and light-proof way; and the second step is to carry out biotin labeling on the paired detection antibodies, carry out purification and enrichment, uniformly mix the biotin labeled antibodies after the end, and store the antibodies in a sealed and light-proof manner.
The invention also provides a preparation method of the kit for monitoring the fibrinolytic state of the human body, which comprises the following steps:
1) activating the fluorescent coding microspheres through EDC and NHS;
2) coupling the activated microspheres with six different monoclonal capture antibodies of human fibrinogen PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer at different concentrations respectively;
3) washing the coupled microspheres by using PBST to obtain fluorescent coding microspheres coupled with the capture antibody;
4) respectively adding biotin reagents into the paired detection antibodies for chemical coupling labeling;
5) and (3) carrying out purification and enrichment after labeling in a dialysis or centrifugal ultrafiltration mode to obtain the biotin-labeled paired detection antibody.
As a preferred scheme, the detection channel selects APC and APC-CY7 channels for efficient resolution of the fluorescent coding microspheres, and the PE channel is selected for detecting the abundance of the target substance, so that the mutual interference among the channels is small.
The invention also protects the application of the kit in detecting the fibrinolytic state of a human body, can simultaneously carry out multiple immunoassay and has no cross reaction interference.
As a preferred embodiment of the application of the kit of the invention in detecting the fibrinolytic state of the human body, when the kit is used for detecting the fibrinolytic state of the human body, the detection sample is from the peripheral blood of the human body, and the human body comprises people of all ages.
As a preferred embodiment of the use of the kit of the present invention for detecting fibrinolytic state in human body, the kit is used for detecting PLG, PAI, α 2-AP, PIC, FIB and D-Dimer.
As a preferred embodiment of the application of the kit in detecting the human body fibrinolysis state, the kit comprises the following steps in detecting the human body fibrinolysis state:
(1) separating peripheral blood plasma;
(2) detecting the expression abundance of the corresponding antigen in the peripheral blood plasma of the sample by a flow cytometer;
(3) and (3) performing dimensionality reduction analysis and operation by using an algorithm according to the abundance of each coagulation factor protein expression in peripheral blood, and determining the fibrinolysis state of the detection sample.
When the kit is applied to detecting the fibrinolysis state of a human body, the used detection instrument is preferably but not limited to a fluorescence flow cytometer.
As a preferred embodiment of the application of the kit of the present invention in detecting the fibrinolytic state of the human body, the flow cytometry detection in the step (2) comprises the following steps:
1) performing instrument calibration verification;
2) performing parameter setting and channel voltage adjustment of a flow cytometer
3) Performing on-machine detection to obtain original data;
4) and importing the data into corresponding calculation software, carrying out data analysis, obtaining a corresponding concentration result and outputting a detection report.
Compared with the prior art, the invention has the beneficial effects that:
bleeding or thrombotic diseases are caused by hereditary/acquired factors leading to reduced hemostasis or coagulation function, hypercoagulability or hyperfibrinolysis in vivo, resulting in bleeding of the body and, in turn, thrombosis. Is a common disease with high disability and mortality rate in clinic, the morbidity of the disease is the first of various diseases, and therefore, the monitoring of bleeding or thrombotic diseases is particularly important. The kit disclosed by the invention can be used for deeply analyzing fibrinolysis related factors in peripheral blood, establishing a system for scoring the fibrinolysis state of an organism, evaluating the risk of organism hemorrhage or thrombus, assisting in diagnosing certain diseases, analyzing pathogenesis, guiding treatment, observing curative effect and detecting prognosis. The kit can find whether the detected person has abnormal fibrinolytic state through detection and evaluate the bleeding or thrombus risk of the detected person.
The kit provided by the invention provides a group of 6-color antibody combination schemes by utilizing 6 antibodies, realizes single-tube detection of a single sample by utilizing the kit provided by the invention on a multi-color flow cytometer platform, can carry out deep analysis on fibrinolysis related factors by bioinformatics analysis, and can effectively monitor the fibrinolysis state of a physical examiner. By adopting the kit, the depth analysis of the coagulation related factors in the peripheral blood can be rapidly and accurately carried out by collecting the peripheral blood through veins; the kit is suitable for monitoring the fibrinolytic state of various conditions.
The kit can quickly and accurately carry out deep analysis on the fibrinolysis state of the human body only by simple operation, the dosage of blood samples is very small, each antibody can be accurately measured only by 0.5-4 muL, and the repeatability reproducibility of measured data is good; the method has the advantages of easily obtained samples, short report period, effectively reduced detection time and cost, high sensitivity of detection results and strong specificity.
Detailed Description
The above-mentioned contents of the present invention are further described in detail by way of examples below, but it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples, and any technique realized based on the above-mentioned contents of the present invention falls within the scope of the present invention.
The kit for monitoring the fibrinolytic state of the human body, provided by the invention, is suitable for people at risk of bleeding or thrombus. The kit analyzes the expression abundance of peripheral blood plasma protein by using a flow cytometer, and evaluates the fibrinolysis state by being assisted with bioinformatics analysis.
The inventor of the application establishes a monitoring scheme based on PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer by analyzing the expression abundance of peripheral blood plasma protein, captures a target substance by coupling a fluorescence coding microsphere with a capture antibody, labels biotin by matching the detection antibody, and amplifies a detection signal by a biotin-streptavidin system. The flow meter is used for detecting the basic samples of hundreds of healthy people, a model which is suitable for Chinese and reflects the fibrinolytic state is established, and the correlation of the fibrinolytic state can be accurately evaluated, so that the bleeding and thrombus risks can be judged, and clinical treatment can be guided.
The monoclonal capture antibody and the detection antibody to be used in the present invention are commercially available, for example, but not limited to, brands such as Biolegend, BD, R & D, etc.
Biotin used in the present invention is commercially available, for example, but not limited to, the brand name ThermoFisher, BD, etc.
Based on a large amount of experimental researches, the inventor monitors the fibrinolysis state, establishes a corresponding flow cytometry scheme, and determines the human fibrinolysis state through a total of 6 antibody combinations of marked PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer.
The human fibrinolysis state monitoring method is suitable for people of all ages, the optimal proportion of the kit is determined through a large number of experiments, high sensitivity and detection specificity can be achieved, and the detection effect can reach an expectation; the sample obtained when the fibrinolysis state monitoring model is established is taken from peripheral blood of a healthy person; and (3) taking 200 healthy human samples, carrying out expression abundance detection on the antigens corresponding to the peripheral fibrinolytic factors, and establishing a biological reference interval through statistical analysis.
When the kit is used for detecting the fibrinolysis state of a human body, the kit firstly detects the peripheral fibrinolysis related factors of a healthy human sample, and the specific method comprises the following steps:
the kit is used for detection, and comprises 6 fluorescent coding microspheres which are respectively coupled with six different monoclonal antibodies, namely PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer, and are mixed in a certain amount and matched with a biotin-labeled detection antibody.
The preparation method of the kit comprises the following steps:
1) the fluorescent-encoded microspheres were activated by EDC and NHS.
2) The activated microspheres are coupled with six different monoclonal capture antibodies of human fibrinogen PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer at different concentrations respectively.
3) And washing the coupled microspheres by using PBST to obtain the fluorescent coding microspheres coupled with the capture antibody.
4) And respectively adding biotin reagents into the paired detection antibodies for chemical coupling labeling.
5) And (3) carrying out purification and enrichment after labeling in a dialysis or centrifugal ultrafiltration mode to obtain the biotin-labeled paired detection antibody.
After the preparation of the kit is finished, the storage condition is 2-8 ℃, and the kit needs to be stored in a dark place. After the preparation of the kit is completed, the human peripheral blood from the 200 samples is detected. Preferably, the sample to be tested is tested by a flow cytometer, and the preparation for flow test of the sample to be tested comprises the following steps:
1) collecting not less than 100 mu L of human peripheral blood as a detection sample; immediately detecting the sample after collection, or refrigerating at 2-8 ℃ and detecting within 48 h;
2) adding 25 mu L of mixed capture microspheres into the reaction hole;
3) placing the reaction pore plate on a magnetic separator for 1min, and throwing off the supernatant;
4) adding 25 mu L of mixed detection antibody and 25 mu L of plasma sample into the reaction hole, and oscillating for 60min at 37 ℃;
5) repeating the step 2, and washing for 3 times by using the washing buffer solution;
6) adding 50 mu LSA-PE into the reaction hole, and shaking for 5min at 37 ℃;
7) repeating step 4, and finally adding 150 μ L of washing buffer into each well;
8) and oscillating the mixture for 30s at room temperature, and detecting the mixture on a multicolor flow cytometer, a mass spectrum flow cytometer platform or a liquid phase suspension chip detector.
The application of the kit is based on a flow cytometer, and the kit is used for detecting fibrinolysis-related protein. And performing dimensionality reduction analysis and machine learning calculation according to the detection results of the fibrinolysis related proteins of the 200 samples, namely by using the expression abundance of the corresponding proteins detected by 6 antibodies of the testee, and establishing a fibrinolysis state scoring system for detecting and evaluating the fibrinolysis states of various crowds.
The invention also provides application of the kit in monitoring the fibrinolytic state of a human body.
As a preferred embodiment of the application of the kit in detecting the human body fibrinolysis state, the kit comprises the following steps in detecting the human body fibrinolysis state:
(1) separating peripheral blood plasma;
(2) detecting the expression abundance of the corresponding antigen in the peripheral blood plasma of the sample by a flow cytometer;
(3) and (3) performing dimensionality reduction analysis and operation by using an algorithm according to the abundance of each coagulation factor protein expression in peripheral blood, and determining the fibrinolysis state of the detection sample.
When the kit is applied to detecting the fibrinolysis state of a human body, the used detection instrument is preferably but not limited to a fluorescence flow cytometer.
As a preferred embodiment of the application of the kit of the present invention in detecting the fibrinolytic state of the human body, the flow cytometry detection in the step (2) comprises the following steps:
1) performing instrument calibration verification;
2) performing parameter setting and channel voltage adjustment of a flow cytometer
3) Performing on-machine detection to obtain original data;
4) and importing the data into corresponding calculation software, carrying out data analysis, obtaining a corresponding concentration result and outputting a detection report.
Preferably, when the kit is used for monitoring the human fibrinolysis state, the kit can detect the human fibrinolysis state, and the specific detection comprises the following steps:
1) detecting PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer markers of the test sample by flow cytometry;
2) the fibrinolytic state of the test sample is evaluated against the above steady state model based on the expression of each protein.
Target substances in plasma are captured through the marked microspheres, and a detection antibody-SA-PE detection compound of capture antibody microsphere-antigen-biotin mark can be formed after biotin-marked paired detection antibody and streptavidin-phycoerythrin (SA-PE) are added. On a multicolor flow cytometer, a mass spectrum flow cytometer platform or a liquid phase suspension chip detector, a detection compound is excited by two beams of laser with different wavelengths, one beam is used for judging the type of microspheres, and the other beam is used for exciting PE fluorescence in the detection compound. The intensity of the PE fluorescence is positively correlated with the concentration of the cytokine marker in the calibrator or sample. And fitting the concentration of the calibrator and the PE fluorescence signal intensity to form a concentration-fluorescence intensity standard curve, and further calculating the target concentration in the sample by using a curve equation.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (8)
1. A kit for monitoring the fibrinolytic state of a human body, characterized in that: comprises 6 fluorescent coding microspheres which are respectively coupled with six different monoclonal antibodies of human fibrinogen PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer and mixed in a certain quantity, and also comprises a pairing detection antibody marked by biotin.
2. A kit for monitoring the fibrinolytic state of a human according to claim 1, wherein: the preparation process comprises two steps, wherein the first step is to couple the capture antibody to the fluorescent coding microspheres, uniformly mix the microspheres after the coupling is finished, and store the microspheres in a sealed and light-proof way; and the second step is to carry out biotin labeling on the paired detection antibodies, carry out purification and enrichment, uniformly mix the biotin labeled antibodies after the end, and store the antibodies in a sealed and light-proof manner.
3. A method of manufacturing a kit for monitoring the fibrinolytic state of a human according to claim 1 or 2, wherein: the method specifically comprises the following steps:
1) activating the fluorescent coding microspheres through EDC and NHS;
2) coupling the activated microspheres with six different monoclonal capture antibodies of human fibrinogen PLG, PAI, alpha 2-AP, PIC, FIB and D-Dimer at different concentrations respectively;
3) washing the coupled microspheres by using PBST to obtain fluorescent coding microspheres coupled with the capture antibody;
4) respectively adding biotin reagents into the paired detection antibodies for chemical coupling labeling;
5) and (3) carrying out purification and enrichment after labeling in a dialysis or centrifugal ultrafiltration mode to obtain the biotin-labeled paired detection antibody.
4. A method of manufacturing a kit for monitoring the fibrinolytic state of a human according to claim 3, wherein: the detection channel selects the APC channel and the APC-CY7 channel to perform high-efficiency resolution of the fluorescent coding microspheres, the PE channel is selected to perform detection on the abundance of the target substance, and the mutual interference among the channels is small.
5. Use of a kit according to claim 1 or 2 for detecting the fibrinolytic state of a human, characterized in that: can simultaneously carry out multiple immunoassay without cross reaction interference.
6. Use of a kit according to claim 5 for detecting the fibrinolytic state of a human, wherein: 6-fold immunoassays including detection for PLG, PAI, α 2-AP, PIC, FIB, and D-Dimer can be performed simultaneously.
7. Use of a kit according to claim 5 for detecting the fibrinolytic state of a human, wherein: the method comprises the following steps:
(1) separating peripheral blood plasma;
(2) detecting the expression abundance of the corresponding antigen in the peripheral blood plasma of the sample by a flow cytometer;
(3) and (3) performing dimensionality reduction analysis and operation by using an algorithm according to the abundance of each coagulation factor protein expression in peripheral blood, and determining the fibrinolysis state of the detection sample.
8. Use of a kit according to claim 5 for detecting the fibrinolytic state of a human, wherein: the detection instrument used was a fluorescence flow cytometer comprising the following steps:
1) performing instrument calibration verification;
2) performing parameter setting and channel voltage adjustment of a flow cytometer
3) Performing on-machine detection to obtain original data;
4) and importing the data into corresponding calculation software, carrying out data analysis, obtaining a corresponding concentration result and outputting a detection report.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353789A (en) * | 2011-06-22 | 2012-02-15 | 江苏迈迪基因生物科技有限公司 | Joint detection method for heart cerebrovascular disease-related protein marker and diagnostic kit thereof |
| CN108289935A (en) * | 2015-11-03 | 2018-07-17 | 普罗米蒂克生物治疗有限公司 | The plasminogen alternative medicine of plasminogen deficiency disease |
| CN111239401A (en) * | 2020-03-12 | 2020-06-05 | 天津美瑞特医疗科技有限公司 | Quantitative detection method and kit for rapid diagnosis of human respiratory pathogens |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353789A (en) * | 2011-06-22 | 2012-02-15 | 江苏迈迪基因生物科技有限公司 | Joint detection method for heart cerebrovascular disease-related protein marker and diagnostic kit thereof |
| CN108289935A (en) * | 2015-11-03 | 2018-07-17 | 普罗米蒂克生物治疗有限公司 | The plasminogen alternative medicine of plasminogen deficiency disease |
| CN111239401A (en) * | 2020-03-12 | 2020-06-05 | 天津美瑞特医疗科技有限公司 | Quantitative detection method and kit for rapid diagnosis of human respiratory pathogens |
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