An antibody composition for detecting pnh clones and uses thereof

Abstract

This invention relates to an antibody composition for detecting PNH clones and its application. The antibody composition comprises a first antibody group, a second antibody group, and a third antibody group. The first antibody group includes a CD235a antibody, the second antibody group includes a CD235a antibody and a CD59 antibody, and the third antibody group includes a FLAER antibody, a CD157 antibody, a CD15 antibody, a CD64 antibody, and a CD45 antibody. In this invention, the three antibody groups can be used to identify mature erythrocytes, neutrophils, and monocytes in peripheral blood. PNH clones in erythrocytes are detected using CD235a and CD59, and PNH clones in neutrophils and monocytes are detected using FLAER and CD157 antibodies. The FLAER antibody directly binds to the GPI anchor structure, and the CD157 antibody, as an additional GPI anchor protein indicator, can detect potentially missed microclones.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to an antibody composition for detecting PNH clones and its applications. Background Technology

[0002] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired clonal disease of hematopoietic stem cells. Its core pathological mechanism lies in a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene within hematopoietic stem cells. This mutation leads to impaired synthesis of glycosylphosphatidylinositol (GPI) anchors on the cell membrane, resulting in the loss or significant reduction in the expression of various protective proteins that depend on GPI anchoring to the cell membrane surface.

[0003] Currently, flow cytometry based on specific monoclonal antibodies is internationally recognized as the reference method for diagnosing PNH. The core of this method lies in using fluorescently labeled high-affinity antibodies to target and bind to GPI anchoring proteins on the surface of different blood cell populations. Commonly used antibody combinations in clinical practice include anti-CD55 and anti-CD59 antibodies against erythrocytes, and anti-CD16, CD24, anti-CD66b, or FLAER antibodies against granulocytes. Through multicolor flow cytometry analysis, these specific antibodies can accurately distinguish between completely missing (Type III) and partially missing (Type II) cell populations resulting from GPI anchor loss, thereby precisely quantifying the size and biological characteristics of PNH clones. Compared to traditional sucrose hemolysis and acid hemolysis tests, this detection strategy based on antibody-antigen specific binding has extremely high sensitivity and specificity, capable of detecting PNH clones as low as 0.01% or even smaller, providing irreplaceable clinical value for early diagnosis, monitoring disease progression, and evaluating treatment efficacy.

[0004] However, existing methods for detecting PNH clones mostly use CD55 / CD59 to detect erythrocytes and CD16 / CD24 to label granulocytes. This strategy is prone to misdiagnosis in patients who have recently received blood transfusions due to dilution of exogenous normal erythrocytes, making it difficult to accurately identify endogenous micro PNH clones. Furthermore, because immature granulocytes naturally express low levels of CD16 / CD24, current technologies cannot effectively distinguish them from true PNH clones, often leading to false positives. In addition, detection methods relying solely on a single type of anchoring protein lack combined validation with direct targeting of GPI anchors and multidimensional subset typing. This makes it difficult to establish conclusive diagnostic evidence in cases with abnormal expression or rare variants, and the large differences in antibody combinations hinder the achievement of high standardization and cross-center result recognition for microclone detection. Summary of the Invention

[0005] Based on the above description, the present invention provides an antibody composition for detecting PNH clones and its application, aiming to solve the problem of how to improve the accuracy of screening PNH clones.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides an antibody composition for detecting PNH clones, comprising: The first antibody group includes CD235a antibody; The second antibody group includes CD235a antibody and CD59 antibody; The third antibody group includes FLAER antibody, CD157 antibody, CD15 antibody, CD64 antibody and CD45 antibody.

[0007] Furthermore, the volume ratio of the first antibody group, the second antibody group, and the third antibody group is 7:14:26; In the second antibody group, the volume ratio of the CD235a antibody to the CD59 antibody is 1:1; In the third antibody group, the volume ratio of the FLAER antibody, the CD157 antibody, the CD15 antibody, the CD64 antibody, and the CD45 antibody is 1:1:1:5:5.

[0008] Furthermore, all antibodies are fluorescently labeled antibodies.

[0009] Furthermore, the CD235a antibody is labeled with FITC; The CD59 antibody is labeled with PE as a fluorescent dye. The fluorochrome label of the FLAER antibody is iFluor488; The CD157 antibody is labeled with PE as a fluorescent dye. The CD15 antibody is labeled with fluorescein V450; The CD64 antibody is labeled with APC as a fluorescent dye. The CD45 antibody is labeled with the fluorescent dye PerCP.

[0010] The present invention also provides a kit for detecting PNH clones, comprising the antibody composition for detecting PNH clones as described above.

[0011] Furthermore, the kit also includes PBS buffer and red blood cell lysis buffer.

[0012] Furthermore, the concentration of the PBS buffer is 0.02 mol / L, and the pH value is 7.2–7.6.

[0013] The present invention also proposes the application of an antibody composition for detecting PNH clones as described above or a kit for detecting PNH clones as described above in the detection of PNH clones for non-disease diagnosis and treatment purposes.

[0014] Furthermore, the detection of PNH clones includes the following steps: S1. Name the three flow cytometry tubes tube 1, tube 2 and tube 3 respectively. Add CD235a antibody to tube 1, add CD235a antibody and CD59 antibody to tube 2, and add FLAER antibody, CD157 antibody, CD15 antibody, CD64 antibody and CD45 antibody to tube 3. S2. Dilute the sample to be tested with PBS buffer to obtain diluted sample. Add the diluted sample to tube 1 and tube 2 respectively. After incubation in the dark, add PBS and mix well. Centrifuge to remove the supernatant. Resuspend in PBS buffer to obtain samples for tube 1 and tube 2. Add the sample to be tested to tube 3. After incubation in the dark, add red blood cell lysis buffer to tube 3. Mix. Incubate in the dark. Centrifuge. Remove the supernatant. Add PBS and mix well. Centrifuge to remove the supernatant. Resuspend in PBS buffer to obtain samples for tube 3. S3. Place the three-tube sample on a flow cytometer for detection and analysis to determine the proportion of PNH clones of erythrocytes, monocytes, and granulocytes in the sample.

[0015] Further, in step S2, 1 μL of peripheral blood sample is diluted with 100 μL of PBS, divided equally into two tubes (tube 1 and tube 2), and then the concentration of white blood cells in the sample is diluted with PBS to 3 × 10⁻⁶. 7 ~4×10 7 Add cells / mL to tube 3 to make the cell count in tube 3 2 × 10⁻⁶. 6 indivual.

[0016] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: In the technical solution of this invention, by employing the first antibody group and the second antibody group, CD235a antibody is used as a cytoskeletal protein marker with strong red blood cell specificity and stable expression. Mature red blood cells can be circled by FSC / CD235a. Tube 1 serves as a negative control for CD59, and tube 2 circles normal cells (type I cells), partially missing cells (type II cells), and completely missing cells (type III cells) PNH clones by CD235a / CD59 dot plot. By introducing CD15, CD64, and CD45 into the third antibody group, mature neutrophils and monocytes can be distinguished. By employing FLAER antibody and CD157 antibody, where FLAER antibody directly binds to the GPI anchor structure and CD157 antibody serves as an additional GPI anchor protein indicator, potentially missed microclones can be detected. Attached Figure Description

[0017] Figure 1 The flow cytometry analysis of a normal peripheral blood sample using the peripheral blood PNH detection method provided in Example 3 of this application. Figure 2 The flow cytometry analysis of a peripheral blood sample containing a PNH clone is provided by the method for detecting peripheral blood PNH in Example 3 of this application. Figure 3 The flow cytometry analysis of a normal peripheral blood sample using the peripheral blood PNH detection method provided in Example 3 of this application. Figure 4 The flow cytometry analysis of a normal peripheral blood sample using the peripheral blood PNH detection method provided in Comparative Example 3 of this application. Detailed Implementation

[0018] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings, which illustrate embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be thorough and complete.

[0019] Unless otherwise defined, 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0020] Current methods for detecting PNH clones mostly use CD55 / CD59 to detect erythrocytes and CD16 / CD24 to label granulocytes. This strategy is prone to misdiagnosis in patients who have recently received blood transfusions due to dilution of exogenous normal erythrocytes, making it difficult to accurately identify endogenous micro PNH clones. Furthermore, because immature granulocytes naturally express low levels of CD16 / CD24, current technologies cannot effectively distinguish them from true PNH clones, often leading to false positives. In addition, detection methods relying solely on a single type of anchoring protein lack combined validation with direct targeting of GPI anchors and multidimensional subset typing. This makes it difficult to establish conclusive diagnostic evidence in cases with abnormal expression or rare variants, and the large differences in antibody combinations hinder the achievement of high standardization and cross-center result recognition for microclone detection.

[0021] In view of this, the present invention provides an antibody composition for detecting PNH clones, comprising: The first antibody group includes CD235a antibody; The second antibody group includes CD235a antibody and CD59 antibody; The third antibody group includes FLAER antibody, CD157 antibody, CD15 antibody, CD64 antibody and CD45 antibody.

[0022] In the technical solution of this invention, by employing the first antibody group and the second antibody group, CD235a antibody is used as a cytoskeletal protein marker with strong red blood cell specificity and stable expression. Mature red blood cells can be circled by FSC / CD235a. Tube 1 serves as a negative control for CD59, and tube 2 circles normal cells (type I cells), partially missing cells (type II cells), and completely missing cells (type III cells) PNH clones by CD235a / CD59 dot plot. By introducing CD15, CD64, and CD45 into the third antibody group, mature neutrophils and monocytes can be distinguished. By employing FLAER antibody and CD157 antibody, where FLAER antibody directly binds to the GPI anchor structure and CD157 antibody serves as an additional GPI anchor protein indicator, potentially missed microclones can be detected.

[0023] Furthermore, the volume ratio of the first antibody group, the second antibody group, and the third antibody group is 7:14:26; In the second antibody group, the volume ratio of the CD235a antibody to the CD59 antibody is 1:1; In the third antibody group, the volume ratio of the FLAER antibody, the CD157 antibody, the CD15 antibody, the CD64 antibody, and the CD45 antibody is 1:1:1:5:5.

[0024] In the technical solution of the present invention, by using the antibody in the above proportion, based on the differences in antigen expression abundance, antibody affinity characteristics and the principle of flow cytometry optical detection, the problems of signal competition, high background noise and missed detection of microclones commonly found in multicolor flow cytometry are solved, ensuring that the antibody composition can achieve extremely high sensitivity in complex clinical samples while maintaining excellent stability and economy.

[0025] Furthermore, all antibodies are fluorescently labeled antibodies.

[0026] In the technical solution of the present invention, by using fluorescent labeling, there is no need to add secondary antibodies or perform complex enzymatic colorimetric reactions during the experiment. Moreover, all markers in the first, second and third antibody groups can be detected simultaneously in the same reaction tube or in the same run, ensuring a high degree of comparability between erythrocyte, granulocyte and monocyte data.

[0027] Furthermore, the CD235a antibody is labeled with FITC; The CD59 antibody is labeled with PE as a fluorescent dye. The fluorochrome label of the FLAER antibody is iFluor488; The CD157 antibody is labeled with PE as a fluorescent dye. The CD15 antibody is labeled with fluorescein V450; The CD64 antibody is labeled with APC as a fluorescent dye. The CD45 antibody is labeled with the fluorescent dye PerCP.

[0028] In the technical solution of this invention, signal saturation and overflow are avoided by scientifically matching strong and weak dyes, and the orthogonal distribution of spectra is used to achieve non-interference between granulocyte and monocyte data, so as to achieve accurate quantitative diagnosis of PNH clones in a single test with the highest signal-to-noise ratio, the lowest false positive rate and the simplest analysis process.

[0029] The present invention also provides a kit for detecting PNH clones, comprising the antibody composition for detecting PNH clones as described above.

[0030] Since this kit for detecting PNH clones adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be repeated here.

[0031] Furthermore, the kit also includes PBS buffer and red blood cell lysis buffer.

[0032] In the technical solution of this invention, PBS buffer is used for sample washing, resuspending and dilution, maintaining cell osmotic pressure balance and native antigen conformation, ensuring specific antibody binding and reducing non-specific background noise; red blood cell lysis buffer can rapidly and selectively dissolve high-abundance red blood cells in whole blood samples, effectively eliminating their obstruction of the flow cytometry detection field and interference with fluorescence signals, while maximizing the protection of the integrity of fragile PNH clones and preventing the loss of microclones; the two are highly compatible and synergistic with the specific antibody composition, constructing a standardized full-process system from sample pretreatment, enrichment to staining, significantly improving the signal-to-noise ratio, sensitivity and batch-to-batch reproducibility of the detection, and ensuring accurate detection of extremely low proportions of PNH clones.

[0033] Furthermore, the concentration of the PBS buffer is 0.02 mol / L, and the pH value is 7.2–7.6.

[0034] In the technical solution of this invention, by limiting the concentration of PBS buffer to 0.02 mol / L and controlling the pH value to 7.2–7.6, the aim is to construct a physiological microenvironment optimal for flow cytometry detection. This isotonic concentration effectively maintains the morphological integrity of erythrocytes and granulocytes, preventing cell shrinkage or rupture due to osmotic pressure fluctuations, and provides sufficient ionic strength to shield non-specific electrostatic adsorption, significantly reducing background noise. The weakly alkaline pH range of 7.2–7.6 is used to simulate the human physiological environment to maintain the native conformation of CD59, CD157, and FLAER binding sites and antibody binding activity, and also ensures the maximization of fluorescence quantum yield and photostability of fluorescein markers within this range. The synergistic effect of these two factors provides a key physicochemical basis for the accurate quantification of PNH clones with high sensitivity and low false positives.

[0035] The present invention also proposes the application of an antibody composition for detecting PNH clones as described above or a kit for detecting PNH clones as described above in the detection of PNH clones for non-disease diagnosis and treatment purposes.

[0036] Furthermore, the detection of PNH clones includes the following steps: S1. Name the three flow cytometry tubes tube 1, tube 2 and tube 3 respectively. Add CD235a antibody to tube 1, add CD235a antibody and CD59 antibody to tube 2, and add FLAER antibody, CD157 antibody, CD15 antibody, CD64 antibody and CD45 antibody to tube 3. S2. Dilute the sample to be tested with PBS buffer to obtain diluted sample. Add the diluted sample to tube 1 and tube 2 respectively. After incubation in the dark, add PBS and mix well. Centrifuge to remove the supernatant. Resuspend in PBS buffer to obtain samples for tube 1 and tube 2. Add the sample to be tested to tube 3. After incubation in the dark, add red blood cell lysis buffer to tube 3. Mix. Incubate in the dark. Centrifuge. Remove the supernatant. Add PBS and mix well. Centrifuge to remove the supernatant. Resuspend in PBS buffer to obtain samples for tube 3. S3. Place the three-tube sample on a flow cytometer for detection and analysis to determine the proportion of PNH clones of erythrocytes, monocytes, and granulocytes in the sample.

[0037] In the technical solution of this invention, a three-tube stepwise detection strategy is constructed to achieve simultaneous and precise quantification of erythroid and granulocytic / monocytic PNH clones. Tube 1 serves as a negative control tube, used to precisely set the voltage gain and compensation matrix of the flow cytometer, effectively eliminating fluorescence spectral overlap interference. Tube 2 uses CD235a and CD59; the highly expressed CD235a precisely delineates the erythrocyte population, thereby keenly capturing small PNH erythrocyte clones lacking CD59 expression in complex backgrounds. Tube 3 incorporates FLAER and CD157 double negatives. The sex determination criteria, combined with CD45 / CD15 / CD64 multi-parameter typing, can not only specifically identify granulocyte and monocyte subsets, completely eliminating misjudgments caused by differences in maturity, but also maximize the preservation of the natural conformation and binding activity of various cell surface antigens by first adding samples for incubation and then lysing red blood cells. The synergistic effect of these three factors has constructed a standardized detection process that covers the main target cells of whole blood, has endogenous quality control, strong anti-interference ability, and extremely high sensitivity, significantly improving the detection rate and diagnostic accuracy of extremely low proportions of PNH clones in clinical practice.

[0038] Further, in step S2, 1 μL of peripheral blood sample is diluted with 100 μL of PBS, divided equally into two tubes (tube 1 and tube 2), and then the concentration of white blood cells in the sample is diluted with PBS to 3 × 10⁻⁶. 7 ~4×10 7 Add cells / mL to tube 3 to make the cell count in tube 3 2 × 10⁻⁶. 6 indivual.

[0039] In the technical solution of this invention, a precise balance between flow cytometry detection sensitivity and data quality is achieved by setting differentiated cell concentration thresholds for different detection tubes: the sample volume of tube 1 and tube 2 is controlled at 3×10⁻⁶. 7 ~4×10 7The high-density range of individual cells is designed to ensure a sufficient number of erythrocyte events within a limited sample volume, thereby meeting statistical requirements, significantly improving the detection probability of extremely low proportions of PNH erythrocyte microclones, and reducing counting errors; while the sample size of tube 3 is strictly limited to 1×10⁻⁶. 6 ~3×10 6 The appropriate range for each cell effectively avoids poor hydrodynamic focusing caused by high cell concentration, and prevents fluorescence signal quenching or increased background noise caused by excessive cell density, ensuring clear boundaries and accurate grouping of cell subpopulations in the multi-parameter scatter plot.

[0040] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The main reagents are shown in Table 1.

[0042] Table 1 Main Reagents

[0043] Example 1 This embodiment provides an antibody composition for detecting PNH clones, the components and contents of which are shown in Table 2.

[0044] Table 2. Components and content of the antibody composition in Example 1

[0045] Example 2 This embodiment provides a kit for detecting peripheral blood PNH clones.

[0046] This kit contains antibody compositions as shown in Table 2. Each antibody in Table 2 is a fluorescein-labeled antibody. The fluorescein labeling for each antibody is shown in Table 2, with a one-to-one correspondence between the fluorescein and the antibody.

[0047] Furthermore, the kit also contains erythrocyte lysis buffer. Specifically, the erythrocyte lysis buffer is Lysing solution for FACS10. Red blood cell lysis buffer (produced by Hangzhou Lianke Company, product number: LSB03).

[0048] Example 3 This embodiment provides a method for detecting peripheral blood nephropathy (PNH) using the kit provided in Example 2. The steps are as follows: (1) Preparation of test samples: Dilute 1 μL of peripheral blood sample with 100 μL of PBS, divide it into two equal tubes (tube 1 and tube 2), and then dilute the white blood cell concentration in the sample to 3 × 10⁻⁶ with PBS. 7 ~4×10 7 Add the diluted sample to tube 3 to obtain 2 × 10⁶ cells / mL. 6 indivual.

[0049] (2) Add the FITC-labeled CD235a antibody to flow cytometry tube 1 according to the dosages in Table 2. Add the FITC-labeled CD235a antibody and PE-labeled CD59 antibody to flow cytometry tube 2 in sequence. Add the iFluor488-labeled FLAER antibody, PE-labeled CD157 antibody, V450-labeled CD15 antibody, APC-labeled CD64 antibody, and PerCP-labeled CD45 antibody to flow cytometry tube 3 in sequence. Mix well and incubate in the dark for 20 min. Add PBS to tubes 1 and 2, mix well, and centrifuge to remove the supernatant. Add 2-3 mL of 1×Lysing solution for FACS 10 to tube 3. Mix the erythrocyte lysis buffer gently and incubate at room temperature in the dark for 10 min.

[0050] (3) Centrifuge flow cytometry tube 3 at 400 rcf / min for 5 minutes and discard the supernatant. Add 2 mL of PBS buffer to flow cytometry tube 3, mix well, and then centrifuge at 400 rcf / min for 5 minutes and discard the supernatant. Resuspend the tubes in PBS buffer by adding 150-300 μL to tubes 1, 2, and 3. Place the flow cytometry tubes on a flow cytometer for detection and obtain 500,000-1,000,000 events. Export the data in FCS format and analyze the data using flow cytometry software to determine the proportion of PNH clones of erythrocytes, monocytes, and granulocytes in peripheral blood.

[0051] Comparative Example 1 This comparative example provides an antibody composition for detecting PNH clones, the components and contents of which are shown in Table 3.

[0052] Table 3. Components and content of the antibody composition in Comparative Example 1

[0053] Comparative Example 2 This comparative example provides a kit for detecting PNH clones in peripheral blood.

[0054] This kit contains antibody compositions as shown in Table 3. Each antibody in Table 3 is a fluorescein-labeled antibody. The fluorescein labeling for each antibody is shown in Table 3, with a one-to-one correspondence between the fluorescein and the antibody.

[0055] Furthermore, the kit also contains erythrocyte lysis buffer. Specifically, the erythrocyte lysis buffer is a Lysing solution for FACS 10. Red blood cell lysis buffer (produced by Hangzhou Lianke Company, product number: LSB03).

[0056] Comparative Example 3 This comparative example provides a method for detecting peripheral blood PNH using the kit provided in Comparative Example 2. The steps are as follows: (1) Preparation of test samples: Dilute 1 μL of peripheral blood sample with 100 μL of PBS, divide it into two equal tubes (tube 1 and tube 2), and then dilute the white blood cell concentration in the sample to 3 × 10⁻⁶ with PBS. 7 ~4×10 7 Add the diluted sample to tube 3 to obtain 2 × 10⁶ cells / mL. 6 indivual.

[0057] (2) Add the FITC-labeled CD235a antibody to flow cytometry tube 1 according to the dosage in Table 3. Add the FITC-labeled CD235a antibody and PE-labeled CD59 antibody to flow cytometry tube 2 in sequence. Add the antibodies in Table 3 to flow cytometry tube 3 in sequence. Mix well and incubate in the dark for 20 min. Add PBS to tubes 1 and 2, mix well, and centrifuge to remove the supernatant. Add 2-3 mL of 1×Lysing solution for FACS 10 to tube 3. Mix the erythrocyte lysis buffer gently and incubate at room temperature in the dark for 10 min.

[0058] (3) Centrifuge flow cytometry tube 3 at 400 rcf / min for 5 minutes and discard the supernatant. Add 2 mL of PBS buffer to flow cytometry tube 3, mix well, and then centrifuge at 400 rcf / min for 5 minutes and discard the supernatant. Resuspend the tubes in PBS buffer by adding 150-300 μL to tubes 1, 2, and 3. Place the flow cytometry tubes on a flow cytometer for detection and obtain 500,000-1,000,000 events. Export the data in FCS format and analyze the data using flow cytometry software to determine the proportion of PNH clones of erythrocytes, monocytes, and granulocytes in peripheral blood.

[0059] The method described in Example 3 was used to test normal peripheral blood samples and peripheral blood samples containing PNH clones, respectively. The results are as follows: Figure 1 and Figure 2 As shown.

[0060] according to Figure 1It can be seen that no PNH clones were found in normal peripheral blood samples. The LOD of erythrocytes was 0.004%, and the LLOQ was 0.01%; the LOD of monocytes was 0.47%, and the LLOQ was 1.16%; the LOD of granulocytes was 0.03%, and the LLOQ was 0.08%.

[0061] according to Figure 2 It can be seen that 1.37% of PNH clones were detected in monocytes, and low levels of PNH clones were detected in erythrocytes and granulocytes, respectively, at 0.56% and 0.29%. Erythrocyte LOD: 0.004%, LLOQ: 0.01%; monocyte LOD: 0.14%, LLOQ: 0.34%; granulocyte LOD: 0.01%, LLOA: 0.03%.

[0062] Normal peripheral blood samples were tested using the methods of Example 3 and Comparative Example 3, and the results are as follows: Figure 3 and Figure 4 As shown.

[0063] Combination Figure 3 and Figure 4 It can be seen that the basophils are CD33 positive, the SSCs are smaller than the granulocytes, the FLAER binding rate is low and CD14 staining is lacking, which may lead to misjudgment as a mononuclear cell PNH clone.

[0064] Basophils that are CD64 negative will not be misdiagnosed as monocytes using the method in Example 3.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0066] In summary, the technical solution of this application has the following beneficial technical effects: In the technical solution of this invention, by employing the first antibody group and the second antibody group, CD235a antibody is used as a cytoskeletal protein marker with strong red blood cell specificity and stable expression. Mature red blood cells can be circled by FSC / CD235a. Tube 1 serves as a negative control for CD59, and tube 2 circles normal cells (type I cells), partially missing cells (type II cells), and completely missing cells (type III cells) PNH clones by CD235a / CD59 dot plot. By introducing CD15, CD64, and CD45 into the third antibody group, mature neutrophils and monocytes can be distinguished. By employing FLAER antibody and CD157 antibody, where FLAER antibody directly binds to the GPI anchor structure and CD157 antibody serves as an additional GPI anchor protein indicator, potentially missed microclones can be detected.

Claims

1. An antibody composition for detecting PNH clones, characterized in that, include: The first antibody group includes CD235a antibody; The second antibody group includes CD235a antibody and CD59 antibody; The third antibody group includes FLAER antibody, CD157 antibody, CD15 antibody, CD64 antibody and CD45 antibody.

2. The antibody composition for detecting PNH clones according to claim 1, characterized in that, The volume ratio of the first antibody group, the second antibody group, and the third antibody group is 7:14:26; In the second antibody group, the volume ratio of the CD235a antibody to the CD59 antibody is 1:1; In the third antibody group, the volume ratio of the FLAER antibody, the CD157 antibody, the CD15 antibody, the CD64 antibody, and the CD45 antibody is 1:1:1:5:

5.

3. The antibody composition for detecting PNH clones according to claim 1, characterized in that, All antibodies are fluorescently labeled antibodies.

4. The antibody composition for detecting PNH clones according to claim 3, characterized in that, The CD235a antibody is labeled with FITC; The CD59 antibody is labeled with PE as a fluorescent dye. The fluorochrome label of the FLAER antibody is iFluor488; The CD157 antibody is labeled with PE as a fluorescent dye. The CD15 antibody is labeled with fluorescein V450; The CD64 antibody is labeled with APC as a fluorescent dye. The CD45 antibody is labeled with the fluorescent dye PerCP.

5. A kit for detecting PNH clones, characterized in that, Includes the antibody composition for detecting PNH clones as described in any one of claims 1 to 4.

6. The kit for detecting PNH clones according to claim 5, characterized in that, The kit also includes PBS buffer and red blood cell lysis buffer.

7. The kit for detecting PNH clones according to claim 6, characterized in that, The concentration of the PBS buffer is 0.02 mol / L, and the pH value is 7.2–7.

6.

8. The use of an antibody composition for detecting PNH clones as described in any one of claims 1 to 4 or a kit for detecting PNH clones as described in any one of claims 5 to 7 in the detection of PNH clones for non-disease diagnosis and treatment purposes.

9. The application according to claim 8, characterized in that, The detection of PNH clones includes the following steps: S1. Name the three flow cytometry tubes tube 1, tube 2 and tube 3 respectively. Add CD235a antibody to tube 1, add CD235a antibody and CD59 antibody to tube 2, and add FLAER antibody, CD157 antibody, CD15 antibody, CD64 antibody and CD45 antibody to tube 3. S2. Dilute the sample to be tested with PBS buffer to obtain diluted sample. Add the diluted sample to tube 1 and tube 2 respectively. After incubation in the dark, add PBS and mix well. Centrifuge to remove the supernatant. Resuspend in PBS buffer to obtain samples for tube 1 and tube 2. Add the sample to be tested to tube 3. After incubation in the dark, add red blood cell lysis buffer to tube 3. Mix. Incubate in the dark. Centrifuge. Remove the supernatant. Add PBS and mix well. Centrifuge to remove the supernatant. Resuspend in PBS buffer to obtain samples for tube 3. S3. Place the three-tube sample on a flow cytometer for detection and analysis to determine the proportion of PNH clones of erythrocytes, monocytes, and granulocytes in the sample.

10. The application according to claim 9, characterized in that, In step S2, 1 μL of peripheral blood sample is diluted with 100 μL of PBS, divided into two equal tubes (tube 1 and tube 2), and then diluted with PBS to a white blood cell concentration of 3 × 10⁻⁶. 7 ~4×10 7 Add cells / mL to tube 3 to make the cell count in tube 3 2 × 10⁻⁶. 6 indivual.

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