CN116840485A - Detection system, detection method and detection kit for detecting platelet function subpopulation PAC-1 marker, annexin-V marker and CD62P marker - Google Patents

Detection system, detection method and detection kit for detecting platelet function subpopulation PAC-1 marker, annexin-V marker and CD62P marker Download PDF

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CN116840485A
CN116840485A CN202310562050.8A CN202310562050A CN116840485A CN 116840485 A CN116840485 A CN 116840485A CN 202310562050 A CN202310562050 A CN 202310562050A CN 116840485 A CN116840485 A CN 116840485A
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pac
annexin
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王文元
王霄汉
高文成
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Heluo Xintu Biotechnology Shanghai Co ltd
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/7056—Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
    • G01N2333/70564—Selectins, e.g. CD62

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Abstract

The present invention provides a detection system for detecting three markers on platelets in whole blood, comprising: a buffer, a calcium concentrate, and an agonist combination; the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5‑12mM HEPES、5‑12mM NaHCO 3 、0.1‑0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45, agonist comprising 1-2mM ADP and 0.1-1mM TRAP6, calcium concentrate comprising 50-100mM calcium ions. Methods and kit designs for detecting three markers are also provided. The buffer provides a physiological condition more similar to that of plasma and allows for the simultaneous detection of three markers.

Description

Detection system, detection method and detection kit for detecting platelet function subpopulation PAC-1 marker, annexin-V marker and CD62P marker
Technical Field
The invention belongs to the field of detection of platelet functional quality in clinical examination and cell therapy production, and particularly relates to a detection system, a detection method and a detection kit for detecting PAC-1 markers, annexin-V markers and CD62P markers on platelets.
Background
Platelets (platlets) are important cellular components of blood that mediate hemostasis. Platelets are formed by the lysis of megakaryocyte (megakaryocyte) cytoplasm in bone marrow, and undergo continuous maturation and lysis after entering blood circulation to form mature platelets [1]. When bleeding occurs, platelets activate and interact with the coagulation system to form a physiological thrombus for hemostasis [2]. However, various causes such as hematological malignancy, autoimmune diseases, radiotherapy and chemotherapy, etc. induce thrombocytopenia (thrombic cytopenia) leading to bleeding tendency by affecting bone marrow function, peripheral platelet status and platelet clearance [3,4]. Platelet infusion is therefore an important alternative treatment to supplement platelets in vivo in cases of indication and satisfaction of infusion criteria [4-6]. Furthermore, the platelet function detection method is very important, and can be used for detecting the function of the infused platelets and providing basis for the diagnosis and treatment of platelet diseases.
Platelets undergo three functional changes upon activation, 1, aggregation, 2, procoagulant, 3, secretion [2,7] to form platelet thrombi to promote hemostasis. Platelet aggregation is judged by PAC-1 expression levels before and after platelet activation, procoagulant function is judged by Annexin-V binding before and after platelet activation, and secretory function is evaluated by CD62P expression levels before and after platelet activation [7-11]. The current flow of platelet function detection is shown in FIG. 1. Briefly, blood is drawn from a human body and stored and diluted 10-fold with detection buffer (Modified HEPES-Tyrode buffer), and activators and other additives required for the test are added, platelets are fixed after platelet activation is completed, and marker analysis and counting are performed using a flow cytometer [8-10].
However, the three label assays currently suffer from the following disadvantages:
1. the detection method has the defects that: as can be seen from table 4, the three markers each have a detection method and have no correlation [8,9, 11], and do not reflect platelet function under physiological conditions;
2. the assay buffer also had the following disadvantages: the assay Buffer (Modified HEPES-Tyrode Buffer) formulation is shown in Table 5, which suffers from the following disadvantages:
1. the concentration of potassium ions, bicarbonate and calcium ions are different from the concentration of plasma electrolyte under physiological conditions, and can influence the functional state of platelets;
HEPES is used for preparing artificial tissue buffer solution because HEPES has high content, HEPES has high cell edema prevention and strong buffer function, but the buffer substance naturally existing in non-blood is needed to be reduced;
3. the colloidal component adopts bovine serum albumin as a heterogeneous protein component, wherein heterogeneous antibodies can influence the functional state of platelets.
The presence of these deficiencies and deviations can have an impact on the accuracy of the test results and do not reflect platelet function in normal physiological conditions. Therefore, there is a need in the art for a platelet function detection system with correlation and simulated physiological environment to achieve simple, rapid, and accurate detection.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, it is an object of the present invention to provide a detection system, method and kit for detecting PAC-1, annexin-V and CD62P markers on platelets, as well as a detection buffer having electrolyte and colloidal components similar to plasma, to solve the problems of the prior art.
To achieve the above and other related objects, the present invention provides a detection system, method and kit for detecting PAC-1 markers on platelets, an detection system, method and kit for detecting Annexin-V and CD62P markers on platelets, and a detection system, method and kit for detecting PAC-1 markers, annexin-V markers and CD62P markers on platelets.
In a first aspect the present invention provides a detection system for detecting PAC-1 markers on platelets, the detection system comprising the following components: buffers, calcium concentrates and agonists; the saidThe buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45; the calcium concentrate comprises 50-100mM calcium ions; agonists comprise 1-2mM ADP and 0.1-1mM TRAP6. The NaCl content in the buffer is selected from 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140mM.
KCl content was selected from 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0mM.
MgCl 2 The content is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2mM.
HEPES levels were selected from 5, 6, 7, 8,9, 10, 11, 12mM.
NaHCO 3 The content is selected from 5, 6, 7, 8,9, 10, 11, 12mM.
Na 2 HPO 4 The content is selected from 0.1, 0.2, 0.3, 0.4, 0.5mM.
The human serum albumin content is selected from 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% w/v.
The glucose content is selected from 2, 3,4, 5, 6, 7, 8mM.
The pH is selected from 7.35, 7.36, 7.37, 7.38, 7.39, 7.40, 7.41, 7.42, 7.43, 7.44, 7.45.
The ADP content is selected from 1.0, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.28 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.0mM.
The TRAP6 content is selected from 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0mM.
The calcium concentrate content is selected from 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM.
PAC-1 markers are platelet membrane glycoprotein IIb/IIIa complexes, contain specific sites for binding to adhesion proteins such as fibrinogen (Fg), fibronectin (Fn) and vWF factors, exist in a monomer form under normal conditions, have no ability to bind to ligands, change configuration when platelets are activated, expose receptors on PAC-1 molecules, specifically bind to various adhesion proteins, accelerate platelet fibrinogen and platelet aggregation, and are the final common way for causing platelet thrombus.
Annexin V is widely used in biomedical research due to its ability to bind phosphatidylserine. Phosphatidylserine is a phospholipid, usually located in the inner layer of the cell membrane. In activated parts of platelets, phosphatidylserine will migrate to the outer layer of the cell membrane and Annexin V can recognize and bind to surface-exposed phosphatidylserine as one of the markers for detecting procoagulant platelets.
The CD62P marker is a membrane glycoprotein located in the weibe1-palade minibody of platelet specific granules and vascular endothelial cells. Under normal conditions, CD62P is only expressed in small amounts on the surface of platelets, and when platelets are stimulated, the CD62P is rapidly expressed in large amounts on the surface of platelet membranes, and through the action of N-terminal lectin domains, the adhesion function of activated endothelial cells to monocytes, neutrophils and the like is mediated, and the cells not only promote fibrin deposition, but also participate in inflammatory reactions and thrombosis.
ADP, also known as adenosine diphosphate or adenosine diphosphate, is a very important class of platelet-activating substances in the body, playing an important role in platelet function. Mainly because ADP is usually present in high-density particles in platelet cells, while atpase is present on platelets, ADP inhibits atpase activity and ADP can participate in inducing aggregation of platelets after being released by platelets, thereby promoting hemostasis.
TRAP6 is a peptide fragment (42-47 residues) of protease activated receptor 1 and is also a PAR1 hexapeptide agonist. TRAP6 can stimulate transient mobilization of free intracellular calcium, platelet aggregation (EC 50 = 0.8 μm) and rapidly phosphorylate PDE3A.
As certain embodiments of the invention, the buffer comprises: the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4;
agonists comprise 1.34mM ADP and 0.54mM TRAP6;
the calcium concentrate contained 100mM calcium ions.
As some embodiments of the invention, the detection system further comprises Pacific Blue anti-human CD41PerCP anti-human CD42b/>And antibodies against PAC-1 markers. As certain embodiments of the present invention, the antibody against PAC-1 marker is selected from FITC anti-human PAC-1Or FITC anti-human PAC-1->
As certain embodiments of the present invention, the detection system further comprises a detection device or detection reagent that detects the combination of the expressed PAC-1 marker and an antibody against the PAC-1 marker. As some embodiments of the invention, the detection device comprises a flow cytometer, and the detection reagent fluoresces to detect the reagent. As certain embodiments of the invention, the calcium ions are derived from calcium chloride, calcium gluconate.
The invention also provides a method for detecting PAC-1 markers on platelets by using the detection system, which comprises the following steps: (1) diluting a sample to be detected with a buffer solution; (2) Mixing the diluted sample in the step (1) with an agonist and an antibody against a PAC-1 marker, incubating, and quantitatively detecting; (3) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting PAC-1 marker, incubating and quantitatively detecting; and (4) calculating the ratio of the detection value of the step (3) to the detection value of the step (2).
As some embodiments of the present invention, the sample to be detected in the step (1) is diluted 10 to 40 times with a buffer. As some embodiments of the present invention, the sample to be detected in the step (1) is diluted 20-fold with a buffer.
As certain embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation at room temperature for 15-30 minutes. As some embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation for 20 minutes at room temperature.
As some embodiments of the invention, the diluted sample, agonist, antibody volume ratio in step (2) is 50:10:6.
as some embodiments of the invention, the diluted sample, agonist, antibody, calcium concentrate in step (3) has a volume ratio of 50:10:6:0.3-1. The invention also provides a detection kit for detecting the PAC-1 marker on the blood platelets, and the detection kit comprises the components.
The invention also provides an application of the detection system or the detection kit in preparing a detection reagent for detecting PAC-1 markers on platelets.
In another aspect, the present invention provides a detection system for detecting Annexin-V and CD62P markers on platelets, the detection system comprising the following components: buffers, calcium concentrates and agonists; the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45; the calcium concentrate comprises 50-100mM calcium ions; agonists comprise 1-2mM ADP and 0.1-1mM TRAP6.
As certain embodiments of the invention, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4; agonists comprise 1.34mM ADP and 0.54mM TRAP6; the calcium concentrate contained 100mM calcium ions.
As some embodiments of the invention, the detection system further comprises Pacific Blue anti-human CD41PerCP anti-human CD42b/>And antibodies to Annexin-V markers and antibodies to CD62P markers.
As certain embodiments of the invention, the antibody against an Annexin-V marker is selected from the group consisting of PE/Cy7-Annexin V
As certain embodiments of the invention, the antibody against the CD62P marker is selected from the group consisting of APC anti-human CD42b
As certain embodiments of the invention, the detection system further comprises a detection device or detection reagent that detects the combination of the expressed Annexin-V marker and the antibody against the Annexin-V marker, and a detection device or detection reagent that detects the combination of the expressed CD62P marker and the antibody against the CD62P marker.
As some embodiments of the invention, the detection device comprises a flow cytometer and the detection reagent comprises a fluorescent detection reagent. As certain embodiments of the invention, the calcium ions are derived from calcium chloride, calcium gluconate.
The invention also provides a method for detecting an Annexin-V marker and a CD62P marker on platelets by using the detection system, wherein the detection method comprises the following steps of: (1) diluting a sample to be detected with the buffer solution; (2) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting an Annexin-V marker, incubating, and quantitatively detecting; (3) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting the CD62P marker, incubating and quantitatively detecting.
As some embodiments of the present invention, the sample to be detected in the step (1) is diluted 10 to 40 times with a buffer. As some embodiments of the present invention, the sample to be detected in the step (1) is diluted 20-fold with a buffer. As certain embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation at room temperature for 15-30 minutes. As some embodiments of the present invention, the incubation conditions in steps (2) and (3) are incubation for 20 minutes at room temperature.
As some embodiments of the present invention, the diluted sample, agonist, antibody, calcium concentrate in the steps (2) and (3) has a volume ratio of 50:10:5-6:1. the invention also provides a method for simultaneously detecting the Annexin-V marker and the CD62P marker on the blood platelet, the Annexin-V marker and the CD62P marker are simultaneously detected by using the method, and the detection equipment is a flow cytometer.
The invention also provides a detection kit for detecting an Annexin-V marker and a CD62P marker on platelets, and the detection kit comprises the components.
The invention also provides an application of the detection system or the detection kit in preparing detection reagents for detecting an Annexin-V marker and a CD62P marker on blood platelets.
In another aspect of the present invention, there is provided a detection system for detecting PAC-1 markers, annexin-V markers and CD62P markers on platelets, the detection system comprising the following components: buffers, calcium concentrates and agonists; the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45; the calcium concentrate comprises 50-100mM calcium ions; agonists comprise 1-2mM ADP and 0.1-1mM TRAP6.
Preferably, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4. Agonists comprise 1.34mM ADP and 0.54mM TRAP6; the calcium concentrate contained 100mM calcium ions.
As some embodiments of the invention, the detection system further comprises Pacific Blue anti-human CD41PerCP anti-human CD42b/>And antibodies to PAC-1 marker, antibodies to Annexin-V marker and antibodies to CD62P marker.
As certain embodiments of the present invention, the antibody against PAC-1 marker is selected from FITC anti-human PAC-1Or FITC anti-human PAC-1/>
As certain embodiments of the invention, the antibody against an Annexin-V marker is selected from the group consisting of PE/Cy7-Annexin V
As certain embodiments of the invention, the antibody against the CD62P marker is selected from the group consisting of APC anti-human CD62P
As certain embodiments of the present invention, the detection system further comprises a detection device or detection reagent that detects the combination of the expressed PAC-1 marker and the antibody against PAC-1 marker, a detection device or detection reagent that detects the combination of the expressed Annexin-V marker and the antibody against Annexin-V marker, and a detection device or detection reagent that detects the combination of the expressed CD62P marker and the antibody against CD62P marker.
As some embodiments of the invention, the detection device comprises a flow cytometer and the detection reagent comprises a fluorescent detection reagent.
As certain embodiments of the invention, the calcium ions are derived from calcium chloride, calcium gluconate.
The invention also provides a method for detecting PAC-1 markers, annexin-V markers and CD62P markers on platelets by using the detection system, wherein the detection method comprises the following steps of: (1) diluting a sample to be detected with a buffer solution; (2) Mixing the diluted sample in the step (1) with an agonist and an antibody against a PAC-1 marker, incubating, and quantitatively detecting; (3) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting PAC-1 marker, incubating and quantitatively detecting; (4) Calculating the ratio of the detection value of the step (3) to the detection value of the step (2) to realize the detection of the PAC-1 marker; (5) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting an Annexin-V marker, incubating, and quantitatively detecting to realize detection of the Annexin-V marker; and (6) mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting the CD62P marker, incubating, quantitatively detecting and detecting the CD62P marker.
As some embodiments of the present invention, the sample to be detected in the step (1) is diluted 10 to 40 times with a buffer. As some embodiments of the present invention, the sample to be detected in the step (1) is diluted 20-fold with a buffer.
As some embodiments of the present invention, the incubation conditions in step (2), step (3), step (5) and step (6) are incubation at room temperature for 15-30 minutes. As some embodiments of the present invention, the incubation conditions in step (2), step (3), step (5) and step (6) are incubation for 20 minutes at room temperature.
As some embodiments of the invention, the diluted sample, agonist, antibody volume ratio in step (2) is 50:10:6.
as some embodiments of the invention, the diluted sample, agonist, antibody, calcium concentrate in steps (3), (5) and (6) has a volume ratio of 50:10:5-6:1.
the invention also provides a method for simultaneously detecting the PAC-1 marker, the Annexin-V marker and the CD62P marker on the blood platelet, the PAC-1 marker, the Annexin-V marker and the CD62P marker are simultaneously detected by using the method, and the detection equipment is selected from a flow cytometer.
The invention also provides a detection kit for detecting PAC-1 markers, annexin-V markers and CD62P markers on platelets, and the detection kit comprises the components. The invention also provides an application of the detection system or the detection kit in preparing detection reagents for detecting PAC-1 markers, annexin-V markers and CD62P markers on blood platelets.
Another aspect of the present invention is to provide a method for detecting PAC-1, annexin-V and CD62P markers on plateletsA buffer, the buffer comprising: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45. Agonists comprise 1-2mM ADP and 0.1-1mM TRAP6; the buffer contains no calcium ions or also contains 1.5mM calcium ions.
As certain embodiments of the invention, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4. Agonists comprise 1.34mM ADP and 0.54mM TRAP6; the buffer contains no calcium ions or also contains 1.5mM calcium ions.
As described above, the detection system, method and kit for detecting PAC-1 marker on platelets, the detection system, method and kit for detecting Annexin-V and CD62P markers on platelets, and the detection system, method and kit for detecting PAC-1 marker, annexin-V marker and CD62P marker on platelets of the present invention have the following
The beneficial effects are that:
a platelet function detection method with correlation and simulated physiological environment is provided to evaluate platelet in-vivo functions;
providing a buffer solution with electrolyte components and colloid components similar to plasma;
the provided buffer solution can detect three markers of PAC-1, annexin-V and CD 62P;
the method for simultaneously detecting the PAC-1 marker, the Annexin-V marker and the CD62P marker of the sample variety is provided, so that the detection of the three markers is realized rapidly and efficiently;
provides a basic buffer solution and a calcium concentrate, and can realize detection of three markers.
Drawings
FIG. 1 shows a prior art flow of platelet activation state detection work;
FIG. 2 shows a new flow of optimized platelet activation state detection established for the present invention. The innovation point has been highlighted in red;
FIG. 3 is a schematic diagram showing the platelet activation state detection scheme of the present invention, wherein the calcium ion concentration varies with the detection purpose; WB is whole blood, PLT is platelets;
FIG. 4 shows the average levels of PAC-1, annexin-V and CD62P after activation in HT buffer 1 and HT buffer 2, for calcium concentrations of the present invention ranging from 0mM to 1.5 mM;
FIG. 5 shows the average PAC-1 levels of the present invention after activation in HT buffer 1 (HT 1) and HT buffer 2 (HT 2);
FIG. 6 shows Annexin-V levels after activation in HT buffer 1 (HT 1) and HT buffer 2 (HT 2) according to the invention;
FIG. 7 shows CD62P levels after activation in HT buffer 1 (HT 1) and HT buffer 2 (HT 2) according to the present invention;
FIG. 8 shows normalized levels of PAC-1 and Annexin-V after activation in HT buffer 1 (HT 1) and HT buffer 2 (HT 2) according to the present invention.
Detailed Description
The invention is further illustrated below in connection with specific examples, which are to be understood as being illustrative of the invention and not limiting the scope of the invention. Other advantages and effects of the present invention will be readily apparent to those skilled in the art from the present disclosure. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
It is to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range.
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 invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, all experimental methods, detection methods, and preparation methods disclosed herein employ techniques conventional in the art and those conventional in the relevant arts.
Detailed Description
The flow of platelet activation function detection developed by the present invention is shown in FIG. 2. The blood collection was collected using a blood collection tube, which was approved by ethical authorities in hospitals. ACD-A anticoagulant was pre-filled into the blood collection tube, 15 ml of fresh venous blood was collected, and whole blood samples were diluted 20-fold using HEPES-Tyrode buffer developed by the present invention as buffer (formula see Table 1). The assay buffer developed according to the invention was then used to formulate an activation reaction system with HEPES-Tyrode buffer (HT 2, see Table 1 for the individual components) as assay buffer or as a major component of an assay buffer:
activation reaction system = diluted blood sample + activator + antibody + calcium concentrate
The volumes of diluted blood sample, activator, antibody and calcium concentrate are respectively: 50. Mu.L, 10. Mu.L, antibody volumes were added according to Table 2, 0-1. Mu.L. The activator adopts ADP and TRAP6 to be combined, calcium chloride is added into an activation reaction system to adjust the concentration of calcium ions between 0mM and 1.5mM for testing, the activation and antibody (primary antibody) incubation are carried out at the same time for 20 minutes at room temperature, and then HBS buffer is added to directly carry out quantitative analysis without fixation, and flow cytometry and analysis are used, as shown in figure 3.
TABLE 1 composition of optimized HEPES-Tyrode buffer (HT 2) of the invention
Component (A) Concentration (mM)
NaCl 137
KCl 4
MgCl 2 1
HEPES 5
NaHCO 3 10
Na 2 HPO 4 0.3
Human serum albumin 0.35％(w/v)
Glucose 5.5
pH 7.4
The antibodies tested and their concentrations are shown in Table 2.
Table 2 antibodies used in the assays
Antibody name Manufacturer(s) Volume (mu L) Working concentration (μg/mL)
Pacific Blue anti-human CD41 Biolegend 2 2.5
PerCP anti-human CD42b Biolegend 2 12
FITC anti-human PAC-1 Invitrogen 0.5 NA
FITC anti-human PAC-1 Biolegend 2 3
PE/Cy7-Annexin V Biolegend 1 0.2
APC anti-human CD62P Biolegend 2 6
The technology aims to solve the defects of the prior art so as to improve the platelet function detection quality, and mainly solves the problems in two aspects:
a platelet function detection method with correlation and simulated physiological environment is provided to evaluate platelet in-vivo functions;
a detection buffer is provided having an electrolyte component and a colloidal component similar to plasma.
According to the test design and the test result, the patent is hopeful to develop a platelet activating function detection kit.
TABLE 3 ingredients of platelet activation state detection kit
The kit included a buffer and a platelet agonist, and a calcium ion concentrate, as shown in table 3. After blood is collected through the blood collection tube, the blood sample is diluted 20-fold with buffer. The diluted blood samples were then added to different label buffers and incubated at room temperature for 20 minutes with platelet activators and corresponding label antibodies (not included in the kit) for flow cytometry detection and analysis.
Example 1
For comparison purposes, the conventional Modified HEPES-Tyrode buffer (referred to herein as HT1, whose composition and concentration are shown in Table 5) is used as a control, while the optimized buffer developed by the present invention, HEPES-Tyrode buffer, is referred to herein as HT2 (whose composition and concentration are shown in Table 1). Table 4 shows the detection systems of three existing markers PAC-1, annexin-V and CD 62P. The following detection test results respectively describe the three in detail:
TABLE 4 Current methods for detecting platelet activation markers [8,9, 11]
TABLE 5 Components of the Modified HEPES-Tyrode buffer (HT 1)
1. Determination of the detection method of the present invention
PAC-1
PAC-1 is used to detect alpha IIb beta III following conformational change following platelet activation for assessing platelet aggregation function. As shown in FIGS. 4 and 5, PAC-1 expression levels were affected by the level of calcium ions in the buffer, with the highest level of activated platelet PAC-1 in the 0mM calcium (the concentration of calcium ions in conventional Modified HT buffer) environment. PAC-1 expression ratios (%) were 54.6.+ -. 20.8%, 38.5.+ -. 17.1%, 35.5.+ -. 17.4% and 33.9.+ -. 17% at 0.5mM,1mM,1.5mM, respectively, calcium concentration 0mM,0.5mM,1mM, and 56.2.+ -. 22.5%, 39.1.+ -. 17.1%, 37.2.+ -. 18.3%, and 36.2.+ -. 17.1% in HT2, respectively, when HT1 was used.
Within HT1 and HT2, there was a significant difference between PAC-1 expression in the 0mM calcium group and the 1.5mM calcium group (p<0.05,) see fig. 8. However, considering that the environment in which the platelets are located is blood and 0mM calcium ions are in a non-physiological state, a ratio of 1.5mM calcium ions PAC-1% to 0mM calcium ions PAC-1%, i.e., PAC-1@1.5 Ca, is proposed 2+ ]/PAC-1@[0Ca 2+ ]To comprehensively evaluate PAC-1 expression in reserve and near physiological states, rather than reflecting only functional states in the case of single detection. This ratio is 0.62 at HT1 and 0.64 at HT2.
Annexin-V
Anexin-V was used to detect phosphotidyleriene after platelet activation. The expression of Annexin-V is correlated with calcium ion levels. As shown in FIGS. 4 and 6, the level of Annexin-V expression was affected by the level of calcium ions in the buffer, and the level of activated platelet Annexin-V was highest in the high-calcium environment. Using HT1, the expression ratios (%) of Annexin-V were 14.8.+ -. 1.3%, 19.6.+ -. 4%, 18.1.+ -. 2.1% and 21.3.+ -. 3.4% at 0mM,0.5mM,1mM,1.5mM, respectively, of calcium ion concentration of 0mM,0.5mM,1mM, 14.8.+ -. 1.3% and 20.6.+ -. 1.5% at calcium ion concentration of 0mM,0.5mM,1mM,1.5mM, respectively.
There was a significant difference between 0mM calcium group and 1.5mM calcium group Annexin-V expression in HT1 (p < 0.05), and between 0mM calcium group and 1.5mM calcium group Annexin-V expression in HT2 (p < 0.05), see FIG. 8. However, considering that the environment in which platelets are located is blood and 0mM calcium ion is in a non-physiological state, detection of Annexin-V needs to be performed at a concentration of 1.5mM calcium ion that is near physiological.
CD62P
CD62P is expressed on platelet granule vesicles, which fuse with the envelope to release the active substance in the case of platelet activation, so that the expression level of CD62P before and after platelet activation evaluates the platelet secretion function. As shown in fig. 4 and 7, the CD62P expression level was affected by the calcium ion level in the buffer, and the level of activated platelet CD62P was the lowest in the 0mM calcium environment. Using HT1, CD62P expression ratios (%) were 87.1.+ -. 4%, 89.6.+ -. 2.2%, 90.2.+ -. 0.2% and 89.9.+ -. 0.1% at calcium ion concentrations of 0mM,0.5mM,1mM,1.5mM, respectively, 88.7.+ -. 2%, 90.5.+ -. 1.1%, 90.9.+ -. 0.3% and 90.9.+ -. 0.3% at calcium ion concentrations of 0mM,0.5mM,1mM,1.5mM, respectively, in HT2.
However, considering that the environment in which platelets are located is blood and 0mM calcium ions are non-physiological, the detection of CD62P needs to be performed at a concentration of 1.5mM calcium ions that is near physiological.
2. Comparison of the detection effect of HT1, activation System and HT2, activation System of the invention
HT1 and HT2 were evaluated from the expression level status of PAC-1, annexin-V and CD 62P. As shown in FIGS. 4 and 5, the relationship between the expression trend of CD62P and PAC-1 and calcium ion is smaller than that of HT type, however, variation and oscillation of the expression trend are larger in HT1 for Annexin-V, and the trend is linear and gentle in HT2. In order to detect the three in the same system, HT2 buffer solution is selected as test buffer solution according to test results. The detection system for the three activation markers is shown in Table 6.
Table 6PAC-1, annexin-V and CD62P detection system
Markers Buffer solution Agonists Calcium ion concentration (mM)
PAC-1 HT2 ADP+TRAP 6 0，1.5
Annexin-V HT2 ADP+TRAP 6 1.5
CD62P HT2 ADP+TRAP 6 1.5
By combining the results, the method of the invention can detect PAC-1, annexin-V and CD62P expression respectively by improving the buffer composition to be closer to the physiological state and setting different calcium ion concentrations in the activation reaction system compared with the traditional Modified HT buffer (HT 1) and the activation system, so as to conveniently evaluate platelet aggregation, coagulation promoting and secretion functions.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
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Claims (27)

1. A detection system for detecting PAC-1 markers on platelets, the detection system comprising the following components: buffers, calcium concentrates and agonists;
the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45;
agonists comprise 1-2mM ADP and 0.1-1mM TRAP6;
the calcium concentrate comprises 50-100mM calcium ions;
preferably, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4.
Agonists comprise 1.34mM ADP and 0.54mM TRAP6;
the calcium concentrate contained 100mM calcium ions.
2. The detection system of claim 1, wherein theThe detection system also comprises Pacific Blue anti-human CD41PerCP anti-human CD42b/>Antibody FITC anti-human PAC-1 +.>Or FITC anti-human PAC-1->Preferably, the antibody against PAC-1 marker is selected from FITC anti-human PAC-1 +.>
3. The detection system according to any one of claims 1 to 2, further comprising a detection device or detection reagent for detecting a combination of the expressed PAC-1 marker and an antibody against the PAC-1 marker; preferably, the detection device comprises a flow cytometer and the detection reagent comprises a fluorescent detection reagent.
4. A detection system according to any one of claims 1 to 3, wherein the calcium ions are derived from calcium chloride or calcium gluconate.
5. A method of detecting PAC-1 marker on platelets using the detection system of any one of claims 1-4, the detection method comprising the steps of:
(1) The sample to be tested is diluted with a buffer,
(2) Mixing the diluted sample obtained in the step (1), an agonist and an antibody for resisting the PAC-1 marker, incubating and quantitatively detecting;
(3) Mixing the diluted sample obtained in the step (1) with an agonist, a calcium concentrate and an antibody for resisting the PAC-1 marker, incubating and quantitatively detecting;
(4) Calculating the ratio of the detection value of the step (3) to the detection value of the step (2).
6. The method according to claim 5, wherein the sample to be detected in step (1) is diluted 10 to 40 times with a buffer; preferably, the sample to be detected in step (1) is diluted 20-fold with a buffer;
the incubation conditions in the steps (2) and (3) are that the incubation is carried out for 15-30 minutes at room temperature; preferably, the incubation conditions in steps (2) and (3) are incubation for 20 minutes at room temperature.
7. A test kit for detecting PAC-1 markers on platelets, characterized in that the test kit comprises the components of any one of claims 1 to 4.
8. Use of the detection system according to any one of claims 1 to 4, or the detection kit according to claim 7, for the preparation of a detection reagent for detecting PAC-1 markers on platelets.
9. A detection system for detecting Annexin-V and CD62P markers on platelets, the detection system comprising the following components: buffers, calcium concentrates and agonists;
the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45;
agonists comprise 1-2mM ADP and 0.1-1mM TRAP6;
the calcium concentrate comprises 50-100mM calcium ions;
preferably, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4;
agonists comprise 1.34mM ADP and 0.54mM TRAP6; the calcium concentrate contained 100mM calcium ions.
10. The detection system according to claim 9, further comprising Pacific Blue anti-human CD41PerCP anti-human CD42b/>And antibodies to an Annexin-V marker and antibodies to a CD62P marker;
preferably, the antibody against an Annexin-V marker is selected from the group consisting of PE/Cy7-Annexin V
Preferably, the antibody against the CD62P marker is selected from the group consisting of APC anti-human CD62P
11. The detection system according to any one of claims 9 to 10, further comprising a detection device or a detection reagent for detecting a combination of the expressed Annexin-V marker and an antibody against the Annexin-V marker, and
a detection device or detection reagent for detecting the combination of the expressed CD62P marker and an antibody against the CD62P marker;
preferably, the detection device comprises a flow cytometer and the detection reagent comprises a fluorescent detection reagent.
12. The detection system according to any one of claims 9 to 11, wherein the calcium ions are derived from calcium chloride or calcium gluconate.
13. A method of detecting Annexin-V markers and CD62P markers on platelets using the detection system of any one of claims 9-12, the detection method comprising the steps of:
(1) Diluting a sample to be detected with the buffer solution,
(2) Mixing the diluted sample obtained in the step (1) with an agonist, a calcium concentrate and an antibody for resisting an Annexin-V marker, incubating, and quantitatively detecting;
(3) Mixing the diluted sample obtained in the step (1) with an agonist, a calcium concentrate and an antibody for resisting the CD62P marker, incubating and quantitatively detecting.
14. The method according to claim 13, wherein the sample to be tested in step (1) is diluted 10-40 times with a buffer; preferably, the sample to be detected in step (1) is diluted 20-fold with a buffer;
the incubation conditions in the steps (2) and (3) are that the incubation is carried out for 15-30 minutes at room temperature; preferably, the incubation conditions in steps (2) and (3) are incubation for 20 minutes at room temperature.
15. A method for simultaneous detection of an Annexin-V marker and a CD62P marker on platelets, characterized in that the Annexin-V marker and the CD62P marker are detected simultaneously using the method according to any one of claims 13-14, and the detection device is selected from the group consisting of flow cytometry.
16. A test kit for detecting an Annexin-V marker and a CD62P marker on platelets, the test kit comprising the components of any one of claims 9-12.
17. Use of a detection system according to any one of claims 9 to 12, or a detection kit according to claim 16, for the preparation of a detection reagent for detecting an Annexin-V marker and a CD62P marker on platelets.
18. A detection system for detecting PAC-1, annexin-V and CD62P markers on platelets, the detection system comprising the following components: buffers, calcium concentrates and agonists;
the buffer comprises: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45;
agonists comprise 1-2mM ADP and 0.1-1mM TRAP6;
the calcium concentrate comprises 50-100mM calcium ions;
preferably, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4; agonists comprise 1.34mM ADP and 0.54mM TRAP6; the calcium concentrate contained 100mM calcium ions.
19. The detection system of claim 18, further comprising Pacific Blue anti-human CD41PerCP anti-human CD42b/>And antibodies to PAC-1 marker, antibodies to Annexin-V marker and antibodies to CD62P marker;
preferably, the antibody against PAC-1 marker is selected from FITC anti-human PAC-1
Preferably, the antigenAntibodies to Annexin-V markers are selected from PE/Cy7-Annexin V
Preferably, the antibody against the CD62P marker is selected from the group consisting of APC anti-human CD62P
20. The detection system according to any one of claims 18 to 19, wherein the detection system further comprises a detection device or a detection reagent for detecting a combination of the expressed PAC-1 marker and an antibody against the PAC-1 marker,
a detection device or a detection reagent for detecting a conjugate of the expressed Annexin-V marker and an antibody against the Annexin-V marker, and
a detection device or detection reagent for detecting the combination of the expressed CD62P marker and an antibody against the CD62P marker;
preferably, the detection device comprises a flow cytometer and the detection reagent comprises a fluorescent detection reagent.
21. The detection system according to any one of claims 18 to 20, wherein the calcium ions are derived from calcium chloride or calcium gluconate.
22. A method of detecting PAC-1, annexin-V, and CD62P markers on platelets using the detection system of any one of claims 18-21, the detection method comprising the steps of:
(1) Diluting a sample to be detected by using a buffer solution;
(2) Mixing the diluted sample agonist and the anti-PAC-1 marker antibody in the step (1), incubating and quantitatively detecting;
(3) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting PAC-1 marker, incubating and quantitatively detecting;
(4) Calculating the ratio of the detection value of the step (3) to the detection value of the step (2) to realize the detection of the PAC-1 marker;
(5) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting an Annexin-V marker, incubating, and quantitatively detecting to realize detection of the Annexin-V marker;
(6) Mixing the diluted sample obtained in the step (1) with calcium concentrate, an agonist and an antibody for resisting the CD62P marker, incubating, and quantitatively detecting to realize detection of the CD62P marker.
23. The method according to claim 22, wherein the sample to be tested in step (1) is diluted 10-40 times with a buffer; preferably, the sample to be detected in step (1) is diluted 20-fold with a buffer;
the incubation conditions in the step (2), the step (3), the step (5) and the step (6) are that incubation is carried out for 15-30 minutes at room temperature; preferably, the incubation conditions in step (2), step (3), step (5) and step (6) are incubation for 20 minutes at room temperature.
24. A method for simultaneous detection of PAC-1, annexin-V and CD62P markers on platelets, characterized in that PAC-1, annexin-V and CD62P markers are detected simultaneously using the method of any one of claims 22-23, the detection device being selected from a flow cytometer.
25. A test kit for detecting PAC-1, annexin-V and CD62P markers on platelets, the test kit comprising the components of any one of claims 18-21.
26. Use of a detection system according to any one of claims 18 to 21, or a detection kit according to claim 25, in the manufacture of a detection reagent for detecting PAC-1, annexin-V and CD62P markers on platelets.
27. A buffer for detecting PAC-1, annexin-V, and CD62P markers on platelets, the buffer comprising: 130-140mM NaCl, 2.5-4mM KCl, 0.5-2mM MgCl 2 、5-12mM HEPES、5-12mM NaHCO 3 、0.1-0.5mM Na 2 HPO 4 0.1% -0.4% w/v human serum albumin, 2-8mM glucose, pH 7.35-7.45;
agonists comprise 1-2mM ADP and 0.1-1mM TRAP6;
the buffer contains no calcium ions or also contains 1.5mM calcium ions;
preferably, the buffer comprises: 137mM NaCl, 4mM KCl and 1mM MgCl 2 、5mM HEPES、10mM NaHCO 3 、0.3mM Na 2 HPO 4 0.35% w/v human serum albumin, 5.5mM glucose, pH 7.4; agonists comprise 1.34mM ADP and 0.54mM TRAP6; the buffer contains no calcium ions or also contains 1.5mM calcium ions.
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