Human Fc receptor composite sealant for flow cytometry as well as preparation method and application thereof
By using a combination of phosphorylated oligodeoxynucleotides, human serum, and ProClin 300 as a blocking agent, the problem of non-specific antibody binding during flow cytometry was solved, achieving efficient blocking of anthocyanin-labeled antibodies and reducing false positive results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies in flow cytometry suffer from non-specific binding of antibodies to Fc receptors on the cell surface, leading to increased background signal and false positive results. In particular, the binding of CD64 to anthocyanin-based fluorescent dyes is difficult to inhibit with traditional blocking agents, and the blocking effect of using phosphate thioester-modified oligodeoxynucleotides alone is limited.
A composite blocking agent consisting of phosphorylated oligodeoxynucleotides, human serum, and ProClin 300 was formulated into a 10× human Fc receptor composite blocking agent by adjusting the concentration, which was used to block the non-specific binding of fluorescently labeled antibodies to cells.
It significantly improved the blocking effect on monocyte populations, inhibited the non-specific binding of lymphocyte populations, maintained the specific binding of antibodies to target antigens, and had no cytotoxicity to human leukocytes.
Smart Images
Figure CN121856537A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a human Fc receptor complex blocking agent for flow cytometry, its preparation method and application, which effectively inhibits the non-specific binding of anthocyanin tandem dyes. Background Technology
[0002] Flow cytometry is a single-cell analysis technique widely used in immunology, cell biology, and clinical diagnostics. In this technique, the specific binding of fluorescently labeled antibodies to specific antigens on the cell surface or within the cell is crucial for data acquisition. However, non-specific antibody binding, particularly through the binding of the Fc fragment to Fc receptors on the cell surface, is one of the main causes of increased background signal and false positive results. Fc receptors are expressed on monocytes, macrophages, granulocytes, B cells, and NK cells. Monocytes express various Fcγ receptor molecules such as CD16, CD32, and CD64 on their surface. Fcγ receptors can bind to the Fc structure of IgG antibodies, leading to non-specific antibody binding and thus false positive results. It has been reported that CD64 binds with high affinity to the Fc structure of IgG monomers and also to anthocyanin-based fluorescent dyes, similarly affecting the accuracy of flow cytometry experiments. Furthermore, this binding cannot be inhibited by traditional Fc blocking. Therefore, when using anthocyanin-based fluorescent dyes (such as Pe-Cy5, Pe-cy7, AF647, and Percp-cy5.5) to label antibodies for flow cytometry experiments, it is necessary to block both the non-specific binding of the Fc terminus of IgG to monocytes and the non-specific binding of anthocyanin-based fluorescent dyes to monocytes.
[0003] While there have been reports of using phosphate thioester-modified oligodeoxynucleotides (PS-ODNs) to block the non-specific binding of anthocyanin dyes, the use of PS-ODNs alone has drawbacks, including significant individual variability and limited effectiveness in blocking certain lymphocyte subsets. Therefore, there is an urgent need to develop a more efficient, stable, and universally applicable blocking agent. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a human Fc receptor composite blocking agent for flow cytometry, its preparation method, and its application.
[0005] The present invention adopts the following technical solution: In one aspect, the present invention provides a human Fc receptor complex blocker for flow cytometry, comprising thiophosphorylated oligodeoxynucleotides, human serum, and ProClin 300.
[0006] Preferably, the thiophosphorylated oligodeoxynucleotide is a product obtained by thiophosphorylating three deoxynucleotides at each end of the oligodeoxynucleotide sequence, and its nucleotide sequence is shown in SEQ ID No. 1.
[0007] Preferably, the ProClin 300 has a volume percentage concentration of 0.005% in the working solution; the working concentration of the thiophosphorylated oligodeoxynucleotide is 10 µg / mL; and the human serum has a volume percentage concentration of 5% in the working solution.
[0008] In another aspect, the present invention provides a method for preparing the above-mentioned composite sealant, characterized by comprising the following steps: Step 1): Synthesis and purification of thiophosphorylated oligodeoxynucleotides; Step 2): Dissolve the purified phosphorylated oligodeoxynucleotides in DEPC water, then add human serum and ProClin 300, and adjust the concentration of phosphorylated oligodeoxynucleotides to 100 μg / mL, the volume percentage concentration of human serum to 50%, and the volume percentage concentration of ProClin 300 to 0.05%, respectively, to prepare a 10× human Fc receptor complex blocking agent.
[0009] In another aspect, the present invention provides an application of the above-mentioned composite blocking agent for use in flow cytometry to inhibit the non-specific binding of fluorescently labeled antibodies to cells.
[0010] Preferably, the nonspecific binding is caused by anthocyanin tandem dyes in the fluorescently labeled antibody.
[0011] Preferably, the anthocyanin tandem dye is selected from one or more of PE-Cy5, PE-Cy7, APC-Cy7 or PerCP-Cy5.5.
[0012] This Fc receptor blocking agent exhibits good blocking effect against anthocyanin dye-labeled antibodies, with a blocking effect superior to blocking the non-specific binding of the Fc receptor to the Fc terminus of the antibody alone.
[0013] This Fc receptor blocking agent only blocks the non-specific binding of the Fc receptor to the anthocyanin dye labeled with the antibody and the Fc terminus of the antibody, without affecting the binding of the antibody to its target antigen epitope.
[0014] This Fc receptor blocker is non-cytotoxic to human leukocytes and does not affect the normal cell populations of PBMC lymphocytes and monocytes.
[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The human Fc receptor complex blocking agent provided by this invention significantly improves the blocking effect on monocyte populations when applied to anthocyanin-based dye fluorescently labeled antibodies.
[0016] 2. The human Fc receptor complex blocking agent provided by this invention, when applied to anthocyanin-based dye fluorescently labeled antibodies, also inhibits the appearance of abnormal cell populations caused by non-specific binding of lymphocyte populations. Attached Figure Description
[0017] Figure 1 This is a schematic diagram of the thiophosphorylation modification of deoxynucleotides in this invention; Figure 2 Flow cytometry diagram showing the blocking effect of the human Fc receptor complex blocking agent provided by this invention on anthocyanin dye-labeled isotype control. Figure 3 Flow cytometry diagram showing the blocking effect of the human Fc receptor complex blocking agent provided by this invention on anthocyanin dye-labeled antibodies; Figure 4 This is a flow cytometry diagram showing the comparison of the effects of the PS-ODN and human Fc receptor composite blocking agent of the present invention and the accelerated stability verification. Detailed Implementation
[0018] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the present invention without creative effort are within the protection scope of the present invention.
[0019] This invention provides a human Fc receptor composite blocking agent for flow cytometry, composed of phosphorylated oligodeoxynucleotides, human serum, and ProClin 300. The phosphorylated oligodeoxynucleotides are obtained by phosphorylation of three deoxynucleotides at each end of the oligodeoxynucleotide sequence. This composite blocking agent effectively blocks the non-specific binding of human Fc receptors to anthocyanin-labeled antibodies without affecting the specific binding of antibodies to target antigens. The composite blocking agent provided by this invention has advantages such as synergistic effect, high stability, and ease of operation, and is suitable for multicolor flow cytometry analysis.
[0020] Phosphorylated oligodeoxynucleotides: synthesized and purified by Azenta Life Sciences; human serum: FSU-MIX, ORiCELLS; DEPC-treated water: D1455161, Shanghai Aladdin Biochemical Technology Co., Ltd.; ProClin 300: P406395, Shanghai Aladdin Biochemical Technology Co., Ltd.; phosphate-buffered saline (PBS): T494526, Shanghai Aladdin Biochemical Technology Co., Ltd.; fetal bovine serum (FBS): FND500, ExCell; human PBMCs: XFB-HP025B, SaiLi Biotechnology; other products were all conventional commercially available products. In the embodiments of this invention, unless otherwise specified, the technical means used are all conventional means well known to those skilled in the art.
[0021] Example 1: Preparation of human Fc receptor complex blocking agent The designed and synthesized thiophosphorylated oligodeoxynucleotide sequence is shown in SEQ ID No. 1 (SEQ ID No. 1: 5'-GGGGGAGCATGCTGGGGGGG-3', with 3 deoxynucleotides at each end of the sequence modified by thiophosphorylation, as shown in SEQ ID No. 1). Figure 1 (As shown). The purified phosphorylated oligodeoxynucleotides were first dissolved in DEPC water, and then human serum and ProClin 300 were added to adjust the concentration of phosphorylated oligodeoxynucleotides to 100 μg / mL, the volume percentage concentration of human serum to 50%, and the volume percentage concentration of ProClin 300 to 0.05%, respectively, to prepare a human Fc receptor complex blocking agent (10×).
[0022] Example 2: Flow Cytometry Verification of the Blocking Effect of Human Fc Receptor Complex Blocker The specific steps are as follows: (1) Resuspend resuscitated human PBMCs, centrifuge at 500g for 5 min, wash twice with PBS, discard the supernatant, and repeat at 1×10 7 After resuspending the cells in PBS at a cell / mL density, add 100 μL of cell suspension to each well of a 96-well plate, centrifuge at 500g for 5 min, and discard the supernatant; (2) Add 10 μL of 10× human Fc receptor complex blocking agent and 90 μL of flow buffer (flow buffer is 2% FBS dissolved in PBS) to each well, resuspend and mix the cells, block at 4℃ for 30 min, centrifuge at 500g for 5 min, and discard the supernatant. (3) Add 100 μL of 1 μg / mL fluorescently labeled primary antibody without washing, resuspend and mix the cells, incubate at 4°C in the dark for 1 h, centrifuge at 500g for 5 min, and discard the supernatant; (4) Resuspend the cells in 200 μL of flow buffer at room temperature, wash twice by centrifugation at 500g, add 100 μL of 1 μg / mL DAPI nucleic acid dye to distinguish between dead and live cells, incubate at room temperature in the dark for 10 min, centrifuge at 500g, wash twice with flow buffer, and then analyze by flow cytometry.
[0023] The blocking effect of human Fc receptor complex blocker on anthocyanin PE-cy5 labeled Mouse IgG2c isotype control is as follows: Figure 2 As shown, the human Fc receptor complex blocking agent has a better blocking effect on human PBMC monocytes than the conventional Fc blocking method (5% human serum blocking), and also has a significant blocking effect on lymphocyte populations; in addition, the human Fc receptor complex blocking agent does not affect the population of human PBMC monocytes and lymphocytes.
[0024] The blocking effect of the human Fc receptor complex blocking agent on mouse anti-Human CD3 antibody labeled with anthocyanin dyes PE-cy5 / PE-cy7 / AF647 is as follows: Figure 3 As shown in the figure: the red and black scatter plots represent the cell populations of PBMCs co-stained with Mouse anti-Human CD3 antibody and Mouse anti-Human CD14 antibody when blocking is performed and when not blocking is performed, respectively. CD14 is used to label the monocyte population. The human Fc receptor complex blocking agent blocks the non-specific binding of various anthocyanin dyes to monocytes and does not affect the specific binding of the antibody to the lymphocyte population.
[0025] Accelerated stability results of human Fc receptor complex blocker are as follows: Figure 4 As shown: For blocking with PE-cy5 labeled antibody, the stability of the human Fc receptor complex blocking agent is better than that of PS-ODN alone.
[0026] It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and variations made by those skilled in the art without departing from the spirit and scope of the present invention, based on the disclosed technical content, are equivalent embodiments of the present invention; furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims
1. A human Fc receptor complex blocking agent for flow cytometry, characterized in that, Including thiophosphorylated oligodeoxynucleotides, human serum, and ProClin 300.
2. The composite sealant according to claim 1, characterized in that, The thiophosphorylated oligodeoxynucleotide is the product of thiophosphorylation modification of three deoxynucleotides at each end of the oligodeoxynucleotide sequence, and its nucleotide sequence is shown in SEQ ID No.
1.
3. The composite sealant according to claim 1, characterized in that, The working concentration of the thiophosphorylated oligodeoxynucleotide is 10 µg / mL; the volume percentage concentration of the human serum in the working solution is 5%; and the volume percentage concentration of ProClin 300 in the working solution is 0.005%.
4. A method for preparing a composite sealant as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1): Synthesis and purification of thiophosphorylated oligodeoxynucleotides; Step 2): Dissolve the purified phosphorylated oligodeoxynucleotides in DEPC water, then add human serum and ProClin 300, and adjust the concentration of phosphorylated oligodeoxynucleotides to 100 μg / mL, the volume percentage concentration of human serum to 50%, and the volume percentage concentration of ProClin 300 to 0.05%, respectively, to prepare a 10× human Fc receptor complex blocking agent.
5. The application of a composite sealant as described in any one of claims 1-3, characterized in that, Used to inhibit the non-specific binding of fluorescently labeled antibodies to cells during flow cytometry.
6. The application according to claim 5, characterized in that, The nonspecific binding is caused by anthocyanin tandem dyes in the fluorescently labeled antibody.
7. The application according to claim 6, characterized in that, The anthocyanin tandem dyes are selected from one or more of PE-Cy5, PE-Cy7, APC-Cy7, or PerCP-Cy5.5.