Antibody compositions and uses thereof

Abstract

The application provides an antibody composition and application thereof, wherein the antibody composition comprises a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies and a sixth group of antibodies. The application forms a "diagnosis+differentiation+prognosis" integrated marker system, significantly enhances the differentiation ability for atypical CLL and other B-CLPD, and especially improves the interpretation of cases with MS scores in the "gray zone" of 0-3 points.

Description

Technical Field

[0001] This invention belongs to the field of antibody pharmaceutical technology, specifically relating to an antibody composition and its application. Background Technology

[0002] B-cell chronic lymphocytic leukemia (B-CLL) is a clonal proliferative disorder originating from mature B lymphocytes. Clinically, it is characterized by the abnormal accumulation of mature lymphocytes in the peripheral blood and bone marrow, often accompanied by lymphadenopathy, hepatosplenomegaly, and involvement of multiple tissues and organs. Due to the overlap between morphological and clinical manifestations, relying solely on morphological and clinical data is insufficient for accurate subtyping and differential diagnosis. Immunophenotyping analysis has become an important tool for the diagnosis, differential diagnosis, and prognostic assessment of CLL. Among these methods, multicolor flow cytometry, due to its high throughput, quantification, and ability to detect multiple indicators simultaneously, is widely used in the laboratory subtyping of B-CLL and other B-cell lymphoproliferative disorders (B-CLPD).

[0003] To improve the consistency and reproducibility of immunological diagnosis of CLL, in 1994, Mattes et al. proposed the MoreauScore (MS) flow cytometry scoring system based on five biomarkers: CD5, CD23, FMC-7, CD22, and surface immunoglobulin (sIg). This system achieved high overall accuracy (approximately 91.8%) when the score was ≥4. In 1997, Moreau et al. introduced CD79b into the system, reporting an further improvement in overall accuracy to approximately 96.8%. However, despite the high overall accuracy of these scoring systems, their specificity remains limited (approximately 53.8%). Furthermore, in clinical practice, some atypical CLL and other B-CLPDs (such as diseases phenotypically overlapping with CLL) can present immunophenotypes similar to or overlapping with typical CLL, leading to difficulties in differentiating cases with MS scores in the 0-3 range. This phenotypic overlap and "gray zone" problem makes scoring systems relying solely on limited biomarkers insufficient to fully meet the precise diagnostic needs in complex clinical situations.

[0004] Furthermore, existing technologies face common challenges in practical applications: to compensate for deficiencies in specificity and discrimination, multiple rounds of testing or parallel application of other methods (such as cytogenetics, FISH, or molecular biology assays) are often required for comprehensive interpretation, leading to lengthy testing procedures, increased overall time consumption, higher reagent and labor costs, increased energy consumption, and larger sample volumes. Multiple tube triage and repeated staining introduce operational complexity and potential contamination risks. Differences in antibody selection, fluorescence channel configuration, and gating strategies among different laboratories also affect the consistency and comparability of results. Simultaneously, traditional small panel assemblies primarily for diagnosis struggle to simultaneously cover prognostic biomarkers, failing to integrate diagnosis, differentiation, and risk stratification in a single test, thus limiting their immediate guidance value for clinical treatment strategies and efficacy monitoring. Summary of the Invention

[0005] In view of this, the present invention provides an antibody composition and its application, forming an integrated biomarker system of "diagnosis + identification + prognosis", which significantly enhances the ability to distinguish atypical CLL and other B-CLPD, especially improving the interpretation of cases with MS scores in the "gray zone" of 0-3.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an antibody composition comprising: a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies, and a sixth group of antibodies. The first group of antibodies comprises anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody, and anti-CD45 antibody; the second group of antibodies comprises anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody; the third group of antibodies comprises anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody; the fourth group of antibodies comprises anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody, and anti-CD45 antibody; the fifth group of antibodies comprises anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody; and the sixth group of antibodies comprises anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody.

[0008] Preferably, the anti-membrane Kappa antibody and the anti-membrane Lambda antibody are polyclonal antibodies, and the remaining antibodies are monoclonal antibodies.

[0009] Preferably, the fluorescent labeling order of the anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody and anti-CD45 antibody in the first group of antibodies is FITC, PE, APC, PE-CY7, BV421 and PerCP, respectively.

[0010] The fluorescent labeling order of the anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody in the second group of antibodies is PE, APC-Cy7, BV421, and PerCP, respectively.

[0011] The fluorescent labeling order of the anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody in the third group of antibodies is FITC, PE, APC, BV421, and PerCP, respectively.

[0012] The fluorescent labeling order of the anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody and anti-CD45 antibody in the fourth group of antibodies is FITC, PE, PE-CY7, APC and PerCP, respectively.

[0013] The fluorescent labeling order of the anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody in the fifth group of antibodies is PE, APC, BV421, and PerCP, respectively.

[0014] The fluorescent labeling order of the anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody in the sixth group of antibodies is PE, BV421, and PerCP, respectively.

[0015] Preferably, in the first antibody composition, the volume ratio of anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:3:2:5;

[0016] In the second antibody composition, the volume ratio of anti-CD81 antibody, anti-CD20 antibody, and anti-CD45 antibody is 5:3:2:5;

[0017] In the third antibody composition, the volume ratio of anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:2:5;

[0018] In the fourth antibody composition, the volume ratio of anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:3:5;

[0019] In the fifth antibody composition, the volume ratio of anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:3:2:5;

[0020] In the sixth antibody composition, the volume ratio of anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody is 5:2:5.

[0021] Secondly, the present invention provides the application of the antibody composition described above in the preparation of flow cytometry samples for immunophenotyping detection of chronic lymphocytic leukemia.

[0022] Thirdly, the present invention provides the application of the antibody composition described above in the preparation of an immunophenotyping detection product for chronic lymphocytic leukemia.

[0023] Fourthly, the present invention provides a reagent kit, comprising a first container, a second container, a third container, a fourth container, a fifth container, and a sixth container;

[0024] The first container contains the first group of antibodies, the second container contains the second group of antibodies, the third container contains the third group of antibodies, the fourth container contains the fourth group of antibodies, the fifth container contains the fifth group of antibodies, and the sixth container contains the sixth group of antibodies.

[0025] Preferably, it also includes other containers for holding hemolysin and PBS buffer, respectively.

[0026] Preferably, it also includes flow cytometry tubes for use with the flow cytometer.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) Based on retaining the core markers of MS (CD5, CD23, FMC-7, CD22 / sIg, and introducing CD79b), this invention adds CD200, CD81, CD43, CD49d, CD38, sIgM, etc., forming an integrated marker system of "diagnosis + differentiation + prognosis", which significantly enhances the ability to distinguish atypical CLL and other B-CLPD, especially improving the interpretation of cases with MS scores in the "gray zone" of 0-3 points.

[0029] (2) The comprehensive pattern interpretation of CD200 (CLL is often positive, MCL is often negative), CD81 (CLL is often reduced), FMC-7 / CD79b / CD22 (CLL is often weak / negative, MCL is often strong positive) and sIgM, CD20 intensity in this invention can quickly distinguish CLL and MCL phenotypically overlapping entities and reduce the risk of misjudgment.

[0030] (3) By replacing the “single score of a few biomarkers” with “multi-feature joint decision-making”, the specificity and consistency of non-CLL and B-CLPD are significantly improved while maintaining high sensitivity, and the passive dependence on morphology and additional molecular detection is reduced. Attached Figure Description

[0031] Figure 1 The FSC / SSC scatter plot provided in Embodiment 3 of the present invention is mainly to remove cell debris or impurities and circle a single live cell.

[0032] Figure 2 The FSC-A / FSC-H scatter plot provided in Embodiment 3 of the present invention is mainly a plot after removing adherent cells;

[0033] Figure 3 The CD45 / SSC scatter plot provided in Embodiment 3 of the present invention mainly uses CD45 / SSC gating to circle all white blood cell populations;

[0034] Figure 4 The cell population diagram provided in Embodiment 3 of the present invention first uses CD45 / CD19 gating to delineate the CD19+ cell population;

[0035] Figure 5 The diagram provided in Example 3 of this invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of FMC-7 / CD23 is observed.

[0036] Figure 6 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD5 / CD19 is observed.

[0037] Figure 7 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD5 / CD10 is observed.

[0038] Figure 8 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD81 / CD20 is observed.

[0039] Figure 9 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD19 / CD200 is observed.

[0040] Figure 10 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD79b / CD19 is observed.

[0041] Figure 11The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD22 / CD19 is observed.

[0042] Figure 12 The cell population diagram provided in Embodiment 3 of the present invention first uses CD45 / CD19 gating to delineate the CD19+ cell population;

[0043] Figure 13 The diagram provided in Example 3 of this invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of Kappa / Lambda is observed.

[0044] Figure 14 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD19 / CD38 is observed.

[0045] Figure 15 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD19 / CD43 is observed.

[0046] Figure 16 The diagram provided in Embodiment 3 of the present invention shows that first, a CD19+ cell population is gated by CD45 / CD19, and then the expression of CD19 / CD49d is observed.

[0047] Figure 17 The diagram provided in Example 3 of this invention shows that first, a CD19+ cell population is delineated by gating with CD45 / CD19, and then the expression of CD19 / sIgM is observed. Detailed Implementation

[0048] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0049] To improve the consistency and reproducibility of immunological diagnosis of CLL, in 1994, Mattes et al. proposed the MoreauScore (MS) flow cytometry scoring system based on five biomarkers: CD5, CD23, FMC-7, CD22, and surface immunoglobulin (sIg). This system achieved high overall accuracy (approximately 91.8%) when the score was ≥4. In 1997, Moreau et al. introduced CD79b into the system, reporting an further improvement in overall accuracy to approximately 96.8%. However, despite the high overall accuracy of these scoring systems, their specificity remains limited (approximately 53.8%). Furthermore, in clinical practice, some atypical CLL and other B-CLPDs (such as diseases phenotypically overlapping with CLL) can present immunophenotypes similar to or overlapping with typical CLL, leading to difficulties in differentiating cases with MS scores in the 0-3 range. This phenotypic overlap and "gray zone" problem makes scoring systems relying solely on limited biomarkers insufficient to fully meet the precise diagnostic needs in complex clinical situations.

[0050] Furthermore, existing technologies face common challenges in practical applications: to compensate for deficiencies in specificity and discrimination, multiple rounds of testing or parallel application of other methods (such as cytogenetics, FISH, or molecular biology assays) are often required for comprehensive interpretation, leading to lengthy testing procedures, increased overall time consumption, higher reagent and labor costs, increased energy consumption, and larger sample volumes. Multiple tube triage and repeated staining introduce operational complexity and potential contamination risks. Differences in antibody selection, fluorescence channel configuration, and gating strategies among different laboratories also affect the consistency and comparability of results. Simultaneously, traditional small panel assemblies primarily for diagnosis struggle to simultaneously cover prognostic biomarkers, failing to integrate diagnosis, differentiation, and risk stratification in a single test, thus limiting their immediate guidance value for clinical treatment strategies and efficacy monitoring.

[0051] To address the aforementioned technical problems, the present invention provides an antibody composition comprising: a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies, and a sixth group of antibodies. The first group of antibodies comprises anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody, and anti-CD45 antibody; the second group of antibodies comprises anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody; the third group of antibodies comprises anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody; the fourth group of antibodies comprises anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody, and anti-CD45 antibody; the fifth group of antibodies comprises anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody; and the sixth group of antibodies comprises anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody.

[0052] In the above technical solution, the beneficial effects of the present invention are as follows: First, while retaining the core biomarkers of the MS system (CD5, CD23, FMC-7, CD22, CD79b, sIg), it introduces key molecules such as CD200, CD81, CD43, CD49d, and CD38, significantly enhancing the ability to differentiate atypical CLL and other B-CLPDs with MS scores of 0-3, and reducing misjudgments caused by phenotypic overlap. Second, by combining CD200 (CLL is usually positive, MCL is mostly negative), FMC-7 (CLL is mostly negative, MCL is mostly positive), CD79b / CD22 intensity (CLL is mostly weak, MCL is mostly strong), CD23 (CLL is mostly positive, MCL is mostly negative), and CD20 and sIgM intensity patterns, it can quickly differentiate CLL from MCL, a common clinical challenge. Third, CD10 is used to suggest / exclude follicular lymphoma (FL). Combined with FMC-7, CD23, CD200, CD43, and other patterns, it helps to initially differentiate FL from marginal zone lymphoma (MZL) and atypical CLL, narrowing the scope of further molecular testing. Fourth, the inclusion of prognostic markers such as CD38, CD49d, and CD81 allows for risk stratification while completing diagnosis / differentiation (e.g., high expression of CD49d and CD38 usually indicates a poor prognosis; reduced CD81 is associated with IGHV mutation status), reducing the need for additional testing. Clonality assessment is more reliable: Surface light chain κ / λ and sIgM are set, combined with CD19 gating, to achieve simultaneous assessment of B cell clonality and immunoglobulin intensity; κ / λ uses a polyclonal antibody to improve the identification of variant epitopes and the detection rate of weakly expressed light chains, reducing false negatives. Fifth, the panel includes commonly expressed weak antigens of CLL (such as CD22, CD79b, CD20, sIgM, and CD81 reduction), improving the confidence of weak expression detection through multi-tube complementarity and signal integration, and reducing subjective differences in positive and negative boundary samples. Sixth, each group is equipped with CD19 and CD45 as a backbone, which is conducive to consistent gating and population alignment across tubes, improving reproducibility and comparability of results between different laboratories / operators. Balancing diagnosis, differentiation, and monitoring: The panel design covers the entire chain of information from "diagnosis to differentiation to prognosis to clonal variation," reducing additional supplementary tests and multiple rounds of retesting, providing a one-time, structured output for clinical decision-making. Seventh, it reduces passive reliance on cytogenetics, FISH, or molecular testing and the frequency of additional tests, saving sample volume and manpower, shortening report generation time, and reducing overall costs and energy consumption. Eighth, fixed tube groups and preset combinations reduce multiple preparation and repeated staining steps, reducing pipetting errors and contamination risks, and improving the stability and safety of daily instrumentation procedures. Ninth, the biomarker combination is clear and the structural grouping is well-defined, which facilitates the preparation of standardized reagent kits and their mass production and clinical validation, promoting consistent application across platforms and centers.Tenth, the selected biomarkers are compatible with mainstream multi-laser flow cytometry platforms, facilitating direct deployment under existing laboratory conditions without significant adjustments to instrument configuration and operating procedures. Eleventh, clinicians can initially use diagnostic / differential assessment groups, adding prognostic and clonal correlation groups when results are uncertain or stratification is required, improving throughput and cost-effectiveness. Twelfth, by replacing single-marker scoring with multi-feature joint interpretation, specificity and inter-laboratory consistency are significantly improved while maintaining high sensitivity, better meeting the needs for accurate diagnosis in complex clinical scenarios.

[0053] Furthermore, the anti-membrane Kappa antibody and the anti-membrane Lambda antibody are polyclonal antibodies, while the remaining antibodies are monoclonal antibodies. In this technical solution, the beneficial effects of using polyclonal antibodies for the anti-membrane Kappa antibody and the anti-membrane Lambda antibody are as follows: First, CLL is often accompanied by weak expression of surface immunoglobulins and light chains. Using polyclonal κ / λ antibodies can simultaneously recognize multiple variant epitopes, improving the signal intensity and detection rate when expression is weak, and reducing false negatives and double negative light chain results. Second, obtaining κ / λ restriction more stably helps to make a clear judgment on B cell clonality in conjunction with CD19 gating, improving consistency with molecular clonality results, and reducing the need for subsequent additional testing. Adaptable to various processing procedures: Polyclonal light chain antibodies are more tolerant to epitope effects caused by sample processing (such as hemolysis, fixation / non-fixation, storage time), maintaining a high positive rate and signal-to-noise ratio. Third, in the case of low light chain background and sparse BCR, polyclonal antibodies can achieve clear typing under conventional fluorescent dye configurations due to the signal accumulation effect generated by multi-point binding, thereby improving the overall robustness of the panel.

[0054] The advantages of using monoclonal antibodies are as follows: First, monoclonal antibodies are used for CD5, CD23, FMC-7, CD22, CD79b, CD200, CD20, CD81, CD38, CD43, CD49d, CD10, and sIgM, resulting in high specificity, low background, and clear positive / negative markers, facilitating the differentiation between CLL and MCL, FL, and MZL. Second, monoclonal antibodies exhibit minimal batch-to-batch variation and stable affinity and brightness, promoting comparability and quality control across batches, instruments, and laboratories, meeting the standardization requirements of in vitro diagnostic products. Third, monoclonal antibodies provide more uniform signals and stable fluorescence intensity distribution. Combined with predetermined dye allocation, this helps maintain the long-term stability of the compensation matrix and gating strategy, reducing operator subjectivity. Fourth, using polyclonal antibodies for light chains maximizes sensitivity, while using monoclonal antibodies for other subtyping and prognostic key molecules ensures specificity and resolution, achieving optimal overall panel performance. Fifth, the higher detection rate of light chain restriction and clearer phenotypic definition directly improve the diagnostic accuracy of atypical CLL and gray zone cases, reducing the need for repeated testing and additional molecular tests.

[0055] Furthermore, the fluorescent labeling order of the anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody and anti-CD45 antibody in the first group of antibodies is FITC, PE, APC, PE-CY7, BV421 and PerCP, respectively.

[0056] The fluorescent labeling order of the anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody in the second group of antibodies is PE, APC-Cy7, BV421, and PerCP, respectively.

[0057] The fluorescent labeling order of the anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody in the third group of antibodies is FITC, PE, APC, BV421, and PerCP, respectively.

[0058] The fluorescent labeling order of the anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody and anti-CD45 antibody in the fourth group of antibodies is FITC, PE, PE-CY7, APC and PerCP, respectively.

[0059] The fluorescent labeling order of the anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody in the fifth group of antibodies is PE, APC, BV421, and PerCP, respectively.

[0060] The fluorescent labeling order of the anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody in the sixth group of antibodies is PE, BV421, and PerCP, respectively.

[0061] Furthermore, in the first antibody composition, the volume ratio of anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:3:2:5;

[0062] In the second antibody composition, the volume ratio of anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:3:2:5;

[0063] In the third antibody composition, the volume ratio of anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:2:5;

[0064] In the fourth antibody composition, the volume ratio of anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:3:5;

[0065] In the fifth antibody composition, the volume ratio of anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:3:2:5;

[0066] In the sixth antibody composition, the volume ratio of anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody is 5:2:5.

[0067] Secondly, the present invention also provides the application of the antibody composition described above in the preparation of flow cytometry samples for immunophenotyping detection of chronic lymphocytic leukemia.

[0068] Thirdly, the present invention also provides the application of the antibody composition described herein in the preparation of an immunophenotyping detection product for chronic lymphocytic leukemia.

[0069] Fourthly, the present invention also provides a reagent kit, comprising a first container, a second container, a third container, a fourth container, a fifth container, and a sixth container;

[0070] The first container contains the first group of antibodies, the second container contains the second group of antibodies, the third container contains the third group of antibodies, the fourth container contains the fourth group of antibodies, the fifth container contains the fifth group of antibodies, and the sixth container contains the sixth group of antibodies.

[0071] Furthermore, it also includes other containers for holding hemolysin and PBS buffer, respectively.

[0072] Furthermore, it also includes flow cytometry tubes used with flow cytometers.

[0073] In some embodiments, the detection steps are as follows: (1) Prepare six flow cytometry tubes A, B, C, D, E and F, and adjust the concentration of the peripheral blood or cerebrospinal fluid sample to be tested to 1×10⁻⁶. 6 -1×10 7 / mL, and add it to four test tubes A, B, C, and E. The amount added is 70-100μL, and the maximum amount should not exceed 300μL.

[0074] (2) After washing the sample three times with PBS, add it to tubes D and F at a concentration of 1×10⁻⁶. 6 -1×10 7 / mL, the amount added is 70-100μL, and the maximum should not exceed 300μL.

[0075] (3) Add the antibody composition to each flow cytometry tube and mix thoroughly. Incubate at room temperature in the dark for 10-30 min. The amount of antibody added to each flow cytometry tube is 23-60 μL for the first group, 15-55 μL for the second group, 20-55 μL for the third group, 21-45 μL for the fourth group, 15-35 μL for the fifth group, and 12-30 μL for the sixth group. The volume ratio of each antibody in each group is as described above.

[0076] (4) Add 1-2 mL of hemolysin to each incubated test tube, mix well, and let stand at room temperature in the dark for 8-10 min; centrifuge at 300 g and wash for 5 min, then discard the supernatant.

[0077] (5) Add 1-2 mL of PBS wash buffer to each flow cytometry tube from step (4), centrifuge at 1500-2000 rpm for 5 min, and discard the supernatant. Resuspend the cells in 0.5 mL of PBS buffer;

[0078] (6) Perform flow cytometry analysis on the resuspended cells.

[0079] Example 1 Reagent Information

[0080] This invention provides a fluorescently labeled antibody composition, comprising a first group of antibodies, a second group of antibodies, a third group of antibodies, a fourth group of antibodies, a fifth group of antibodies, and a sixth group of antibodies. The first group of antibodies includes anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody, and anti-CD45 antibody; the second group of antibodies includes anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody; the third group of antibodies includes anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody; the fourth group of antibodies includes anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody, and anti-CD45 antibody; the fifth group of antibodies includes anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody; and the sixth group of antibodies includes anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody.

[0081] The fluorescein labeling order of the anti-FMC-7, anti-CD23, anti-CD5, anti-CD10, anti-CD19, and anti-CD45 antibodies in the first group is FITC, PE, APC, PE-CY7, BV421, and PerCP, respectively. The fluorescein labeling order of the anti-CD81, anti-CD20, anti-CD19, and anti-CD45 antibodies in the second group is PE, APC-Cy7, BV421, and PerCP, respectively. The fluorescein labeling order of the anti-CD79b, anti-CD22, anti-CD200, anti-CD19, and anti-CD45 antibodies in the third group is FITC, PE, APC-CY7, BV421, and PerCP, respectively. The fluorescent labeling order of the fourth group of antibodies (anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody, and anti-CD45 antibody) is FITC, PE, PE-CY7, APC, and PerCP, respectively; the fluorescent labeling order of the fifth group of antibodies (anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody) is PE, APC, BV421, and PerCP, respectively; the fluorescent labeling order of the sixth group of antibodies (anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody) is PE, BV421, and PerCP, respectively.

[0082] These fluorescently labeled antibodies can be purchased directly through public channels; see Table 1 for specific sources.

[0083] Table 1

[0084]

[0085] Example 2 Sample Processing

[0086] Bone marrow or peripheral blood samples stored in heparin or EDTA anticoagulant tubes were adjusted to a cell density of 1-5 × 10⁻⁶ cells / mL. 6 / mL, then filtered through a 200-mesh industrial sieve to remove clumps of material from the sample, ensuring a single-cell suspension, and stored at 2-8℃ to obtain the processed sample.

[0087] Example 3 Sample typing detection

[0088] (1) Prepare six flow cytometry tubes A, B, C, D, E, and F, and adjust the concentration of peripheral blood sample to be tested to 1×10⁻⁶. 6 / mL, and added to four test tubes A, B, C, and E, with an addition volume of 100μL.

[0089] (2) After washing the peripheral blood sample three times with PBS, add it to tubes D and F at a concentration of 1×10⁻⁶. 6 / mL, the amount added is 100μL.

[0090] (3) Add the antibody composition to each flow cytometry tube and mix thoroughly. Incubate at room temperature in the dark for 15 min. The amount of antibody added to each flow cytometry tube is 25 μL for the first group, 25 μL for the second group, 25 μL for the third group, 25 μL for the fourth group, 25 μL for the fifth group, and 25 μL for the sixth group. The volume ratio of each antibody in each group is as described above.

[0091] (4) Add 2 mL of hemolysin to each incubated test tube, mix well, and let stand at room temperature in the dark for 8-10 min; centrifuge at 300 g and wash for 5 min, then discard the supernatant.

[0092] (5) Add 2 mL of PBS wash buffer to each flow cytometry tube from step (4), centrifuge at 2000 rpm for 5 min, and discard the supernatant. Resuspend the cells in 0.5 mL of PBS buffer;

[0093] (6) Perform flow cytometry analysis on the resuspended cells.

[0094] When resuspending cells for flow cytometry analysis, tube A is gated as follows: using scatter plots of FSC / SSC, FSC-A / FSC-H, and CD45 / SSC, single viable cells are circled and adherent cells are removed. All leukocyte populations are circled using CD45 / SSC. Lymphocytes are then circled using CD45 / SSC among all leukocyte gates. CD19+ lymphocytes are then circled using CD45 / CD19. The marker combinations formed by FMC-7 / CD23, CD5 / CD10, and CD5 / CD19 within CD19+ lymphocytes are observed to detect CD19+ lymphocytes.

[0095] The gates in tube B are set up as follows: using scatter plots of FSC / SSC, FSC-A / FSC-H, and CD45 / SSC, single live cells are circled and adherent cells are removed. All white blood cell populations are circled using CD45 / SSC. Lymphocytes are then circled using CD45 / SSC among all white blood cell gates. CD20+ lymphocytes are then circled using CD45 / CD20. The marker combination formed by CD81 / CD20 within CD20+ lymphocytes is observed to detect CD19+ lymphocytes.

[0096] C-tube is gated as follows: using scatter plots of FSC / SSC, FSC-A / FSC-H, and CD45 / SSC, single live cells are circled and adherent cells are removed. All leukocyte populations are circled using CD45 / SSC. Lymphocytes are then gated using CD45 / SSC among all leukocytes. CD19+ lymphocytes are then gated using CD45 / CD19. The marker combinations formed by CD79b / CD19, CD19 / CD200, and CD19 / CD22 within CD19+ lymphocytes are observed to detect CD19+ lymphocytes.

[0097] The D tube is gated as follows: using scatter plots of FSC / SSC, FSC-A / FSC-H, and CD45 / SSC, single live cells are circled and adherent cells are removed. All white blood cell populations are circled using CD45 / SSC. Lymphocytes are then gated using CD45 / SSC among all white blood cell gates. CD19+ lymphocytes are then gated using CD45 / CD19. The marker combination formed by Kappa / Lambda and CD19 / CD38 within CD19+ lymphocytes is observed to detect CD19+ lymphocytes.

[0098] The E tube is gated as follows: using scatter plots of FSC / SSC, FSC-A / FSC-H, and CD45 / SSC, single live cells are circled and adherent cells are removed. All leukocyte populations are circled using CD45 / SSC. Lymphocytes are then gated using CD45 / SSC among all leukocytes. CD19+ lymphocytes are then gated using CD45 / CD19. The marker combinations formed by CD43 / CD19 and CD19 / CD49d within CD19+ lymphocytes are observed to detect CD19+ lymphocytes.

[0099] The F-tube is gated as follows: using scatter plots of FSC / SSC, FSC-A / FSC-H, and CD45 / SSC, single live cells are circled and adherent cells are removed. All leukocyte populations are circled using CD45 / SSC. Lymphocytes are then circled using CD45 / SSC among all leukocyte gates. CD19+ lymphocytes are then circled using CD45 / CD19. The marker combination formed by sIgM / CD19 within CD19+ lymphocytes is observed to detect CD19+ lymphocytes.

[0100] Based on the attached diagram, the conclusion is: B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma (B-CLL / SLL).

[0101] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0102] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody composition for distinguishing atypical CLL and other B-CLPDs, characterized in that, include: The first group of antibodies includes anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody, and anti-CD45 antibody; The second group of antibodies includes anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody; The third group of antibodies includes anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody; The fourth group of antibodies includes anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody, and anti-CD45 antibody; The fifth group of antibodies includes anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody; and, The sixth group of antibodies includes anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody.

2. The antibody composition for distinguishing atypical CLL and other B-CLPDs according to claim 1, characterized in that, Of the six groups of antibodies, the anti-membrane Kappa antibody and the anti-membrane Lambda antibody are polyclonal antibodies, while the remaining antibodies are monoclonal antibodies.

3. The antibody composition for distinguishing atypical CLL and other B-CLPDs according to claim 1, characterized in that, The fluorescent labeling order of the anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody and anti-CD45 antibody in the first group of antibodies is FITC, PE, APC, PE-CY7, BV421 and PerCP, respectively. The fluorescent labeling order of the anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody, and anti-CD45 antibody in the second group of antibodies is PE, APC-Cy7, BV421, and PerCP, respectively. The fluorescent labeling order of the anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody, and anti-CD45 antibody in the third group of antibodies is FITC, PE, APC, BV421, and PerCP, respectively. The fluorescent labeling order of the anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody and anti-CD45 antibody in the fourth group of antibodies is FITC, PE, PE-CY7, APC and PerCP, respectively. The fluorescent labeling order of the anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody, and anti-CD45 antibody in the fifth group of antibodies is PE, APC, BV421, and PerCP, respectively. The fluorescent labeling order of the anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody in the sixth group of antibodies is PE, BV421, and PerCP, respectively.

4. The antibody composition for distinguishing atypical CLL and other B-CLPDs according to claim 1, characterized in that, In the first antibody composition, the volume ratio of anti-FMC-7 antibody, anti-CD23 antibody, anti-CD5 antibody, anti-CD10 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:3:2:5; In the second antibody composition, the volume ratio of anti-CD81 antibody, anti-CD20 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:3:2:5; In the third antibody composition, the volume ratio of anti-CD79b antibody, anti-CD22 antibody, anti-CD200 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:2:5; In the fourth antibody composition, the volume ratio of anti-membrane Kappa antibody, anti-membrane Lambda antibody, anti-CD38 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:5:3:3:5; In the fifth antibody composition, the volume ratio of anti-CD49d antibody, anti-CD43 antibody, anti-CD19 antibody and anti-CD45 antibody is 5:3:2:5; In the sixth antibody composition, the volume ratio of anti-sIgM antibody, anti-CD19 antibody, and anti-CD45 antibody is 5:2:

5.

5. The use of the antibody composition according to any one of claims 1-4 in the preparation of flow cytometry samples for immunophenotyping of chronic lymphocytic leukemia.

6. The use of the antibody composition according to any one of claims 1-4 in the preparation of an immunophenotyping assay for chronic lymphocytic leukemia.

7. A kit for differentiating atypical CLL and other B-CLPDs, characterized in that, This includes a first container, a second container, a third container, a fourth container, a fifth container, and a sixth container; The first container contains the first group of antibodies according to any one of claims 1-4, the second container contains the second group of antibodies according to any one of claims 1-4, the third container contains the third group of antibodies according to any one of claims 1-4, the fourth container contains the fourth group of antibodies according to any one of claims 1-4, the fifth container contains the fifth group of antibodies according to any one of claims 1-4, and the sixth container contains the sixth group of antibodies according to any one of claims 1-4.

8. The kit for differentiating atypical CLL and other B-CLPDs according to claim 7, characterized in that, It also includes other containers for holding hemolysin and PBS buffer, respectively.

Images