Method for detecting a cell surface antigen and use thereof

Abstract

The application discloses a detection method of cell surface antigens and application thereof, and relates to the detection field. The detection method of cell surface antigens comprises the following steps: mixing liposome-treated cells to be detected with capture microspheres and fluorescent microspheres, detecting the fluorescence intensity, and analyzing the expression of cell surface antigens. The surface of the capture microspheres is coated with a first antibody against a specific antigen on the surface of target cells. The surface of the fluorescent microspheres is coated with a second antibody against a target antigen on the surface of target cells. The detection method has low cost and can be used for directly quantitatively and qualitatively analyzing the expression of surface antigens of specific types of cells.

Description

Technical Field

[0001] This invention relates to the field of detection, and in particular to a method for detecting cell surface antigens and its application. Background Technology

[0002] Dendritic cells are among the most important cells in the immune system, functioning throughout the early stages of pathogen recognition and later acquired immune activation, reflecting the immunogenicity of vaccines early on. Related techniques often employ flow cytometry to separate specific lymphocyte populations from whole blood samples or human peripheral blood mononuclear cells (PBMCs) for analysis and detection of cell surface antigens. The principle is as follows: fluorescently labeled or stained single-cell suspensions are pumped into a flow chamber filled with sheath fluid by a peristaltic pump. Enclosed by the sheath fluid and subject to relative motion, single-cell droplets are uniformly arranged and ejected. When these droplets are charged with positive and negative charges, they change direction under a high-voltage electric field and fall into their respective collection containers, thus achieving cell sorting. However, this method suffers from high equipment costs. Furthermore, it can only sort and collect specific cell types for further experiments, and cannot perform fluorescent labeling and quantitative analysis of cell surface markers. Further cell processing, fluorescent labeling, and testing using other immunological platforms are still required, making the process complex and time-consuming. Furthermore, since dendritic cells are present in relatively small amounts in the blood, and current flow cytometry instruments are expensive and cannot analyze the early immune response of dendritic cells in the blood of vaccinated individuals.

[0003] Therefore, there is a need for a low-cost method that facilitates the analysis of surface antigen expression in specific cell types. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for detecting cell surface antigens that is low in cost and can be used for direct quantitative and qualitative analysis of the expression of surface antigens in specific cell types.

[0005] The present invention also provides a kit for detecting cell surface antigens.

[0006] The present invention also provides applications of the above-described detection method or kit.

[0007] A method for detecting cell surface antigens according to a first aspect of the present invention includes the following steps: mixing liposome-modified test cells with capture microspheres and fluorescent microspheres, detecting fluorescence intensity, and analyzing cell surface antigen expression.

[0008] The surface of the capture microspheres is coated with a first antibody against a target cell surface-specific antigen.

[0009] The surface of the fluorescent microspheres is coated with a second antibody against the target antigen on the surface of the target cells.

[0010] The detection method according to embodiments of the present invention has at least the following beneficial effects:

[0011] The detection method described in this embodiment combines liposome technology, flow cytometry, and fluorescence immunoassay. It employs a double-antibody sandwich method to achieve fluorescent labeling of target cells, enabling direct detection of the expression of markers such as target cell surface antigens in samples (e.g., whole blood samples) without separation and purification. This method offers advantages such as high sensitivity and short experimental time. Compared to flow cytometry sorting technology, this detection method only requires a single fluorescence channel from a commonly used single-laser four-channel flow cytometer, reducing instrument costs by tens of times.

[0012] When used to detect dendritic cells and other cells in whole blood samples, this detection method can achieve highly sensitive and rapid determination of various markers on the surface of dendritic cells without separation using a flow cytometer, which is a standard laboratory equipment. This is of great significance for studying the immune response of dendritic cells in systems vaccinology and revealing the progress of the immune response of the entire immune system. It can also rapidly quantify the immunogenicity of vaccines in the short term after vaccination.

[0013] According to some embodiments of the present invention, the test cells may be cells from whole blood.

[0014] According to some embodiments of the present invention, the test cells may include dendritic cells. The target cell surface-specific antigen is a marker with strong target cell surface specificity, and the first antibody can specifically enrich the target cells. For example, the target cells are dendritic cells, and the target cell surface-specific antigen may be CD209. The second antibody is used to analyze the expression of the target cell surface antigen. For example, the target cells are dendritic cells, and the target cell surface antigen may be CD180 or CD169. CD180 can be used to assess the Toll-like receptor signaling pathway, and CD169 can be used to assess the interferon-induced immune response.

[0015] According to some embodiments of the present invention, the liposome-treated test cells refer to test cells processed to the size of liposomes.

[0016] According to some embodiments of the present invention, the ratio of the number of target cells in the liposomed test cells to the number of the capture microspheres and the fluorescent microspheres is (5-15):1:(4000-6000).

[0017] According to some embodiments of the present invention, the method for preparing the liposome-mediated test cells includes the following steps:

[0018] The liposome-based test cells were prepared by extrusion technology from a solution containing the test cells.

[0019] According to some embodiments of the present invention, the extrusion technology can be specifically implemented using a liposome extruder.

[0020] According to some embodiments of the present invention, the solution containing the test cells further includes a protease inhibitor. The concentration of the protease inhibitor is 0.5% to 2%.

[0021] According to some embodiments of the present invention, the protease inhibitor includes at least one of trypsin inhibitor, pepsin inhibitor, leucine inhibitor peptide, ethylenediaminetetraacetic acid (EDTA), and benzyl sulfonyl fluoride (PMSF).

[0022] According to some embodiments of the present invention, the cell density of the solution containing the cells to be tested is 1000 cells / μL to 3000 cells / μL.

[0023] According to some embodiments of the present invention, the diameter of the liposomalized test cells is 200 nm to 500 nm. After liposomalization, the resulting liposomalized cells, with a diameter of several hundred nanometers, exhibit significantly higher antigen-antibody binding efficiency than cells with diameters of several micrometers or even tens of micrometers, resulting in stronger antigen-antibody reactivity.

[0024] According to some embodiments of the present invention, the first antibody and the second antibody each independently comprise at least one of monoclonal antibodies and polyclonal antibodies. It is understood that any antibody capable of specifically binding to the target cell surface antigen is acceptable.

[0025] According to some embodiments of the present invention, the particle size of the captured microspheres is 3 μm to 10 μm.

[0026] According to some embodiments of the present invention, the particle size of the fluorescent microspheres is 100 nm to 500 nm.

[0027] According to some embodiments of the present invention, the fluorescein in the fluorescent microspheres is selected from fluorescein isothiocyanate (FITC), Alexa Fluor 488, phycoerythrin (PE), PE-Cy5, PE-Cy5.5, PE-Cy7, PerCP-Cy5.5, APC-Cy7, propidium iodide (PI) / 7-aminoactinomycin D (7-AAD), allophycocyanin (APC), Cy5, and Alexa Fluor 647.

[0028] According to some embodiments of the present invention, the analytical basis of the detection method is that the higher the fluorescence intensity, the higher the expression level of the target antigen on the surface of the liposomed test cell.

[0029] According to some embodiments of the present invention, the materials of the capturing microspheres and the fluorescent microspheres are each independently selected from at least one of polystyrene and silica.

[0030] According to some embodiments of the present invention, the method for preparing the captured microspheres includes the following steps:

[0031] A1. Activate the carboxylated microspheres;

[0032] A2. React the activated carboxylated microspheres from step A1 with the first antibody to couple the first antibody to the activated carboxylated microspheres.

[0033] A3. Close the enclosure to obtain the captured microspheres.

[0034] According to some embodiments of the present invention, the temperature at which the activated carboxylated microspheres are reacted with the first antibody is 2°C to 6°C.

[0035] According to some embodiments of the present invention, the method for preparing the fluorescent microspheres includes the following steps:

[0036] B1. Activate the carboxylated fluorescent microspheres;

[0037] B2. React the activated carboxylated fluorescent microspheres from step B1 with the second antibody to couple the second antibody onto the activated carboxylated fluorescent microspheres.

[0038] B3. Seal the microspheres to obtain the fluorescent microspheres.

[0039] According to some embodiments of the present invention, the temperature at which the activated carboxylated fluorescent microspheres are reacted with the second antibody is 2°C to 6°C.

[0040] According to some embodiments of the present invention, step A1 or B1 may specifically include the following steps:

[0041] Prepare a buffer solution containing the carboxylated microspheres or the carboxylated fluorescent microspheres and an activator, and react at room temperature;

[0042] The activators include carbodiimide (EDAC) and N-hydroxysuccinimide (NHS).

[0043] According to some embodiments of the present invention, the amounts of the EDAC and the NHS are 8,000 to 20,000 times the number of moles of carboxyl groups contained in the carboxylated microspheres or the carboxylated fluorescent microspheres, respectively.

[0044] According to some embodiments of the present invention, the blocking in step A3 or B3 refers to blocking the microspheres after antibody conjugation by adding a blocking solution. The blocking solution comprises: 40mM–60mM Tris, 0.8%–1% NaCl, 0.03%–0.07% PC300, 0.3%–0.7% BSA, and 0.03%–0.07% Tween 20; the pH of the blocking solution is 7–8.

[0045] A kit for detecting cell surface antigens according to a second aspect of the present invention, the kit comprising:

[0046] Capture microspheres, the surface of which is coated with a first antibody against a target cell surface-specific antigen;

[0047] Fluorescent microspheres, wherein the surface of the fluorescent microspheres is coated with a secondary antibody against the target antigen on the surface of the target cells.

[0048] According to some embodiments of the present invention, the method of using the detection kit is as shown in the method for detecting cell surface antigens in the embodiments of the first aspect described above.

[0049] Application of the above-described detection method according to a third aspect of the present invention in biomedicine.

[0050] The specific applications may include vaccine immunogenicity analysis.

[0051] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0052] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0053] Figure 1 This is a schematic diagram of the detection process of the present invention;

[0054] Figure 2 This is a schematic diagram of the detection principle of the present invention;

[0055] Figure 3 This is a graph showing the particle size detection results of liposome-mediated cells in Example 1 of the present invention;

[0056] Figure 4 This is a diagram showing the detection results of THP-1 cells and iDC cells using the method of Embodiment 1 of the present invention;

[0057] Figure 5 This is a diagram showing the detection results of THP-1 cells, iDC cells, and mDC cells using the method of Embodiment 1 of the present invention. Detailed Implementation

[0058] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0059] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0060] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0061] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0062] Unless otherwise specified, "about" in this invention means that the allowable error is within ±10%.

[0063] Unless otherwise specified, "room temperature" in this invention refers to 25℃±5℃.

[0064] The formulation of solution A used in the following examples is: 0.1M MES, 0.9% NaCl, pH 5.0 (solvent is water).

[0065] Solution B is formulated as follows: 0.1M PB, 0.05% PC300, pH 8.0 (solvent is water).

[0066] Solution C is formulated as follows: 0.1M PB, 5% BSA, 0.05% PC300, pH 8.0 (solvent is water).

[0067] The formulation of solution D is: 1M glycine, pH 8.0 (solvent is water).

[0068] Solution E is formulated as follows: 50 mM Tris, 0.9% NaCl, 0.05% PC300, 0.5% BSA, 0.05% Tween 20, pH 7.5 (solvent is water).

[0069] The preparation method of the captured microspheres consists of three steps: activation, coating, and blocking, as detailed below:

[0070] 1) Activation:

[0071] ① Add 9 times the volume of solution A to the original microsphere solution, centrifuge at 3000×g at room temperature for 5 min, and discard the supernatant.

[0072] The microspheres in the microsphere stock solution are carboxylated polystyrene microspheres with a diameter of 5 μm (standard particles for flow cytometry), provided by Weidu Bio.

[0073] ② Add an equal volume of solution A from step ① to the microspheres, vortex until fully resuspended, centrifuge at 3000×g at room temperature for 5 min, and discard the supernatant.

[0074] ③ Add solution A (containing approximately 10,000 times the total number of carboxyl groups of the microspheres) in an amount equal to that in step ① to the microspheres, vortex for 1 min to mix thoroughly, open the lid and let stand for 1 min, seal, and rotate and shake at room temperature for 40 min to obtain the activated microsphere solution.

[0075] 2) Wrapped in blankets:

[0076] ① Centrifuge the activated microsphere solution from step 1) at 4℃ and 5500×g for 5 min, and discard the supernatant;

[0077] ② Add solution B to the microspheres in the same volume as the microsphere solution in step ①, centrifuge at 4℃ and 5500×g for 5 min, and discard the supernatant. Repeat three times.

[0078] ③ Add solution B (containing antibody with 4 times the surface saturation constant of the microspheres) to the microspheres in step ①, vortex for 1 min to mix thoroughly, and then invert and shake at 4℃ for 24 h to obtain the coated microsphere solution.

[0079] 3) Enclosure:

[0080] ① Centrifuge the coated microsphere solution from step 2) at 4℃ and 5500×g for 5 min, and discard the supernatant;

[0081] ② Add an appropriate amount of a mixture of solution C and solution D (by volume, solution C: solution D = 20:1), mix well for 1 min, and then invert and shake at 4℃ for 24 h.

[0082] ③ Centrifuge the microsphere solution obtained in step ② at 4℃ and 5500×g for 5 min, and discard the supernatant; add solution E (volume the same as the microsphere solution obtained in step ②) to the microspheres. Repeat 3 times to obtain the concentrated microsphere capture solution, which can be stored at 2℃~8℃ for later use.

[0083] The preparation method of fluorescent microspheres is basically the same as that of capture microspheres, except that the inversion and shaking reaction in step 2) is "inverted and shaken at room temperature for 2 hours". The microsphere stock solution used for preparing fluorescent microspheres contains FITC-containing carboxylated polystyrene microspheres (FG0300CA) with a diameter of 300 nm, a maximum excitation light of 488 nm, and a maximum emission light of 520 nm, provided by Weidu Bio.

[0084] In the following examples, the cells used for detection are:

[0085] (1) THP-1 cells (provided by Kunming Cell Bank, Chinese Academy of Sciences);

[0086] (2) Immature dendritic cells (iDCs) are obtained as follows:

[0087] After centrifuging the exponentially growing THP-1 cells, they were resuspended in RPMI 1640 induction medium (containing 10% FBS, recombinant human IL-4 (100 ng / mL = 1500 IU / mL), and recombinant human GM-CSF (100 ng / mL = 1500 IU / mL) to obtain a cell concentration of 5 × 10⁻⁶ cells / mL. 5 Cell suspension of [number] cells / mL. Change the culture medium every 2 days during induction, for a total of 6 days, during which the cell concentration should not exceed 2 × 10⁶ cells / mL. 6 Cells / mL. Centrifuge and collect iDCs.

[0088] (3) The vaccine stimulates mature dendritic cells (mDCs), which are obtained as follows:

[0089] iDCs were resuspended in RPMI 1640 medium and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL. After diluting the YF-17D vaccine (provided by Tiantan Biological Products) 2000 times, iDCs were treated for 24 hours to obtain mDCs.

[0090] Example 1

[0091] This embodiment provides a method for detecting dendritic cell surface markers in whole blood. The detection process and reaction principle are as follows: Figure 1 and Figure 2 As shown, the steps are as follows:

[0092] Take the test cells (THP-1 cells or iDC cells), wash twice with solution C, and then add 1% (v / v) PMSF (0.01M PBS, pH=7.4) to a cell suspension of 2000 cells / μL. Then, extrude the cells through a liposome extruder equipped with an 800nm ​​pore size filter to obtain liposomalized test cells with an average hydrated particle size of 367.8nm, a standard deviation of 137.9nm, and a peak intensity percentage of 82.8% (the particle size detection results of the liposomalized cells are shown in the figure). Figure 3 As shown in the figure. Liposomed test cells were mixed with capture microsphere reagent and fluorescent microsphere reagent at a ratio of 10:1:5000. The fluorescence intensity emitted by the fluorescent microspheres was detected by flow cytometry using a FITC channel and filter parameters 530 / 30 to analyze the expression of markers on the surface of dendritic cells.

[0093] Among them, in the capture microsphere reagent, the surface antibody of the microsphere is an anti-CD209 monoclonal antibody (12-2099-42, Invitrogen); CD209 is the surface marker with the strongest specificity for dendritic cells; in the fluorescent microsphere reagent, the surface antibody of the microsphere is an anti-CD180 antibody.

[0094] Test results as follows Figure 4 As shown.

[0095] THP-1 cells are cells that express low levels of the cell surface antigen CD209. After induction, THP-1 cells differentiate into immature dendritic cells (iDCs), which highly express the CD209 cell surface antigen. The diameter of capture microspheres coated with anti-CD209 monoclonal antibody significantly increased after capturing iDCs. For example... Figure 4 As shown.

[0096] Cells were collected, washed with PBS (0.01M, containing 2% BSA), and then liposomed (prepared by extrusion using a liposome extruder equipped with an 800nm ​​pore size filter membrane) to obtain liposome-modified cells. The liposome-modified cells were fixed with 4% paraformaldehyde at 4°C for 15 minutes. The fixed cells were washed and mixed with anti-CD180 antibody-coated fluorescent microspheres (cells to fluorescent microspheres at a ratio of 1:5000) at 4°C in the dark for 30 minutes. The cells were then washed twice with 0.5 × 10⁻⁶ ppm. 6 ~1×10 6 Cells were resuspended at a density of 10 cells / mL, and the fluorescence intensity of the fluorescent microspheres was detected by flow cytometry using a FITC channel with filter parameters of 530 / 30 to analyze the expression of markers on the surface of dendritic cells. A control group (Blank) was used, consisting of cells without liposomes.

[0097] The cells to be tested are THP-1 cells, iDCs, or mDCs.

[0098] The results are as follows Figure 5 As shown.

[0099] After reaction with fluorescent microspheres coated with anti-CD180 antibody, the FITC channel signaling of THP-1 cells, iDCs, and mDCs was significantly enhanced, especially the FITC channel signaling of mDCs that matured after vaccine stimulation. Figure 5 As shown, the Blank contains a small amount of free fluorescent microspheres, but this does not affect the detection of target cells and their surface antigen CD180 expression levels. This indicates that the fluorescent microspheres can indeed be used to bind to the target cell surface, and the expression level of the target antigen on the cell surface can be characterized by fluorescence intensity. This demonstrates that the double-antibody sandwich method provided by this invention can be used to quantitatively detect protein expression on the surface of target dendritic cells, thereby enabling rapid quantitative analysis of vaccine-induced immunogenicity shortly after vaccination.

[0100] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for detecting a cell surface antigen, characterized by, The method comprises the following steps: mixing the liposome-treated test cells with the capture microspheres and the fluorescent microspheres, detecting the fluorescence intensity, and analyzing the expression of cell surface antigens; the surface of the capture microspheres is coated with a first antibody against a specific antigen on the surface of target cells; the surface of the fluorescent microspheres is coated with a second antibody against a target antigen on the surface of target cells; the liposome-treated test cells refer to test cells treated to be of liposome size; the diameter of the liposome-treated test cells is 200 nm to 500 nm; the number ratio of the liposome-treated test cells to the capture microspheres and the fluorescent microspheres is (5-15):1:(4000-6000); the preparation method of the liposome-treated test cells comprises the following steps: the liposome-treated test cells are prepared by extrusion technology from a solution containing test cells.

2. The detection method according to claim 1, characterized in that, The solution containing test cells also contains a protease inhibitor.

3. The method of claim 1, wherein, The material of the capture microspheres and the fluorescent microspheres is independently selected from at least one of polystyrene and silica.

4. The method of claim 1, wherein The cell density of the solution containing test cells is 1000 cells / μL to 3000 cells / μL.

5. The method of claim 1, wherein The fluorescent material in the fluorescent microspheres is selected from FTIC, Alexa Fluor 488, PE, PE-Cy5, PE-Cy5.5, PE-Cy7, PerCP-Cy5.5, APC-Cy7, PI / 7-AAD, APC, Cy5 and Alexa Fluor 647.

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