Platelet marker detection reagent and detection method

Abstract

The invention relates to the field of biological detection technology, and specifically to a platelet marker detection reagent and a detection method; the detection reagent is used to solve the shortcomings of the prior art and achieve more accurate detection results; the invention provides a marker detection reagent, comprising a fluorescently labeled antibody and a phosphate buffer; a method for detecting platelet markers using the detection reagent comprises the following steps: 1) adding anticoagulant blood to the bottom of a flow tube; 2) adding a fluorescently labeled antibody reagent to the flow tube; 3) gently vortexing and mixing and incubating at room temperature in the dark for a period of time; 4) adding or not adding a buffer solution containing paraformaldehyde, fixing and standing in the dark; 5) centrifuging, transferring the supernatant to a new test tube, and detecting on a flow cytometer; the invention adopts a method of centrifugation after staining to remove red blood cells. Compared with the traditional lysis method, the invention can remove red blood cells without affecting platelets and their activation state, and can obtain more accurate detection results.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a platelet marker detection reagent and a detection method. Background Art

[0002] Platelet-related antibody detection usually uses flow cytometry to detect the positive rate of platelet surface membrane glycoproteins and the positive rate of platelet activation markers. The reduction or absence of the expression of membrane glycoproteins CD41 / CD61 / 42a / CD42b indicates congenital or acquired platelet function defects, which will increase the risk of bleeding. The measurement value of CD62P can reflect the degree of platelet activation. Activation is the initial cause of thrombosis. The higher the measurement value, the higher the degree of activation, and the higher the risk of thrombosis. The measurement value of platelet-leukocyte aggregates can evaluate the cellular immune status of infected patients. The higher the measurement value, the progress of bacterial or viral infection; combined with the measurement value of CD62P, the risk of thrombosis in infected patients can be jointly evaluated. The higher the measurement value, the higher the risk. In the process of using anti-platelet drugs, it is necessary to regularly monitor indicators such as platelet activation and evaluate the risk of ischemia or bleeding, so as to help clinical practice achieve individualized medication.

[0003] Flow cytometry is a new technology developed in recent years. It can perform multi-parameter high-throughput detection on a sample with fast detection speed, high precision and good accuracy. Micro-sample multi-index flow protein quantification technology is a multiple protein quantification detection method based on the flow cytometric detection system. It can detect multiple indicators in a single sample at the same time. It is an advanced version of flow cytometry. It uses tiny, dispersed particles to capture liquid analytes, and uses flow cytometers to detect the fluorescence emitted by the "sandwich"-like particles-"capture microspheres-analyte-detection antibody sandwich complex" to determine the number of analytes. This method requires less samples and is faster and more accurate.

[0004] Since the number of red blood cells in general blood is dozens of times more than that of platelets, if red blood cells are not removed, the detection of platelets will be affected when the flow cytometer is used for detection. The current method basically uses the traditional red blood cell lysis method to lyse red blood cells, and the lysis process will affect the activation state of platelets. Summary of the invention

[0005] The purpose of the present invention is to provide a detection reagent and a detection method for detecting platelet markers without affecting the activation state of platelets, so as to solve the shortcomings of the prior art and achieve more accurate detection results.

[0006] The technical solution of the present invention is as follows:

[0007] A platelet marker detection reagent comprises a fluorescent labeled antibody and a phosphate buffer.

[0008] The fluorescently labeled antibody described in the present invention is an antibody against CD41, CD42a, CD42b, CD61, CD62p, CD63, or other platelet markers. That is, the detection reagent of the present invention can be used to detect CD41, CD42a, CD42b, CD61, CD62p, CD63, or other platelet markers according to the different types of fluorescently labeled antibodies contained.

[0009] The method for detecting platelet markers using the platelet marker detection reagent of the present invention comprises the following steps:

[0010] 1) Add blood sample to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0011] 2) Adding fluorescent labeled antibody reagent to the flow tube;

[0012] 3) Gently vortex to mix and incubate at room temperature in the dark;

[0013] 4) Keep it in a fixed place away from light;

[0014] 5) After standing, centrifuge to remove red blood cells, transfer the supernatant to a new test tube, and detect on a flow cytometer.

[0015] A further optimized scheme, the method for detecting platelet markers of the present invention comprises the following steps:

[0016] 1) Add 5 μL of blood sample to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0017] 2) Add 5 μL of fluorescently labeled antibody reagent to the flow tube;

[0018] 3) Gently vortex to mix and incubate at room temperature in the dark for 10-40 minutes;

[0019] 4) Add 0.5-0.8 mL of PBS buffer containing paraformaldehyde and place in a dark place for 10-40 min;

[0020] 5) Centrifuge at 10-1000 g for 2-20 minutes, transfer the supernatant to a new test tube, and analyze by flow cytometry.

[0021] It is further required that the cell concentration of the blood sample used is 100×10 8 / L-280×10 9 / L.

[0022] Preferably, the blood sample used contains citrate anticoagulant.

[0023] It is further required that the supernatant is thoroughly mixed by low-speed vortexing before running the flow cytometer assay.

[0024] For further requirements, flow cytometric analysis was performed by first creating an FSC / SSC scatter plot and circling platelets, then selecting platelets, creating a marker / SSC scatter plot, and circling marker-positive platelets.

[0025] Further, the fluorescently labeled antibody reagent in the present invention uses a fluorescently labeled antibody reagent with allophycocyanin as a carrier. Specifically, it can be obtained by coupling purified platelet marker monoclonal antibody with purified allophycocyanin using aminoPEG4 azide. Specifically, the purified platelet marker monoclonal antibody, purified allophycocyanin, and aminoPEG4 azide are evenly mixed in a molar ratio of 1:4:25, and ultrasonically treated three times at room temperature at 50-100W, 30S in sequence, and then allowed to stand for 10min, 30S, and allowed to stand for 10min, 20s, and continued to stand for 20-30min to obtain the platelet marker-APC fluorescently labeled antibody.

[0026] The working principle and beneficial effects of the present invention are as follows: the present invention uses fluorescent antibody staining and then removes red blood cells by centrifugation. Compared with the traditional lysis method, it can remove red blood cells without affecting platelets and their activation state, and can obtain more accurate detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0028] Figure 1 This is a FSC / SSC scatter plot. The circled cells in the figure are platelets in the blood;

[0029] Figure 2 This is a CD41 / SSC scatter plot, and the circled ones are CD41+ platelets in the blood. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] First, the present invention provides a platelet marker detection reagent, which includes a fluorescently labeled antibody against platelet markers such as CD41 and a phosphate buffer.

[0032] The detection reagent of the present invention is used to detect the expression of platelet markers such as platelet CD41, CD42a, CD42b, CD61, CD62p, CD63, etc. in human biological specimens. The following materials need to be prepared during the detection process:

[0033] 1. PBS buffer containing paraformaldehyde;

[0034] 2. Pipettes and disposable tips suitable for the measuring range;

[0035] 3. 12×75mm flow tube;

[0036] 4. Vortex mixer;

[0037] 5. Flow cytometer;

[0038] 6. Centrifuge.

[0039] It is preferred to use blood samples containing citrate anticoagulant, a platelet stabilizer. Blood samples should be stained within 24 hours of collection. The cell concentration is between 100×10 8 / L-280×10 9 / L is the best staining concentration. The sample should be analyzed immediately after staining

[0040] Taking the detection of CD41 as an example, the specific detection method is:

[0041] 1) Add 5 μL of anticoagulated blood sample and 20 μL of PBS to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0042] 2) Add 5 μL of anti-CD41 fluorescently labeled antibody reagent to the flow tube;

[0043] 3) Gently vortex to mix and incubate at room temperature in the dark for 30 minutes;

[0044] 4) Add 0.5 mL of PBS buffer containing 1% paraformaldehyde and keep it in a dark place for 30 min;

[0045] 5) Centrifuge at 150 g for 5 minutes, transfer the supernatant to a new tube, and analyze by flow cytometry.

[0046] Flow cytometric analysis:

[0047] First, create a FSC / SSC scatter plot (such as Figure 1 As shown), circle the platelets as Figure 1 P1 in.

[0048] Select platelets and create a CD41 / SSC scatter plot (such as Figure 2As shown), circle the CD41-positive platelets and analyze the percentage of CD41+ cells in platelets, with a reference interval of ≥70%.

[0049] Taking the detection of CD42a as an example, the specific detection method is:

[0050] 1) Add 5 μL of anticoagulated blood sample and 20 μL of PBS to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0051] 2) Add 5 μL of anti-CD42a fluorescently labeled antibody reagent to the flow tube;

[0052] 3) Gently vortex to mix and incubate at room temperature in the dark for 20 minutes;

[0053] 4) Keep it away from light and keep it still for 30 minutes;

[0054] 5) Centrifuge at 300 g for 10 minutes, transfer the supernatant to a new tube, and analyze by flow cytometry.

[0055] Taking the detection of CD42b as an example, the specific detection method is:

[0056] 1) Add 5 μL of anticoagulated blood sample and 20 μL of PBS to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0057] 2) Add 5 μL of anti-CD42b fluorescently labeled antibody reagent to the flow tube;

[0058] 3) Gently vortex to mix and incubate at room temperature in the dark for 15 minutes;

[0059] 4) Add 0.5 mL of PBS buffer containing 1% paraformaldehyde and keep it in a dark place for 35 min;

[0060] 5) Centrifuge at 200g for 8 minutes, transfer the supernatant to a new tube, and analyze by flow cytometry.

[0061] Taking the detection of CD61 as an example, the specific detection method is:

[0062] 1) Add 5 μL of anticoagulated blood sample and 20 μL of PBS to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0063] 2) Add 5 μL of anti-CD61 fluorescently labeled antibody reagent to the flow tube;

[0064] 3) Gently vortex to mix and incubate at room temperature in the dark for 40 minutes;

[0065] 4) Add 0.5 mL of PBS buffer containing 1.5% paraformaldehyde and keep it in a dark place for 10 min;

[0066] 5) Centrifuge at 1000 g for 2 minutes, transfer the supernatant to a new tube, and analyze by flow cytometry.

[0067] Taking the detection of CD62p as an example, the specific detection method is:

[0068] 1) Add 5 μL of anticoagulated blood sample and 20 μL of PBS to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0069] 2) Add 5 μL of anti-CD62p fluorescently labeled antibody reagent to the flow tube;

[0070] 3) Gently vortex to mix and incubate at room temperature in the dark for 10 minutes;

[0071] 4) Add 0.5 mL of PBS buffer containing 0.5% paraformaldehyde and keep it in a dark place for 40 min;

[0072] 5) Centrifuge at 10g for 20 minutes, transfer the supernatant to a new tube, and analyze by flow cytometry.

[0073] Taking the detection of CD63 as an example, the specific detection method is:

[0074] 1) Add 5 μL of anticoagulated blood sample and 20 μL of PBS to the bottom of the flow tube to prevent the blood sample from touching the tube wall;

[0075] 2) Add 5 μL of anti-CD63-APC fluorescently labeled antibody reagent to the flow tube;

[0076] 3) Gently vortex to mix and incubate at room temperature in the dark for 30 minutes;

[0077] 4) Add 0.5 mL of PBS buffer containing 1% paraformaldehyde and keep it in a dark place for 30 min;

[0078] 5) Centrifuge at 500 g for 3 minutes, transfer the supernatant to a new tube, and analyze by flow cytometry.

[0079] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. The detection method can be adjusted based on the data of a wide range of sample tests according to the different types of labeled antibodies used and the differences in other test materials. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A platelet marker detection reagent, characterized in that: Includes fluorescently labeled antibodies and phosphate-buffered saline.

2. The detection reagent according to claim 1, characterized in that: The fluorescently labeled antibody is an antibody against CD41, CD42a, CD42b, CD61, CD62p, CD63, or other platelet markers.

3. The method for detecting platelet markers using a detection reagent according to claim 1 or 2, characterized in that: The following steps are involved: 1) Add blood sample to the bottom of the flow tube to prevent the blood sample from touching the tube wall; 2) Adding fluorescent labeled antibody reagent to the flow tube; 3) Gently vortex to mix and incubate at room temperature in the dark; 4) Keep it in a fixed place away from light; 5) After standing, centrifuge to remove red blood cells, transfer the supernatant to a new test tube, and detect on a flow cytometer.

4. The method according to claim 3, characterized in that: The following steps are involved: 1) Add 5 μL of blood sample to the bottom of the flow tube to prevent the blood sample from touching the tube wall; 2) Add 5 μL of fluorescently labeled antibody reagent to the flow tube; 3) Gently vortex to mix and incubate at room temperature in the dark for 10-40 minutes; 4) Add 0.5-0.8 mL of PBS buffer containing paraformaldehyde and place in a dark place for 10-40 min; 5) Centrifuge at 10-1000g for 2-20 minutes, transfer the supernatant to a new test tube, and analyze by flow cytometry.

5. The method according to claim 3, characterized in that: The method according to claim 2 is characterized in that the cell concentration of the blood sample used is 100×10 8 / L-280×10 9 / L.

6. The method according to claim 3, characterized in that The blood samples used contained citrate anticoagulant.

7. The method according to claim 3, characterized in that Before running the flow cytometer, the supernatant was thoroughly mixed by vortexing at low speed.

8. The method according to claim 3, characterized in that For flow cytometric analysis, first create an FSC / SSC dot plot and circle the platelets, then select the platelets, create a marker / SSC dot plot, and circle the marker-positive platelets.

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