B cell specific light chain detection kit

Abstract

The invention relates to a B cell specific light chain detection kit, and belongs to the technical field of biology. The B cell specific light chain detection kit comprises an antibody composition, a fixing agent and a permeabilized buffer solution, the antibody composition comprises an A group antibody and a B group antibody, wherein the A group antibody comprises an anti-Kappa antibody and an anti-Lambda antibody; the group B antibody comprises an anti-CD19 antibody, an anti-CD38 antibody, an anti-CD5 antibody, an anti-CD10 antibody, an anti-CD20 antibody and an anti-CD45 antibody; the permeabilization buffer solution comprises amino acid modified saponin. And the contained permeabilized buffer solution is modified by amino acid, so that the permeability of cell membranes can be ensured, the cell morphology cannot be excessively damaged, and the accuracy of B cell specific light chain detection is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a kit for detecting B-cell specific light chains. Background Art

[0002] Flow cytometry can be used to detect B-cell lymphoma. Kappa chain is a 25Kd polypeptide, which is cleaved from the IgG molecule by papain and encoded by the human immunoglobulin kappa locus (IGK) on chromosome 2. Anti-Kappa antibody recognizes the Kappa light chain immunoglobulin expressed on the surface of cell subsets, and this subset corresponds to approximately 2 / 3 of the mature B lymphocytes in peripheral blood. Lambda light chain immunoglobulin is expressed on the surface of cell subsets, corresponding to approximately 1 / 3 of the mature B lymphocytes in peripheral blood. These two light chains are also found on the surface of immature bone marrow B lymphocyte subsets. Detecting the monoclonal light chain expression on the surface of B lymphocyte proliferative diseases is very important for disease diagnosis and residual lesion monitoring. In the normal B cell population, the ratio of Kappa to Lambda is approximately 1.5:1 or 1:1.5, while the tumor cells of B-cell lymphoma usually show clonal expression of a certain light chain. Generally, a kappa:Lambda ratio > 3:1 or < 0.3:1 is considered monoclonal expression. There are difficulties in labeling the cytoplasm and nucleus during the detection process. For example, the kappa or Lambda light chain is physically blocked by the cell membrane and needs to be fully permeabilized to be bound by the antibody. Summary of the Invention

[0003] The object of the present invention is to provide a kit for detecting B-cell specific light chains to improve the accuracy of detecting B-cell specific light chains.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A kit for detecting B-cell specific light chains, comprising an antibody composition, a fixative, and a permeabilization buffer; the antibody composition includes Group A antibodies and Group B antibodies. Group A antibodies include anti-Kappa antibody and anti-Lambda antibody; Group B antibodies include anti-CD19 antibody, anti-CD38 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD20 antibody, and anti-CD45 antibody; the permeabilization buffer includes amino acid-modified saponin.

[0006] Further, the preparation method of the amino acid-modified saponin comprises the following steps:

[0007] S1: Saponin activation: Dissolve saponin in anhydrous DMSO to prepare a saponin solution, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the saponin solution, and then add N-hydroxysuccinimide. Stir at 30 - 40 °C for 2 - 4 h to obtain an activated intermediate;

[0008] S2: Amino acid coupling: Dissolve the amino acid in PBS to obtain an amino acid solution, slowly drip the activated intermediate into the amino acid solution, stir and react at 30 - 40 °C for 12 - 24 h, and use TLC to detect the reaction progress until the reaction ends.

[0009] Furthermore, the molar ratio of the saponin to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:1.1 - 1.5.

[0010] Furthermore, the molar ratio of the saponin to N-hydroxysuccinimide is 1:0.8 - 1.2.

[0011] Furthermore, the pH value of the PBS is 7.5 - 7.8.

[0012] Furthermore, the saponin is quillaja saponin.

[0013] Furthermore, the amino acid is arginine.

[0014] Furthermore, the permeabilization buffer is prepared by dissolving amino acid-modified saponin in PBS.

[0015] Furthermore, the fluorescence labels of the combination of anti-Kappa antibody, anti-Lambda antibody, anti-CD19 antibody, anti-CD38 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD20 antibody, and anti-CD45 antibody are FITC, PE, PE-Cy7, mFluor450, PerCP, APC-Cy7, APC, and mFluor540 respectively.

[0016] Advantages of the present invention:

[0017] The B-cell specific light chain detection kit of the present invention contains a permeabilization buffer that is modified by amino acids. It can not only ensure the permeability of the cell membrane but also not overly damage the cell morphology, improving the accuracy of B-cell specific light chain detection. Description of the Drawings

[0018] Figure 1 Normal result of B-cell specific light chain detected by the reagents and detection method in Example 1 (Case No. 78)

[0019] Figure 2 Normal result of B-cell specific light chain detected by the reagents and detection method in Example 1 (Case No. 356)

[0020] Figure 3 Normal result of B-cell specific light chain detected by the reagents and detection method in Example 1 (Case No. 983)

[0021] Figure 4 Results of abnormal B-cell specific light chain detected using the reagents and detection method in Example 1. Detailed implementation manners

[0022] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0023] Example 1

[0024] The preparation method of the amino acid-modified saponin comprises the following steps:

[0025] 1) Saponin activation: Dissolve the saponin in anhydrous DMSO to prepare a saponin solution with a concentration of 5 mM. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to the saponin solution, and the molar ratio of saponin to EDC is 1:1.2. Then add N-hydroxysuccinimide (NHS), and the molar ratio of saponin to NHS is 1:1. Stir at 30 °C for 2-4 h to obtain an activated intermediate. The saponin is quillaja saponin.

[0026] 2) Amino acid coupling: Dissolve 10 g of amino acid in 100 mL of PBS with a pH of 7.5 to obtain an amino acid solution. Slowly drop the activated intermediate into the amino acid solution, and stir and react at 35 °C for 12 h. Detect the reaction progress by TLC until the reaction ends. The amino acid is arginine.

[0027] 3) Purification: Load the reaction solution in step 2) into a dialysis bag with a cut-off molecular weight of 1-3 kDa, and dialyze in deionized water for 48 h (change water every 6 h) to remove unreacted amino acids and small molecule by-products. Then use column chromatography to separate the target product, and freeze-dry to obtain the amino acid-modified saponin. When specifically used, dissolve the amino acid-modified saponin in PBS to prepare an amino acid-modified saponin solution, which is the permeabilization buffer.

[0028] Example 2

[0029] I. Main materials and instruments for the experiment

[0030] 1. Materials: 10×PBS buffer, red blood cell lysate, fixative, permeabilization buffer;

[0031] 2. Instruments: Cytek NL-3000 full-spectrum flow cytometer.

[0032] II. Methods

[0033] 1. Sample collection:

[0034] Collect peripheral blood and store the specimen at 4 °C. Flow cytometry (FCM) detection must be completed within 24 hours.

[0035] 2. Single-cell suspension preparation process:

[0036] Add 10× volume of red blood cell lysate BD Pharm Lyse TM , incubate at room temperature in the dark for 10 minutes, centrifuge (300g, 5 minutes), and discard the supernatant. Wash twice with PBS and adjust the cell concentration to 1×10 6 / mL.

[0037] 3. Labeling antibodies

[0038] The antibody combination labeling is shown in Table 1.

[0039] Table 1 Antibody combination labeling

[0040]

[0041] Surface antigen labeling: Add 100 μL of single-cell suspension to each tube, add 10 μL of fluorescently labeled Group B antibody mixture, and incubate at room temperature in the dark for 20 min. Add 2 mL of PBS to wash, centrifuge (300g, 5 min), and discard the supernatant.

[0042] Intracellular antigen labeling:

[0043] a) Fixation: Add 100 μL of fixative, i.e., 4% paraformaldehyde, and incubate at room temperature in the dark for 15 min.

[0044] b) Permeabilization: Add permeabilization buffer and incubate for 20 min.

[0045] c) Labeling: Add Group A antibodies and incubate in the dark for 30 min. Wash twice and resuspend in PBS.

[0046] (4) Instrument detection:

[0047] a) Determine the optimal voltage and compensation: Set the voltage according to the conventional operation method of the spectral flow cytometer.

[0048] b) Instrument setup, calibration, and quality control: Turn on Cytek NL-3000 and preheat the machine for more than 20 min, then rinse with deionized water. Detect the internal quality control product to ensure that each detected value is within the control range. Load the sample from AL-PANAL and collect data.

[0049] c) Instrument detection: According to the set instrument conditions, acquire 50,000 - 100,000 cells per tube. If the detection cannot be performed in time, add 0.5 mL of 1% paraformaldehyde, mix well, and store in a 4°C refrigerator. Complete the detection within 24 hours.

[0050] III. Result analysis: Analyze the data using KLuzaa software:

[0051] Cell population strategy:

[0052] FSC-A vs SSC-A: Gate the lymphocyte population (excluding debris and granulocytes).

[0053] CD45 vs SSC-A: Identify the leukocyte population with high CD45 expression.

[0054] CD19+CD20+: Gate the B cell population.

[0055] Light chain analysis:

[0056] Analyze the κ / λ light chain ratio in the B cell population (normal ≈ 2:1, monoclonal imbalance indicates lymphoma).

[0057] Subtype determination:

[0058] CD5+CD10- → CLL / SLL or MCL (Cyclin D1 needs to be combined).

[0059] CD10+BCL-6+ → Follicular lymphoma or germinal center type DLBCL.

[0060] For Comparative Example 1

[0061] I. Main experimental materials and instruments

[0062] 1. Materials: 10×PBS buffer, red blood cell lysate, fixative, permeabilization buffer;

[0063] 2. Instruments: Cytek NL-3000 full-spectrum flow cytometer.

[0064] II. Methods

[0065] 1. Sample collection:

[0066] Collect peripheral blood and store the specimen at 4°C. Flow cytometry (FCM) detection must be completed within 24 hours.

[0067] 2. Process for preparing single-cell suspension:

[0068] Add 10× volume of red blood cell lysate BD Pharm Lyse TM , incubate at room temperature in the dark for 10 minutes, centrifuge (300g, 5 minutes), and discard the supernatant. Wash twice with PBS and adjust the cell concentration to 1×10 6 / mL.

[0069] 4. Labeling antibodies

[0070] The antibody combination labeling is shown in Table 1.

[0071] Table 1 Antibody combination labeling

[0072]

[0073] Surface antigen labeling: Add 100 μL of single-cell suspension to each tube, add 10 μL of fluorescently labeled Group B antibody mixture, and incubate in the dark at room temperature for 20 min. Add 2 mL of PBS for washing, centrifuge (300 g, 5 min), and discard the supernatant.

[0074] Intracellular antigen labeling:

[0075] d) Fixation: Add 100 μL of fixative, namely 4% paraformaldehyde, and incubate in the dark at room temperature for 15 min.

[0076] e) Permeabilization: Add permeabilization buffer and incubate for 20 min. The permeabilization buffer is a PBS solution of saponin.

[0077] f) Labeling: Add Group A antibodies and incubate in the dark for 30 min. After washing twice, resuspend in PBS.

[0078] (4) Instrument detection:

[0079] a) Determine the optimal voltage and compensation: Set the voltage according to the conventional operation method of the spectral flow cytometer.

[0080] b) Instrument setup, calibration, and quality control: Turn on the Cytek NL-3000 and preheat the machine for more than 20 min, then rinse with deionized water. Detect the internal quality control product to ensure that each detected value is within the control range. Retrieve the AL-PANAL for sampling and collect data.

[0081] c) Instrument detection: According to the set instrument conditions, acquire 50,000 - 100,000 cells per tube. If it cannot be detected on the machine in time, add 0.5 mL of 1% paraformaldehyde, mix well, and store in a 4°C refrigerator, and complete the detection within 24 hours.

[0082] III. Result analysis: Analyze the data using KLuzaa software:

[0083] Cell population strategy:

[0084] FSC-A vs SSC-A: Gate the lymphocyte population (exclude debris and granulocytes).

[0085] CD45 vs SSC-A: Identify the leukocyte population with high CD45 expression.

[0086] CD19+CD20+: Gate the B cell population.

[0087] Light chain analysis:

[0088] Analyze the κ / λ light chain ratio in the B cell population (normal ≈ 2:1, monoclonal imbalance indicates lymphoma).

[0089] Subtype determination:

[0090] CD5+CD10- → CLL / SLL or MCL (Cyclin D1 needs to be combined).

[0091] CD10+BCL-6+ → Follicular lymphoma or germinal center type DLBCL.

[0092] Test Example 1

[0093] Using the reagents and methods of Example 1 and Comparative Example 1 respectively, 42,379 case samples of clinically suspected lymphoma were tested. Among the cases with abnormal κ / λ light chain ratio detected by the reagents and test methods of Example 1, there were 373 cases. Among these 373 cases with abnormal κ / λ light chain ratio, final clinical diagnosis tests were conducted, and the results showed that 369 cases were clinically finally diagnosed as B-cell lymphoma cases. For the cases detected by the reagents and test methods of Comparative Example 1, there were 425 cases with abnormal κ / λ light chain ratio. Among these 425 cases with abnormal κ / λ light chain ratio, final clinical diagnosis tests were conducted, and the results showed that 369 cases were clinically finally diagnosed as B-cell lymphoma cases. The accuracy rate measured by the reagents of Example 1 is relatively high.

Claims

1. A B cell-specific light chain detection kit, characterized in that, It includes an antibody composition, a fixative, and a permeabilization buffer; the antibody composition includes Group A antibodies and Group B antibodies, Group A antibodies include anti-Kappa antibodies and anti-Lambda antibodies; Group B antibodies include anti-CD19 antibodies, anti-CD38 antibodies, anti-CD5 antibodies, anti-CD10 antibodies, anti-CD20 antibodies, anti-CD45 antibodies; the permeabilization buffer includes amino acid-modified saponins.

2. The B cell-specific light chain detection kit according to claim 1, wherein The preparation method of the amino acid-modified saponins includes the following steps: S1: Saponin activation: Dissolve saponins in anhydrous DMSO to prepare a saponin solution, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the saponin solution, and then add N-hydroxysuccinimide, stir at 30-40 °C for 2-4 h to obtain an activated intermediate; S2: Amino acid coupling: Dissolve amino acids in PBS to obtain an amino acid solution, slowly drop the activated intermediate into the amino acid solution, stir and react at 30-40 °C for 12-24 h, and detect the reaction progress by TLC until the reaction ends.

3. The B cell-specific light chain detection kit according to claim 2, characterized in that, The molar ratio of the saponins to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:1.1-1.

5.

4. The B cell-specific light chain detection kit according to claim 2, characterized in that, The molar ratio of the saponins to N-hydroxysuccinimide is 1:0.8-1.

2.

5. The B cell-specific light chain detection kit according to claim 2, wherein, The pH value of the PBS is 7.5-7.

8.

6. The B cell-specific light chain detection kit according to claim 2, wherein, The saponins are quillaja saponins.

7. The B cell-specific light chain detection kit according to claim 2, characterized in that, The amino acids are arginine.

8. The B cell-specific light chain detection kit according to claim 1 or 2, characterized in that, The permeabilization buffer is prepared by dissolving amino acid-modified saponins in PBS.

9. The B cell-specific light chain detection kit according to claim 1, wherein The fluorescence labels of the anti-Kappa antibody, anti-Lambda antibody, anti-CD19 antibody, anti-CD38 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD20 antibody, and anti-CD45 antibody in combination are FITC, PE, PE-Cy7, mFluor450, PerCP, APC-Cy7, APC, and mFluor540 respectively.

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