Antibody composition for detecting platelet CD36 deletion type based on flow cytometry and application

Abstract

The invention discloses an antibody composition for detecting platelet CD36 deletion based on flow cytometry and application, and relates to the technical field of blood detection. The antibody composition for detecting the platelet CD36 deletion type comprises a CD61 antibody, a CD14 antibody and a CD36 antibody. The antibody composition provided by the invention can detect the platelet CD36 deletion condition at one time and analyze the deletion type, has the characteristics of simplifying the operation process, improving the detection efficiency and reducing the detection cost, and makes it possible for people to detect the CD36 deletion type in a large sample size.

Description

Technical Field

[0001] This invention belongs to the field of blood testing technology, specifically relating to an antibody composition and its application for detecting CD36-deficient platelets based on flow cytometry. Background Technology

[0002] The CD36 antigen is expressed in platelets, macrophages, capillary endothelial cells, erythroid plasmablasts, and adipocytes. Studies have found that individuals with CD36-deficient blood types can develop CD36 antibodies through blood transfusions, transplants, and pregnancy, leading to adverse transfusion reactions such as platelet transfusion failure (PTR), clinical transfusion-associated acute lung injury (TRALI), and fetal / neonatal alloimmune thrombocytopenic purpura (FNAIT). Diagnosing whether a patient has CD36 antigen deficiency and providing appropriate transfusions can minimize the transfusion risks for platelet transfusion patients.

[0003] CD36-deficient blood types are classified into two categories: type I and type II. Current methods for CD36 testing require two separate tests, one on platelets and the other on monocytes. This process is time-consuming and complex, limiting the number of individuals screened for CD36-deficient blood types. Some recipients with this condition go undetected and receive platelets from CD36-positive donors, developing anti-CD36 antibodies and resulting in ineffective platelet transfusions. For example, the platelet CD36 antigen test strip in patent CN116908434A offers advantages such as short testing time, low cost, and simple operation, but it cannot distinguish between platelet CD36-deficient individuals.

[0004] Patent CN116536401A proposes a method and kit for genotyping platelet and neutrophil antigens and glycoproteins based on mass spectrometry. It utilizes mass spectrometry chip processing and achieves genotyping of platelet-specific antigens, platelet CD36 glycoprotein, and neutrophil antigens by designing primer combinations and improving amplification reaction conditions. However, it relies on mass spectrometry technology, resulting in high detection costs, long processing time, and low detection efficiency, making it unable to achieve large-sample testing.

[0005] Flow cytometry (FCM) is a detection method capable of quantitative analysis of single cells. It boasts advantages such as speed, high precision, and multi-parameter capabilities, making it one of the most advanced cell quantitative analysis methods currently available. FCM plays a crucial role in both clinical and research fields, particularly in clinical in vitro diagnostics, where it has become one of the most mainstream detection methods. Currently, FCM detection results are considered the gold standard for the clinical diagnosis of malignant diseases such as leukemia and lymphoma. However, no studies have been reported on the use of flow cytometry to detect platelet CD36 deficiency. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects and shortcomings of existing platelet CD36 detection technologies and to provide an antibody composition based on flow cytometry for detecting platelet CD36 deficiency, which can detect platelet CD36 deficiency and analyze the deficiency type in one step.

[0007] Another object of the present invention is to provide a kit for detecting platelet CD36 deletion.

[0008] Another object of the present invention is to provide an application of an antibody composition in the preparation of a product for detecting CD36-deficient platelets.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects an antibody composition for detecting CD36-deficient platelets using flow cytometry, the antibody composition comprising CD61 antibody, CD14 antibody and CD36 antibody.

[0010] In some embodiments, the volume ratio of the CD61 antibody, CD14 antibody and CD36 antibody is 1:(0.9-1.1):(1.8-2.2), preferably 1:1:2.

[0011] In some embodiments, the CD61 antibody, CD14 antibody, and CD36 antibody are all monoclonal antibodies.

[0012] In some embodiments, the CD61 antibody, CD14 antibody, and CD36 antibody are each labeled with a different fluorophore, the fluorophore being selected from at least one of APC, PE, FITC, PERCP, PERCP-CY5.5, APC-CY7, PE-CY7, MF540, MF450, V500, and PE-CF594.

[0013] Optionally, the CD61 antibody is labeled with APC-CY7 fluorescein; the CD14 antibody is labeled with PE fluorescein; and the CD36 antibody is labeled with FITC fluorescein.

[0014] This invention protects a kit for detecting CD36-deficient platelets, comprising the antibody composition described above.

[0015] In some implementations, hemolysin and phosphate buffer are also included.

[0016] Optionally, the phosphate buffer solution has a concentration of 0.1 M and a pH of 7.0-7.6.

[0017] This invention protects the use of an antibody composition in the preparation of a product for detecting CD36-deficient platelets.

[0018] In some embodiments, the method for detecting platelet CD36 deletion includes flow cytometry.

[0019] In some implementations, the process of detecting platelet CD36 deficiency includes the following steps: S1. Take the sample to be tested and mix it evenly with the antibody composition, and react it at 2-35℃ for 10-40 min; preferably, react at room temperature for more than 10 min, or react at 2-8℃ for not less than 30 min; S2, add hemolysin to the sample solution obtained in step S1, wherein the volume ratio of the sample solution to the hemolysin is (8-12):1, and react at 2-35℃ for 5-20 min; preferably, react at room temperature for not less than 5 min, or react at 2-8℃ for not less than 10 min; S3. After centrifuging the solution obtained in step S2, remove the supernatant, add phosphate buffer to resuspend the cells, perform flow cytometry on the resuspended cells, and select CD61 antibody, CD14 antibody monocytes and CD36 antibody to respectively combine SSC gating to obtain platelet CD36-deficient type.

[0020] In some embodiments, the sample to be tested is at least one of peripheral blood, capillary blood, or heel blood.

[0021] In some embodiments, CD61 antibody positive expression is platelets, CD14 antibody positive expression is monocytes, and when both the identified platelets and monocytes show CD36 negative expression, it is determined to be CD36 type I deletion.

[0022] Optionally, CD61 antibody positive expression is platelets, CD14 antibody positive expression is monocytes, and when the identified platelets are CD36 negative and the monocytes are all CD36 positive, it is judged to be CD36 type II deletion.

[0023] Optionally, CD61 antibody positive expression is platelets, CD14 antibody positive expression is monocytes, and when both the identified platelets and monocytes show CD36 positive expression, it is judged as normal CD36 expression.

[0024] Compared with the prior art, the beneficial effects of the present invention are: This invention provides an antibody composition for detecting CD36 deficiency in platelets based on flow cytometry. It can detect CD36 deficiency in platelets in a single test and analyze the deficiency type. It also features simplified operation, improved detection efficiency and reduced detection cost, making it possible to detect CD36 deficiency types in large population samples. Attached Figure Description

[0025] Figure 1 This is a gating diagram of flow cytometry analysis according to Embodiment 1 of the present invention.

[0026] Figure 2 This is a gating diagram of flow cytometry analysis according to Embodiment 2 of the present invention.

[0027] Figure 3 This is a gating diagram of flow cytometry analysis according to Embodiment 3 of the present invention. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0029] Example 1 A method for detecting CD36-deficient platelets based on flow cytometry: 1. Preparation of antibody composition: The antibody composition includes CD61 antibody, CD14 antibody and CD36 antibody in a volume ratio of 1:1:2. All of them are monoclonal antibodies obtained by screening mouse single B cells after immunizing mice with recombinant human CD61 / CD14 / CD36.

[0030] The CD61 antibody was labeled with APC-CY7 fluorescent dye and denoted as CD61-APC-CY7.

[0031] The CD14 antibody is labeled with PE fluorescent dye and is denoted as CD14-PE.

[0032] The CD36 antibody is labeled with FITC fluorescent pigment and is denoted as CD36-FITC.

[0033] 2. Sample processing method, including the following steps: 1) Take 200uL of EDTA-anticoagulated peripheral blood as the test sample, and control the cell count to 2×10⁻⁶ cells / mL. 6 indivual; 2) Mix the sample to be tested with 20 μL of the antibody composition and react at room temperature (25°C) for 10 min.

[0034] 3) After the reaction is complete, add hemolysin, the volume ratio of hemolysin to the mixture is 1:10, mix thoroughly and react at room temperature for 5 minutes; 4) Centrifuge for 5 minutes (centrifugal force 500g), remove the supernatant, add 600uL of phosphate buffer (0.1M, pH=7.3) to resuspend, mix thoroughly and it is ready for instrument detection.

[0035] 3. Specimen testing: The processed samples were analyzed in a flow cytometer equipped with a 488µm laser reflector and capable of detecting FITC / PE / APC-CY7 fluorescence signals. Gating was performed as follows, and the results are as follows: Figure 1 As shown: (1) Fixed gating: The cell size and content richness of the sample were analyzed by FSC / SSC to distinguish cells and platelets, but included some cell debris and aggregates; white blood cell (WBC) position signal points and platelet (plat) position signal points were obtained, totaling 97.36%, of which platelets accounted for 72.49%; (2) Multiple sign combination gates: CD61 / SSC-A and CD14 / SSC-A are used to set up gates respectively, and then CD36 / SSC-A is used to set up gates.

[0036] Specifically, CD61 / SSC-A gating: We identified platelets by CD61-APC-CY7 positivity, distinguishing them from other cell debris; CD61 identified 98.52% of the platelets as positive. CD14 / SSC-A gate: monocytes (mon) are identified by positive expression of CD14-PE; these monocytes account for 0.77% of the total. CD36 / SSC-A gating: The fluorescence intensity of CD36-FITC is used to determine whether platelets (plat) and monocytes (mon) are CD36 positive; The experiment showed that in this example, both platelets and monocytes were CD36 negative, and the positive expression of CD61, CD14 and CD36 in the analysis area accounted for only 0.50%. The detection results are shown in Table 1 below. The diagnostic sample was CD36 type I deletion.

[0037] Example 2 A method for detecting CD36-deficient platelets based on flow cytometry includes the following steps: 1. The antibody composition and sample processing method are the same as in Example 1; 2. Specimen testing: The processed samples were analyzed by flow cytometry, and gating was performed as follows. The results are as follows: Figure 2 As shown: (1) Fixed gating: The cell size and contents of the sample were analyzed by FSC / SSC to distinguish cells and platelets, but included some cell debris and aggregates; a total of 96.85% of the white blood cell (WBC) and platelet (plat) position signal points were obtained, with platelets accounting for 70.98%; (2) Multiple sign combination gates: CD61 / SSC-A and CD14 / SSC-A are used to set up gates respectively, and then CD36 / SSC-A is used to set up gates.

[0038] Specifically, CD61 / SSC-A gating: We identified platelets by CD61-APC-CY7 positivity, distinguishing them from other cell debris; CD61 identified 97.84% of the platelets as positive. CD14 / SSC-A gate: Monocytes are identified by positive expression of CD14-PE; these monocytes account for 1.11% of the total. CD36 / SSC-A gating: Determine whether platelets (plat) and monocytes (mon) are CD36 positive by the fluorescence intensity of CD36; The experiment showed that in this embodiment, all monocytes showed positive expression of CD36, while platelets showed negative expression of CD36. In addition, the proportion of positive expression of CD61, CD14 and CD36 in the analysis area was only 1.63%. The detection results are shown in Table 1 below. The diagnostic sample was CD36 type II deletion.

[0039] Example 3 A method for detecting CD36-deficient platelets based on flow cytometry, such as Figure 3 As shown, it includes the following steps: 1. The antibody composition and sample processing method are the same as in Example 1; 3. Specimen testing: The processed samples were analyzed by flow cytometry, and gating was performed as follows. The results are as follows: Figure 3 As shown: (1) Fixed gating: The cell size and content richness of the sample were analyzed by FSC / SSC to distinguish cells and platelets, but included some cell debris and aggregates; a total of 97.17% of the white blood cell (WBC) and platelet (plat) position signal points were obtained, with platelets accounting for 67.82%; (2) Multiple sign combination gates: CD61 / SSC-A and CD14 / SSC-A are used to set up gates respectively, and then CD36 / SSC-A is used to set up gates.

[0040] Specifically, CD61 / SSC-A gating: We identified platelets by CD61-APC-CY7 positivity, distinguishing them from other cell debris; CD61 identified platelets in 93.70% of these platelets. CD14 / SSC-A gate: monocytes (mon) are identified by positive expression of CD14-PE; these monocytes account for 0.95% of the total. CD36 / SSC-A gating: The fluorescence intensity of CD36 is used to determine whether platelets (plat) and monocytes (mon) are CD36 positive; The experiment showed that platelets and monocytes in this embodiment were positive for CD36. In the analysis area, CD61, CD14 and CD36 were positive, accounting for 99.75%. The detection results are shown in Table 1 below. The diagnostic sample was normal for CD36.

[0041] The test results of Examples 1-3 are shown in Table 1, where “-” represents negative and “+” represents negative.

[0042] Table 1

[0043] Kappa consistency test: The Kappa consistency test was performed using the flow cytometry method for detecting CD36 deletion in platelets and the PCR gene detection method for CD36 expression as described in Example 1 of this invention. The number of samples tested was 42 (22 cases of normal expression, 18 cases of type II deletion, and 2 cases of type I deletion). The experimental results are shown in Table 2.

[0044] Table 2

[0045] The results showed that the method of this invention and the PCR gene detection method had good consistency in the sample detection and analysis results. Specifically, this method detected 18 cases of type II deletion and 2 cases of type I deletion, which were consistent with the gene detection results. The Kappa coefficient was 0.956, indicating that the correlation was almost completely consistent.

[0046] The above embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An antibody composition for detecting CD36-deficient platelets by flow cytometry, characterized in that the antibody composition comprises CD61 antibody, CD14 antibody and CD36 antibody.

2. The antibody composition according to claim 1, characterized in that, The volume ratio of the CD61 antibody, CD14 antibody, and CD36 antibody is 1:(0.9-1.1):(1.8-2.2).

3. The antibody composition according to claim 1, characterized in that, The CD61 antibody, CD14 antibody, and CD36 antibody are each labeled with a different fluorophore, which is selected from APC, PE, FITC, PERCP, PERCP-CY5.5, APC-CY7, PE-CY7, MF540, MF450, V500, and PE-CF594.

4. A kit for detecting CD36-deficient platelets, characterized in that, Includes the antibody composition according to any one of claims 1-3.

5. The kit for detecting platelet CD36 deletion type according to claim 4, characterized in that, It also includes hemolysin and phosphate buffer.

6. The use of the antibody composition according to any one of claims 1-3 in the preparation of a product for detecting platelet CD36 deficiency.

7. The application according to claim 6, characterized in that, The detection method for platelet CD36 deficiency using the aforementioned platelet CD36 deficiency detection product includes flow cytometry.

8. The application according to claim 6, characterized in that, The process of detecting CD36-deficient platelets includes the following steps: S1, after mixing the sample to be tested with the antibody composition evenly, react at 2-35℃ for 10-40 min; S2, add hemolysin to the sample solution obtained in step S1, wherein the volume ratio of the sample solution to the hemolysin is (8-12):1, and react at 2-35℃ for 5-20 min; S3. After centrifuging the solution obtained in step S2, remove the supernatant, add phosphate buffer to resuspend the cells, perform flow cytometry on the resuspended cells, and select CD61 antibody, CD14 antibody monocytes and CD36 antibody to respectively combine SSC gating to obtain platelet CD36-deficient type.

9. The application according to claim 8, characterized in that, Includes at least one of the following (1)-(3): (1) CD61 antibody positive expression is platelet, CD14 antibody positive expression is monocyte. When the platelets and monocytes identified are both CD36 negative, it is judged as CD36 type I deletion; (2) CD61 antibody positive expression is platelets, CD14 antibody positive expression is monocytes. When the identified platelets are CD36 negative and the monocytes are all CD36 positive, it is judged to be CD36 type II deletion. (3) CD61 antibody positive expression is platelet, CD14 antibody positive expression is monocyte. When both the platelets and monocytes are identified as CD36 positive, it is judged as CD36 normal expression.

10. The application according to claim 8, characterized in that, The sample to be tested is at least one of peripheral blood, capillary blood, or heel blood.

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