A method for detecting immune killing activity of whole blood without cell separation

Abstract

The application discloses a method for detecting immune killing activity of whole blood without cell separation, and belongs to the technical field of in-vitro immune detection. The method directly adopts fresh peripheral blood anticoagulated by heparin, does not need density gradient centrifugation, immune magnetic bead sorting or any immune cell purification step, and directly obtains the specific killing rate of whole blood immune cells to target cells through cytokine pre-activation, co-culture of fluorescently labeled target cells, apoptosis staining and flow cytometry analysis. The application has the advantages of simple operation, short time consumption, complete cell activity reservation, high physiological correlation, good repeatability and the like, and can truly reflect the comprehensive killing function of whole blood immune cells.

Description

Technical Field

[0001] This invention relates to a method for detecting the immune cytotoxic activity of whole blood without cell separation, belonging to the field of in vitro immunoassay technology. Background Technology

[0002] Chinese patent CN115124627B discloses the in vitro expansion and / or gene modification of NK cells (CAR-NK) and their reinfusion into the patient's body, which has attracted much attention as a new immunotherapy. The tumor microenvironment is an important factor leading to the failure of NK adoptive infusion therapy. It can damage the phenotype, activation, function and persistence of NK cells. The proliferation and anti-tumor activity of NK cells in the tumor are inhibited by various immunosuppressive factors secreted by tumor cells.

[0003] Chinese patent CN119876018B discloses a method for isolating, culturing, activating, and applying NK cells in tumor immunity. The method includes isolating monocytes from peripheral blood of healthy adults, removing non-NK cell populations using negative selection, and enriching and purifying NK cells. Through stimulation with specific cytokines, NK cells are successfully expanded and their cytotoxicity is activated. This method, by optimizing the preparation and activation of NK cells, yields NK cells with highly efficient cytotoxic activity in vitro, and significantly improves the therapeutic effect on tumors when used in combination with immune checkpoint inhibitors.

[0004] Immune killing activity is an important indicator reflecting the functional status of immune cells in the body and is widely used in basic research, immune status evaluation, and intervention effect monitoring. Currently, conventional methods for detecting immune killing activity all require pretreatment of blood samples, including purification steps such as density gradient centrifugation to separate peripheral blood mononuclear cells (PBMCs) and immunomagnetic bead sorting of NK cells.

[0005] The existing technology has obvious flaws: (1) Cell isolation and purification is a complex and time-consuming operation that requires highly skilled operators; (2) The separation process can easily cause activation, loss or damage to the activity of immune cells, affecting the authenticity of the results; (3) It disrupts the natural environment of plasma, cytokines, and intercellular interactions in the blood, resulting in low physiological relevance; (4) The large number of operation steps leads to a large coefficient of variation and poor detection repeatability; (5) The cost of reagents and equipment is high, making it difficult to achieve standardization and large-scale screening.

[0006] Therefore, developing a method that does not require cell separation and can directly use whole blood for the detection of immune killing activity has important application value. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical defects of the existing technology, solve the above-mentioned technical problems, and propose a whole blood immune killing activity detection method without cell separation, so as to achieve simple, rapid, stable and highly physiologically relevant immune killing function detection.

[0008] The present invention specifically adopts the following technical solution: a method for detecting the immune cytotoxic activity of whole blood without cell separation, comprising the following steps: Step SS1: Collect heparin-anticoagulated peripheral blood, do not perform cell separation and purification, and directly add cytokine mixture to pre-activate immune cells; Step SS2: Co-culture the pre-activated whole blood with a fluorescently labeled target cell suspension; Step SS3: Collect the co-cultured cells from step SS2 and stain them with apoptosis dyes; Step SS4: Differentiate target cells and calculate the specific killing rate using flow cytometry.

[0009] In a preferred embodiment, the cytokine mixture in step SS1 includes IL-2, IL-18, and IL-21.

[0010] As a preferred embodiment, step SS1 specifically includes: taking 200 μL of heparin-anticoagulated peripheral blood, adding a mixed solution of IL-2, IL-18, and IL-21 cytokines, gently mixing, and then letting it stand at room temperature for 10–20 minutes to complete the pre-activation.

[0011] In a preferred embodiment, the fluorescently labeled target cells in step SS2 are K562-GFP cells.

[0012] As a preferred embodiment, step SS2 specifically includes: setting up an experimental group and a control group in a multi-well culture plate, adding 100 μL of pre-activated whole blood and 100 μL of fluorescently labeled target cell suspension to the experimental group, and adding 100 μL of complete culture medium and 100 μL of fluorescently labeled target cell suspension to the control group, with replicates set for each group.

[0013] As a preferred embodiment, the co-culture conditions in step SS2 are: 37°C, 5% CO2 for 18–24 hours.

[0014] In a preferred embodiment, the apoptosis dyes in step SS3 include Annexin V-APC and 7-AAD.

[0015] As a preferred embodiment, the specific killing rate in step SS4 is calculated as follows: Specific killing rate (%) = [(experimental group mortality rate - control group mortality rate) / (1 - control group mortality rate)] × 100%; where the mortality rate is the sum of the proportions of early apoptosis, late apoptosis and necrotic cells.

[0016] The beneficial effects achieved by this invention are as follows: First, this invention eliminates the need for density gradient centrifugation, magnetic bead sorting, and immune cell enrichment or purification throughout the entire process, thus preserving the state and microenvironment of immune cells in vivo to the greatest extent possible. Second, this invention eliminates cell separation throughout the entire process, simplifying the operation steps by more than 50%, saving time and effort. Third, this invention does not damage the activity of immune cells, preserving the true immune microenvironment in vivo to the greatest extent possible. Fourth, this invention preserves whole blood plasma, cytokines, and cell interaction networks, significantly improving physiological relevance. Fifth, this invention exhibits low operational variability, stronger repeatability and stability, and is easy to standardize. Sixth, this invention has a wide range of applications and can be used in scenarios such as scientific research screening, immune status assessment, and intervention effect monitoring. Seventh, this invention has low requirements for equipment and cost, making it suitable for routine laboratory and clinical use. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the delineation and evolution of healthy donor 1 in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the delineated evolution of the healthy donor 2 in Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the delineated evolution of the healthy donor 3 in Embodiment 1 of the present invention; Figure 4 This is the FSC / SSC scatter plot of the present invention, used to characterize cell population delineation; Figure 5 This is a GFP / SSC scatter plot of the present invention, used to distinguish GFP-positive target cells; Figure 6 This is a scatter plot of Annexin V-APC / 7-AAD double staining; Figure 7 This is a distribution map of spontaneous apoptosis in target cells of the control group; Figure 8 This is a distribution chart of whole blood immune killing results in the experimental group; Figure 9 This is a graph showing the immune killing results of subject 1; Figure 10 This is a graph showing the immune killing results of subject 2. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0019] Materials and reagents: Heparin-anticoagulated fresh human peripheral blood; K562-GFP cells (stable expression of green fluorescent protein); recombinant human IL-2, IL-18, and IL-21; Annexin V-APC and 7-AAD apoptosis detection kits; RPMI 1640 complete culture medium. Flow cytometer, high-speed centrifuge, biosafety cabinet, and CO2 incubator.

[0020] Example 1: Detection of whole blood immune killing activity in healthy individuals (1) Take 200 μL of fresh heparin-anticoagulated whole blood, add IL-2 (final concentration 100 IU / mL), IL-18 (final concentration 100 ng / mL), and IL-21 (final concentration 50 ng / mL), mix well and let stand at room temperature for 15 minutes. (2) Collect K562-GFP cells in the logarithmic growth phase and adjust the concentration to 2×10⁻⁶. 5 cells / mL; (3) Grouping in 96-well plates: Experimental group: 100 μL pre-activated whole blood + 100 μL target cell suspension; Control group: 100 μL complete culture medium + 100 μL target cell suspension. Figure 4 It is an FSC / SSC scatter plot. Figure 5 This is the GFP / SSC scatter plot of the present invention; FSC / SSC are two basic scattering light parameters in flow cytometry: FSC (Forward Scatter): reflects cell size, and the signal intensity is positively correlated with cell volume; SSC (Side Scatter): reflects the complexity of the internal structure of the cell (such as granules, organelles, nuclei, etc.), and the signal intensity increases with the increase of internal granularity or heterogeneity; GFP / SSC usually refers to a two-parameter scatter plot with SSC as the vertical axis and GFP fluorescence intensity as the horizontal axis, used to observe the position of GFP positive cell populations in the SSC (granularity) distribution; (4) Incubate at 37℃ and 5% CO2 for 20 hours; (5) Transfer all cells in the well to a flow cytometer tube, wash with PBS, and centrifuge at 600g for 5 minutes; (6) Discard the supernatant, add 100 μL of 1×Binding Buffer to resuspend, add 5 μL of Annexin V-APC and 5 μL of 7-AAD, and incubate in the dark for 15 minutes; (7) Add 400 μL Binding Buffer and perform flow cytometry analysis. Figure 6 This is a scatter plot of Annexin V-APC / 7-AAD double staining; (8) Delineate GFP-positive target cells, analyze the ratio of apoptosis to necrosis, and calculate the killing rate according to the formula.

[0021] Experimental results: such as Figure 1 , Figure 2 and Figure 3 As shown, the percentages for healthy donor 1, 2, and 3 were 16.43%, 17.85%, and 25.95%, respectively. These results indicate that this method can stably detect whole blood immune cytotoxic activity with good reproducibility. Figure 7 This is a distribution map of spontaneous apoptosis in target cells of the control group; Figure 8 This is a distribution chart of the whole blood immune killing results in the experimental group.

[0022] Example 2: Method Stability Testing When the same healthy donor was tested using this method for three consecutive days, the results were 18.2%, 17.9%, and 18.5%, respectively, with an intra-assay coefficient of variation (CV) of <10%, indicating that the method has high stability and reliability.

[0023] Example 3: Monitoring Changes in Immune Status This method was used to compare the immune killing activity of subjects before and after intervention: such as Figure 9 and Figure 10 As shown, Subject 1: 10.31% before intervention, 22.32% after intervention. Subject 2: 17.23% before intervention, 30.33% after intervention. The results indicate that this method can sensitively reflect changes in immune function and is suitable for dynamic monitoring of immune status. Figure 7 , Figure 8 , Figure 9 and Figure 10 In this context, Yellow-B Fluoresence refers to the yellow fluorescence channel, and Red-B Fluoresence refers to the red fluorescence channel.

[0024] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A method for detecting the immune cytotoxic activity of whole blood without cell separation, characterized in that, Includes the following steps: Step SS1: Collect heparin-anticoagulated peripheral blood, do not perform cell separation and purification, and directly add cytokine mixture to pre-activate immune cells; Step SS2: Co-culture the pre-activated whole blood with a fluorescently labeled target cell suspension; Step SS3: Collect the co-cultured cells from step SS2 and stain them with apoptosis dyes; Step SS4: Differentiate target cells and calculate the specific killing rate using flow cytometry.

2. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 1, characterized in that, The cytokine mixture in step SS1 includes IL-2, IL-18, and IL-21.

3. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 2, characterized in that, The specific steps of SS1 include: taking 200 μL of heparin-anticoagulated peripheral blood, adding a mixed solution of IL-2, IL-18, and IL-21 cytokines, gently mixing, and then letting it stand at room temperature for 10–20 minutes to complete the pre-activation.

4. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 1, characterized in that, The fluorescently labeled target cells in step SS2 are K562-GFP cells.

5. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 4, characterized in that, Step SS2 specifically includes: setting up an experimental group and a control group in a multi-well culture plate. The experimental group is added with 100 μL of pre-activated whole blood and 100 μL of fluorescently labeled target cell suspension, and the control group is added with 100 μL of complete culture medium and 100 μL of fluorescently labeled target cell suspension. Each group is set up with replicates.

6. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 4, characterized in that, The co-culture conditions in step SS2 are: 37℃, 5% CO2 for 18–24 hours.

7. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 1, characterized in that, The apoptosis dyes in step SS3 include Annexin V-APC and 7-AAD.

8. The method for detecting whole blood immune cytotoxic activity without cell separation according to claim 1, characterized in that, The specific killing rate in step SS4 is calculated as follows: Specific killing rate (%) = [(experimental group mortality rate - control group mortality rate) / (1 - control group mortality rate)] × 100%; where the mortality rate is the sum of the proportions of early apoptosis, late apoptosis and necrotic cells.

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