Semen leukocyte population flow quantitative detection reagent, kit and detection method

Abstract

The present invention discloses a flow cytometry quantitative detection reagent, a kit and a detection method for semen leukocyte population. The detection reagent and the kit include: 1) a sample treatment agent: containing Na2HPO4, KH2PO4, and NaCl; 2) a sample diluent: containing Na2HPO4, KH2PO4, NaCl and KCl; 3) a sample separation agent: containing polysucrose and diatrizoate; 4) an antibody: an IgG type monoclonal antibody of anti-human CD45 with a fluorescent marker; 5) a sample fixative: containing paraformaldehyde. The detection method of the present invention is based on the principle of flow cytometry, using a sample treatment agent to remove impurities and non-target cells in semen, and then using a leukocyte CD45 antibody to specifically bind to the surface of leukocytes in semen, and finally using a flow cytometer to detect the specific marker fluorescence, thereby obtaining the number of leukocytes. This method is detected and recorded by a machine, which is more objective and stable than the peroxidase method, and is easy to standardize and has strong specificity; the antibodies used for staining are stable in nature, have a long storage time, and are convenient to use.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, kits and detection methods, and in particular to a flow cytometry quantitative detection reagent, kit and detection method for semen leukocyte population. Background Art

[0002] In recent years, due to the improvement of productivity and the use of a large number of mechanized and automated instruments and equipment, men have gradually become the main force of sedentary life. Reproductive tract infections caused by long-term sitting have become an important cause of male infertility. With the spread of sexually transmitted diseases, the male infertility rate has increased year by year. The requirements for the specificity and accuracy of male reproductive tract infection diagnosis are getting higher and higher, and the calls are getting stronger and stronger. Leukocytospermia is an important cause of male infertility, which refers to the concentration of white blood cells in semen exceeding 1×10 6 / ml, indicating possible infection in the reproductive system.

[0003] There are two commonly used methods for detecting white blood cells in semen. One is the leukocyte peroxidase method recommended by WHO. This method uses peroxidase to stain granulocytes in semen, and after counting the stained cells, the concentration of white blood cells in semen is calculated. This method is relatively simple to detect, but this method can only detect neutrophils, and lymphocytes, monocytes / macrophages cannot be detected. In addition, since the microscopic counting is performed by technicians, there are subjective factors, which will cause data loss and instability of results. The other is the enzyme immunohistochemical staining method, which is also recommended by WHO. This method uses the specific antigen CD45 on the surface of white blood cells to detect white blood cells. Therefore, this method is the most accurate for detecting leukocytospermia, but the color substrate in the existing enzyme immunohistochemical staining reagent is extremely unstable and needs to be prepared and used immediately, which is relatively cumbersome. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a flow cytometry quantitative detection reagent, kit and detection method for semen leukocyte population, which are used to solve the problems of strong subjective factors, non-specificity and cumbersome operation in the prior art quantitative detection method of semen leukocyte population.

[0005] In order to achieve the above-mentioned and other related purposes, the present invention provides a first aspect of a flow cytometry quantitative detection reagent for semen leukocyte population, comprising:

[0006] 1) Sample treatment agent: containing Na2HPO4, KH2PO4, NaCl;

[0007] 2) Sample diluent: containing Na2HPO4, KH2PO4, NaCl and KCl;

[0008] 3) Sample separation agent: containing polysucrose and diatrizoate;

[0009] 4) Antibody: fluorescently labeled anti-human CD45 IgG monoclonal antibody;

[0010] 5) Sample fixative: contains paraformaldehyde.

[0011] Furthermore, the sample treatment agent, sample diluent, sample separation agent, antibody, and sample fixative in the detection reagent are all solutions, including:

[0012] 1) Sample treatment solution: a solution containing Na2HPO4, KH2PO4, and NaCl;

[0013] 2) Sample diluent: a solution containing Na2HPO4, KH2PO4, NaCl and KCl;

[0014] 3) Sample separation solution: a solution containing polysucrose and diatrizoate;

[0015] 4) Antibody solution: a solution of fluorescently labeled anti-human CD45 IgG monoclonal antibody;

[0016] 5) Sample fixative: a solution containing paraformaldehyde.

[0017] Furthermore, the sample treatment solution, sample diluent, sample separation solution, antibody solution, and sample fixative are all prepared with deionized water; preferably, the antibody solution also contains fetal bovine serum (FBS); more preferably, the antibody solution contains 0.01% fetal bovine serum (FBS). The deionized water is EW-I to EW-III grade deionized water.

[0018] Optionally, the concentrations of Na2HPO4, KH2PO4, and NaCl in the sample treatment solution are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, and 7 g / L-9 g / L, respectively.

[0019] Optionally, the concentrations of Na2HPO4, KH2PO4, NaCl and KCl in the sample diluent are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, 7 g / L-9 g / L, and 0.1-0.3 g / L, respectively.

[0020] Optionally, the sample separation solution comprises, by mass percentage: 1-6% diatrizoate, 1-5% polysucrose, 0.3-5% hydrophilic high molecular polymer of non-glycosyl units, and the remainder is deionized water without heat source.

[0021] Optionally, the hydrophilic high molecular polymer of the non-glycosyl unit is selected from at least one of polyethylene glycol, polyvinyl pyrrolidone and polystyrene sulfonic acid.

[0022] Optionally, the concentration of the antibody in the antibody solution is 0.01 μg-0.05 μg / 10 6 cells.

[0023] Optionally, the content of paraformaldehyde in the sample fixative is 0.5%-4%.

[0024] Furthermore, the sample treatment agent, sample diluent, sample separation agent, antibody, and sample fixative in the detection reagent are all lyophilized powders, including:

[0025] 1) Sample treatment agent: freeze-dried powder containing Na2HPO4, KH2PO4, and NaCl;

[0026] 2) Sample diluent: freeze-dried powder containing Na2HPO4, KH2PO4, NaCl and KCl;

[0027] 3) Sample separation agent: lyophilized powder containing polysucrose and diatrizoate;

[0028] 4) Antibody: Lyophilized powder of fluorescently labeled anti-human CD45 IgG monoclonal antibody;

[0029] 5) Sample fixative: freeze-dried powder containing paraformaldehyde.

[0030] Furthermore, when the detection reagent is used to detect the semen leukocyte population, the freeze-dried powder is first reconstituted with deionized water to prepare a sample treatment solution, a sample diluent, a sample separation solution, an antibody solution and a sample fixative solution. The deionized water is EW-I to EW-III grade deionized water.

[0031] Optionally, the concentrations of Na2HPO4, KH2PO4, and NaCl in the sample treatment solution are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, and 7 g / L-9 g / L, respectively.

[0032] Optionally, the concentrations of Na2HPO4, KH2PO4, NaCl and KCl in the sample diluent are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, 7 g / L-9 g / L, and 0.1-0.3 g / L, respectively.

[0033] Optionally, the sample separation solution comprises, by mass percentage: 1-6% diatrizoate, 1-5% polysucrose, 0.3-5% hydrophilic high molecular polymer of non-glycosyl units, and the remainder is deionized water without heat source.

[0034] Optionally, the hydrophilic high molecular polymer of the non-glycosyl unit is selected from at least one of polyethylene glycol, polyvinyl pyrrolidone and polystyrene sulfonic acid.

[0035] Optionally, the concentration of the antibody in the antibody solution is 0.01 μg-0.05 μg / 10 6 cells.

[0036] Optionally, the content of paraformaldehyde in the sample fixative is 0.5%-4%.

[0037] Furthermore, the fluorescently labeled anti-human CD45 IgG monoclonal antibody is derived from rabbits or other commonly used experimental animals in the biochemical field.

[0038] Furthermore, the fluorescent markers are existing fluorescent markers of various colors, such as FITC, PE, APC, and PerCP-cy5.5.

[0039] The second aspect of the present invention provides a kit for quantitative flow cytometry detection of semen leukocyte populations comprising the detection reagent described in the first aspect.

[0040] Furthermore, the kit also includes: 6) washing solution; 7) absolute counting microspheres.

[0041] Optionally, the washing solution is a 0.01 M PBS solution containing 0.01% Tween-20.

[0042] Optionally, the absolute counting microspheres are absolute counting fluorescent microspheres for flow cytometry.

[0043] The third aspect of the present invention provides a method for quantitatively detecting semen leukocyte populations, using the detection reagent as described in the first aspect and / or the detection kit as described in the second aspect, the method comprising the following steps:

[0044] (1) treating the semen sample with a sample treatment agent;

[0045] (2) diluting the semen sample treated with the sample treating agent with a sample diluent;

[0046] (3) Add sample separation agent, centrifuge, observe the stratification, and absorb the intermediate layer liquid;

[0047] (4) Add the washing solution to the middle layer liquid, mix well, centrifuge, and discard the supernatant to obtain the cell pellet;

[0048] (5) Resuspend the cell pellet with antibody solution and incubate for staining;

[0049] (6) fixing the cell-antibody complex in the solution obtained in step (5) with a sample fixative, centrifuging and discarding the supernatant;

[0050] (7) Add sample diluent to resuspend the cells, then add absolute counting microspheres, and finally detect using a flow cytometer to record and calculate the results.

[0051] Furthermore, in step (1), the amount of sample treatment agent used is the same as that of the semen sample.

[0052] Furthermore, in step (1), the semen sample is treated with a sample treatment agent at room temperature for 5-10 minutes.

[0053] Furthermore, in step (2), the dilution factor is 1-3 times.

[0054] Furthermore, in step (3), the centrifugation conditions are: 1200-1800×g, centrifugation for 20-30 min.

[0055] Furthermore, in step (4), 1-3 mL of washing solution is added to each mL of the intermediate layer liquid.

[0056] Furthermore, in step (4), the centrifugation conditions are: 400-800×g, centrifugation for 5-10 min.

[0057] Furthermore, in step (5), the amount of antibody solution required to resuspend each milliliter of cell pellet is 40-60 μL.

[0058] Furthermore, in step (5), the incubation is carried out in a light-proof condition, and the incubation time is 20-30 minutes.

[0059] Furthermore, in step (6), 0.1-0.5 mL of sample fixative is added to each mL of the solution obtained in step (5).

[0060] Furthermore, in step (6), the fixing time is 10-20 minutes.

[0061] Furthermore, in step (6), the centrifugation conditions are: 300-400×g, centrifugation for 5-10 min.

[0062] Furthermore, in step (7), the flow cytometer can detect PE, FITC, APC and PerCP-cy5.5 channels.

[0063] Further, in step (7), the flow cytometry results were analyzed using flow cytometry data analysis software, and CD45 + The number of cells, including CD45 + The cells are in the FITC channel; open the FITC channel and the PerCP-Cy5.5 channel at the same time and set the gate for the beads.

[0064] Furthermore, CD45 + The calculation formula of cells is: A = (X / Y) * (N * V), where A is CD45 + Absolute number of cells, X is CD45 detected by flow cytometry +The number of cells, Y is the number of flow cytometry beads, N is the concentration of beads used for detection, and V is the volume of beads used for detection.

[0065] As described above, compared with the prior art, the semen leukocyte population flow quantitative detection reagent, kit and detection method of the present invention have the following beneficial effects:

[0066] The present invention provides an objective, stable, specific and easy-to-operate flow cytometry detection scheme for semen leukocyte population. The flow cytometry-based detection method uses a sample treatment agent to remove impurities and non-target cells in semen, then uses a leukocyte CD45 antibody to specifically bind to the surface of leukocytes in semen, and finally uses a flow cytometer to detect the fluorescence of specific markers to obtain the number of leukocytes in semen.

[0067] Compared with the existing peroxidase method, the detection reagent, kit and detection method of the present invention are based on the principle of flow cytometry, detected and recorded by machine, without the need for subjective judgment by technicians, more objective and stable, and easy to standardize; the present invention detects specific marker proteins of leukocytes, and compared with the limitation of the existing peroxidase method that can only detect multinuclear granulocytes, the present invention has higher accuracy and stronger specificity; the antibody used for staining of the present invention is stable in nature, while the dye solution of the commonly used enzyme immunohistochemical staining method is extremely unstable and can only be stable for 1 week at 2-8°C, while the antibody used in the present method can be stored for 2-3 years in small doses at -20°C, which greatly improves the storage time limit and is convenient for use.

[0068] In summary, the antibodies used in the detection of the present invention are specific, the reagents used can be prepared in advance and stored for a long time, and instruments are used for detection and recording. Therefore, the method of the present invention is more objective, specific, and stable than the peroxidase method, and more efficient than the enzyme immunohistochemical staining method. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Shown is a graph of the semen leukocyte detection results obtained in Example 13 of the present invention. DETAILED DESCRIPTION

[0070] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0071] The present invention provides a flow cytometry quantitative detection reagent for semen leukocyte population, comprising:

[0072] 1) Sample treatment agent: containing Na2HPO4, KH2PO4, NaCl;

[0073] 2) Sample diluent: containing Na2HPO4, KH2PO4, NaCl and KCl;

[0074] 3) Sample separation agent: containing polysucrose and diatrizoate;

[0075] 4) Antibody: fluorescently labeled anti-human CD45 IgG monoclonal antibody;

[0076] 5) Sample fixative: contains paraformaldehyde.

[0077] The detection reagent provided by the present invention is specifically divided into two types. The first type, in which the sample treatment agent, sample diluent, sample separation agent, antibody, and sample fixative are all solutions, includes:

[0078] 1) Sample treatment solution: a solution containing Na2HPO4, KH2PO4, and NaCl;

[0079] 2) Sample diluent: a solution containing Na2HPO4, KH2PO4, NaCl and KCl;

[0080] 3) Sample separation solution: a solution containing polysucrose and diatrizoate;

[0081] 4) Antibody solution: fluorescently labeled anti-human CD45 IgG monoclonal antibody;

[0082] 5) Sample fixative: a solution containing paraformaldehyde.

[0083] Specifically, the sample treatment solution, sample diluent, sample separation solution, antibody solution, and sample fixative are all prepared with deionized water; preferably, the antibody solution also contains fetal bovine serum FBS, which is conducive to the long-term storage of the reagent; more preferably, the antibody solution contains 0.01% fetal bovine serum FBS. The deionized water is EW-I to EW-III grade deionized water; in the following examples, the deionized water used is EW-III grade deionized water.

[0084] Specifically, the concentrations of Na2HPO4, KH2PO4, and NaCl in the sample treatment solution are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, and 7 g / L-9 g / L, respectively.

[0085] Specifically, the concentrations of Na2HPO4, KH2PO4, NaCl and KCl in the sample diluent are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, 7 g / L-9 g / L, and 0.1-0.3 g / L, respectively.

[0086] Specifically, the sample separation solution includes, by mass percentage, 1-6% diatrizoate, 1-5% polysucrose, 0.3-5% hydrophilic high molecular polymer with non-glycosyl units, and the remainder is deionized water without heat source.

[0087] Specifically, the hydrophilic high molecular polymer of the non-glycosyl unit is at least one selected from polyethylene glycol, polyvinyl pyrrolidone, and polystyrene sulfonic acid.

[0088] Specifically, the concentration of the antibody solution is 0.01 μg-0.05 μg / 10 6 cells.

[0089] Specifically, the content of paraformaldehyde in the sample fixative is 0.5%-4%.

[0090] The sample treatment agent, sample diluent, sample separation agent, antibody, and sample fixative in the second detection reagent are all lyophilized powders, including:

[0091] 1) Sample treatment agent: freeze-dried powder containing Na2HPO4, KH2PO4, and NaCl;

[0092] 2) Sample diluent: freeze-dried powder containing Na2HPO4, KH2PO4, NaCl and KCl;

[0093] 3) Sample separation agent: lyophilized powder containing polysucrose and diatrizoate;

[0094] 4) Antibody: Lyophilized powder of fluorescently labeled anti-human CD45 IgG monoclonal antibody;

[0095] 5) Sample fixative: freeze-dried powder containing paraformaldehyde.

[0096] When the second detection reagent is used, the freeze-dried powder is first reconstituted with deionized water to prepare a sample treatment solution, a sample diluent, a sample separation solution, an antibody solution and a sample fixative; preferably, the antibody solution also contains fetal bovine serum FBS, which is conducive to the long-term storage of the reagent; more preferably, the antibody solution contains 0.01% fetal bovine serum FBS. The deionized water is EW-I to EW-III grade deionized water; in the following examples, the deionized water used is EW-III grade deionized water.

[0097] Specifically, the concentrations of Na2HPO4, KH2PO4, and NaCl in the sample treatment solution are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, and 7 g / L-9 g / L, respectively.

[0098] Specifically, the concentrations of Na2HPO4, KH2PO4, NaCl and KCl in the sample diluent are 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, 7 g / L-9 g / L, and 0.1-0.3 g / L, respectively.

[0099] Specifically, the sample separation solution includes, by mass percentage, 1-6% diatrizoate, 1-5% polysucrose, 0.3-5% hydrophilic high molecular polymer with non-glycosyl units, and the remainder is deionized water without heat source.

[0100] Specifically, the hydrophilic high molecular polymer of the non-glycosyl unit is at least one selected from polyethylene glycol, polyvinyl pyrrolidone, and polystyrene sulfonic acid.

[0101] Specifically, the concentration of the antibody solution is 0.01 μg-0.05 μg / 10 6 cells.

[0102] Specifically, the content of paraformaldehyde in the sample fixative is 0.5%-4%.

[0103] Furthermore, the fluorescently labeled anti-human CD45 IgG monoclonal antibody is derived from rabbits or other commonly used experimental animals in the biochemical field. The fluorescently labeled anti-human CD45 IgG monoclonal antibody used in the following examples is derived from rabbits.

[0104] Furthermore, the fluorescent marker carried by the antibody is any existing fluorescent marker, such as FITC, PE, APC, and PerCP-cy5.5.

[0105] The present invention also provides a flow cytometry quantitative detection kit for semen leukocyte populations comprising the detection reagent described above, wherein the kit further comprises: 6) washing solution; and 7) absolute counting microspheres.

[0106] Specifically, the washing solution is a 0.01M PBS solution containing 0.01% Tween-20.

[0107] Specifically, the absolute counting microspheres are absolute counting fluorescent microspheres for flow cytometry. In the following examples, the absolute counting microspheres used are from Biolegend, precision count beads, cat: 424902, but are not limited thereto.

[0108] Based on the above detection reagent and detection kit, the present invention also provides a method for quantitative detection of semen leukocyte population, comprising the following steps:

[0109] (1) treating the semen sample with a sample treatment agent;

[0110] (2) diluting the semen sample treated with the sample treating agent with a sample diluent;

[0111] (3) Add sample separation agent, centrifuge, observe the stratification, and absorb the intermediate layer liquid;

[0112] (4) Add the washing solution to the middle layer liquid, mix well, centrifuge, and discard the supernatant to obtain the cell pellet;

[0113] (5) Resuspend the cell pellet with antibody solution and incubate for staining;

[0114] (6) fixing the cell-antibody complex in the solution obtained in step (5) with a sample fixative, centrifuging and discarding the supernatant;

[0115] (7) Add sample diluent to resuspend the cells, then add absolute counting microspheres, and finally detect using a flow cytometer to record and calculate the results.

[0116] Specifically, in step (1), the amount of sample treatment agent used is the same as that of the semen sample.

[0117] Specifically, in step (1), the semen sample is treated with a sample treatment agent at room temperature for 5-10 minutes.

[0118] Specifically, in step (2), the dilution factor is 1-3 times.

[0119] Specifically, in step (3), the centrifugation conditions are: 1200-1800×g, centrifugation for 20-30 min.

[0120] Specifically, in step (4), 1-3 mL of washing solution is added to each mL of the intermediate layer liquid.

[0121] Specifically, in step (4), the centrifugation conditions are: 400-800×g, centrifugation for 5-10 min.

[0122] Specifically, in step (5), the amount of antibody solution required to resuspend each milliliter of cell aggregate is 40-60 μL.

[0123] Specifically, in step (5), the incubation is carried out in a light-proof condition, and the incubation time is 20-30 minutes.

[0124] Specifically, in step (6), 0.1-0.5 mL of the fixing solution is added to each mL of the solution obtained in step (5).

[0125] Specifically, in step (6), the fixing time is 10-20 minutes.

[0126] Specifically, in step (6), the centrifugation conditions are: 300-400×g, centrifugation for 5-10 min.

[0127] Specifically, in step (7), the flow cytometry results were analyzed using standard flow cytometry data analysis software, and the CD45 + The number of cells, including CD45 + The cells are in the FITC channel; the FITC channel and the PerCP-Cy5.5 channel are opened at the same time, and the beads are gated. The standard software for flow cytometry data analysis used in the present invention is Flow Jo software.

[0128] Specifically, CD45 + The calculation formula of cells is: A = (X / Y) * (N * V), where A is CD45 + Absolute number of cells, X is CD45 detected by flow cytometry + The number of cells, Y is the number of flow cytometry beads, N is the concentration of beads used for detection, and V is the volume of beads used for detection.

[0129] Specifically, the number of beads used for detection = beads concentration * beads volume used for detection. If the operation steps are strictly followed, taking the precision count beads of biolegend as an example, the number of beads used for detection = (9.84×10 5 particles / mL)×0.02mL=1.968×10 4 .

[0130] The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range according to the description herein, and are not limited to the specific values ​​​​exemplified below.

[0131] Example 1

[0132] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0133] 1) Sample treatment solution: a solution containing Na2HPO4, KH2PO4, and NaCl, with the concentrations of Na2HPO4, KH2PO4, and NaCl being 1.42 g / L, 0.27 g / L, and 8 g / L, respectively;

[0134] 2) Sample diluent: a solution containing Na2HPO4, KH2PO4, NaCl and KCl, with the concentrations of Na2HPO4, KH2PO4, NaCl and KCl being 1.42 g / L, 0.27 g / L, 8 g / L and 0.2 g / L respectively;

[0135] 3) Sample separation solution: a solution containing polysucrose and diatrizoate, which includes, by mass percentage, 5% diatrizoate, 4.3% polysucrose, 1.7% polyethylene glycol, and the balance is deionized water without pyrogen;

[0136] 4) Antibody solution: A fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit was selected as the antibody, and fetal bovine serum (FBS) and the antibody were dissolved in deionized water to prepare an antibody solution. The FBS concentration was 0.01% and the antibody concentration was 0.02 μg / 10 6 cells, the fluorescent label is FITC;

[0137] 5) Sample fixative: a solution containing 0.5% paraformaldehyde.

[0138] The antibody in the above reagent is used to bind to the CD45 antigen on the surface of leukocytes. Because the antibody carries a fluorescent label, it can be detected by flow cytometry.

[0139] Example 2

[0140] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0141] 1) Sample treatment solution: a solution containing Na2HPO4, KH2PO4, and NaCl, with the concentrations of Na2HPO4, KH2PO4, and NaCl being 1.30 g / L, 0.19 g / L, and 7 g / L, respectively;

[0142] 2) Sample diluent: a solution containing Na2HPO4, KH2PO4, NaCl and KCl, with the concentrations of Na2HPO4, KH2PO4, NaCl and KCl being 1.30 g / L, 0.19 g / L, 7 g / L and 0.1 g / L respectively;

[0143] 3) Sample separation solution: a solution containing polysucrose and diatrizoate, which includes, by mass percentage, 4% diatrizoate, 5.0% polysucrose, 1.0% polyvinylpyrrolidone, and the balance is deionized water without a heat source;

[0144] 4) Antibody: A fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit was selected as the antibody. The antibody solution was prepared by dissolving fetal bovine serum (FBS) and the antibody in deionized water. The FBS concentration was 0.01% and the antibody concentration was 0.01 μg / 10 6 cells, the fluorescent label is FITC;

[0145] 5) Sample fixative: a solution containing 1% paraformaldehyde.

[0146] The antibody in the above reagent is used to bind to the CD45 antigen on the surface of leukocytes. Because the antibody carries a fluorescent label, it can be detected by flow cytometry.

[0147] Example 3

[0148] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0149] 1) Sample treatment solution: a solution containing Na2HPO4, KH2PO4, and NaCl, with the concentrations of Na2HPO4, KH2PO4, and NaCl being 1.50 g / L, 0.39 g / L, and 9 g / L, respectively;

[0150] 2) Sample diluent: a solution containing Na2HPO4, KH2PO4, NaCl and KCl, with the concentrations of Na2HPO4, KH2PO4, NaCl and KCl being 1.50 g / L, 0.39 g / L, 9 g / L and 0.3 g / L respectively;

[0151] 3) Sample separation solution: a solution containing polysucrose and diatrizoate, which includes, by mass percentage, 4% diatrizoate, 4.0% polysucrose, 2.0% polystyrene sulfonic acid, and the balance is deionized water without a heat source;

[0152] 4) Antibody: A fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit was selected as the antibody. The antibody solution was prepared by dissolving fetal bovine serum (FBS) and the antibody in deionized water. The FBS concentration was 0.01% and the antibody concentration was 0.05 μg / 10 6 cells, the fluorescent label is PE;

[0153] 5) Sample fixative: a solution containing 2% paraformaldehyde.

[0154] The antibody in the above reagent is used to bind to the CD45 antigen on the surface of leukocytes. Because the antibody carries a fluorescent label, it can be detected by flow cytometry.

[0155] Example 4

[0156] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0157] 1) Sample treatment solution: a solution containing Na2HPO4, KH2PO4, and NaCl, with the concentrations of Na2HPO4, KH2PO4, and NaCl being 1.45 g / L, 0.35 g / L, and 8.5 g / L, respectively;

[0158] 2) Sample diluent: a solution containing Na2HPO4, KH2PO4, NaCl and KCl, with the concentrations of Na2HPO4, KH2PO4, NaCl and KCl being 1.45 g / L, 0.35 g / L, 8.5 g / L and 0.3 g / L respectively;

[0159] 3) Sample separation solution: a solution containing polysucrose and diatrizoate, which includes, by mass percentage, 1% diatrizoate, 1.0% polysucrose, 0.3% polystyrene sulfonic acid, and the balance is deionized water without a heat source;

[0160] 4) Antibody: A fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit was selected as the antibody. The antibody solution was prepared by dissolving fetal bovine serum (FBS) and the antibody in deionized water. The FBS concentration was 0.01% and the antibody concentration was 0.05 μg / 10 6 cells, the fluorescent label is PE;

[0161] 5) Sample fixative: a solution containing 4% paraformaldehyde.

[0162] The antibody in the above reagent is used to bind to the CD45 antigen on the surface of leukocytes. Because the antibody carries a fluorescent label, it can be detected by flow cytometry.

[0163] Example 5

[0164] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0165] 1) Sample treatment agent: freeze-dried powder containing Na2HPO4, KH2PO4, and NaCl;

[0166] 2) Sample diluent: freeze-dried powder containing Na2HPO4, KH2PO4, NaCl and KCl;

[0167] 3) Sample separation agent: lyophilized powder containing polysucrose and diatrizoate;

[0168] 4) Antibody: Lyophilized powder of fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit, with the fluorescent label being FITC;

[0169] 5) Sample fixative: freeze-dried powder containing paraformaldehyde.

[0170] When in use, firstly reconstitute the freeze-dried powder with EW-III grade deionized water to prepare sample treatment solution, sample diluent, sample separation solution, antibody solution and sample fixative solution of the concentration described in Example 1.

[0171] Example 6

[0172] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0173] 1) Sample treatment agent: freeze-dried powder containing Na2HPO4, KH2PO4, and NaCl;

[0174] 2) Sample diluent: freeze-dried powder containing Na2HPO4, KH2PO4, NaCl and KCl;

[0175] 3) Sample separation agent: lyophilized powder containing polysucrose and diatrizoate;

[0176] 4) Antibody: Lyophilized powder of fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit, with the fluorescent label being FITC;

[0177] 5) Sample fixative: freeze-dried powder containing paraformaldehyde.

[0178] When in use, firstly reconstitute the freeze-dried powder with EW-III grade deionized water to prepare sample treatment solution, sample diluent, sample separation solution, antibody solution and sample fixative solution of the concentration described in Example 2.

[0179] Example 7

[0180] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0181] 1) Sample treatment agent: freeze-dried powder containing Na2HPO4, KH2PO4, and NaCl;

[0182] 2) Sample diluent: freeze-dried powder containing Na2HPO4, KH2PO4, NaCl and KCl;

[0183] 3) Sample separation agent: lyophilized powder containing polysucrose and diatrizoate;

[0184] 4) Antibody: Lyophilized powder of fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit, with the fluorescent label being FITC;

[0185] 5) Sample fixative: freeze-dried powder containing paraformaldehyde.

[0186] When in use, firstly reconstitute the freeze-dried powder with EW-III grade deionized water to prepare sample treatment solution, sample diluent, sample separation solution, antibody solution and sample fixative solution of the concentration described in Example 3.

[0187] Example 8

[0188] A reagent for quantitative flow cytometry detection of semen leukocyte population, comprising:

[0189] 1) Sample treatment agent: freeze-dried powder containing Na2HPO4, KH2PO4, and NaCl;

[0190] 2) Sample diluent: freeze-dried powder containing Na2HPO4, KH2PO4, NaCl and KCl;

[0191] 3) Sample separation agent: lyophilized powder containing polysucrose and diatrizoate;

[0192] 4) Antibody: Lyophilized powder of fluorescently labeled anti-human CD45 IgG monoclonal antibody from rabbit, with the fluorescent label being FITC;

[0193] 5) Sample fixative: freeze-dried powder containing paraformaldehyde.

[0194] When in use, firstly reconstitute the freeze-dried powder with EW-III grade deionized water to prepare sample treatment solution, sample diluent, sample separation solution, antibody solution and sample fixative solution of the concentration described in Example 4.

[0195] Example 9

[0196] The detection reagents of this embodiment are the same as those of Example 5. The sample treatment agent, sample diluent, sample separation agent, antibody, and sample fixative are all lyophilized powders. When used, they are first reconstituted with EW-III grade deionized water. The difference is that 0.01% fetal bovine serum FBS is also added to the antibody solution, which is more conducive to the long-term storage of the reagents.

[0197] Example 10

[0198] A kit for quantitative flow cytometry detection of semen leukocyte populations, comprising the detection reagent in Example 1, and further comprising:

[0199] 6) Washing solution: 0.01 M PBS solution containing 0.01% Tween-20.

[0200] 7) Absolute counting microspheres: Absolute counting fluorescent microspheres for flow cytometry, Biolegend, precision count beads, cat: 424902.

[0201] Embodiment 11

[0202] A kit for quantitative flow cytometry detection of semen leukocyte populations, comprising the detection reagent in Example 2, and further comprising:

[0203] 6) Washing solution: 0.01 M PBS solution containing 0.01% Tween-20.

[0204] 7) Absolute counting microspheres: Absolute counting fluorescent microspheres for flow cytometry, Biolegend, precision count beads, cat: 424902.

[0205] Example 12

[0206] A kit for quantitative flow cytometry detection of semen leukocyte populations, comprising the detection reagent in Example 7, and further comprising:

[0207] 6) Washing solution: 0.01 M PBS solution containing 0.01% Tween-20.

[0208] 7) Absolute counting microspheres: Absolute counting fluorescent microspheres for flow cytometry, Biolegend, precision count beads, cat: 424902.

[0209] Example 13

[0210] A method for quantitative detection of semen leukocytes using the kit described in Example 10 comprises the following steps:

[0211] 1. Take 0.2 mL of semen sample into a 1.5 mL centrifuge tube, add 0.2 mL of sample treatment solution, invert 10 times to mix, and incubate at room temperature for 5 minutes.

[0212] 2. Add 0.6 mL of sample diluent and mix by inverting 10 times.

[0213] 3. Aspirate 200 μL of sample separation solution, extend the pipette tip to the bottom of the solution and slowly add the sample separation solution. Note that at this time, you can see that there is a boundary layer between the sample separation solution and the sample to ensure the separation effect; centrifuge at 1500×g for 25 minutes; after centrifugation, observe obvious stratification, aspirate the middle layer cells (middle layer liquid) and add them to a new 1.5mL centrifuge tube with a volume of about 0.2~0.3mL. Be careful not to aspirate the cells at the bottom.

[0214] 4. Add 0.5 mL of washing solution, invert 10 times, mix well, and centrifuge at 500 g for 5 minutes; after the sample is centrifuged, discard the supernatant to obtain the cell pellet.

[0215] During the centrifugation process, the staining antibody can be prepared: calculate the amount of antibody required for the experiment. Taking 10 samples as an example, take 0.5mL of sample diluent, add 10μL of antibody solution, and mix well to obtain the staining antibody solution. The staining amount is 50μL / tube.

[0216] 5. Resuspend the cell pellet obtained by the above treatment in 50 μL of antibody solution to prepare the sample staining tube; incubate all stained samples at room temperature in the dark for 20 minutes.

[0217] Note: During the cell resuspension process, be sure to thoroughly mix by pipetting to ensure the staining effect.

[0218] 6. Resuspend the cells in 0.2 mL of sample fixative and fix for 10 min; centrifuge at 350 × g for 5 min and discard the supernatant.

[0219] At this time, the volume of the cell suspension is about 20 μL. Add 0.2 mL of sample diluent to resuspend the cells. At this time, the volume of the cell suspension is 240 μL.

[0220] 7. Vortex the absolute counting microspheres for 20 seconds to disperse them evenly and pipette about 1.96×10 4 Absolute counting microspheres are added to the cell suspension obtained in step 6, and after being pipetted and mixed, the cells are tested on a flow cytometer (the test can be completed within 24 hours). The flow cytometer is used for the test, and the instrument is required to be able to detect PE and FITC channels.

[0221] 8. Analyze the flow cytometry results using Flow Jo software and calculate CD45 by absolute bead counting. + The number of cells, including CD45 + The cells are in the FITC channel; opening the FITC channel and the PerCP-Cy5.5 channel at the same time can set a gate for the beads.

[0222] CD45 + The cells are calculated as follows:

[0223] CD45 + Absolute number of cells (A) = (CD45 + Number of cells (X) / Number of flow cytometry beads (Y)) * (Concentration of beads used for detection (N) * Volume of beads used for detection (V))

[0224] The number of beads used for detection = beads concentration * volume of beads used for detection. If the operation steps are strictly followed, the number of beads used for detection = (9.84 × 10 5 particles / mL)×0.02mL=1.968×10 4 .

[0225] Figure 1 The results of the semen leukocyte detection obtained in the embodiment of the present invention are shown in FIG. Figure 1 It can be seen that the content of CD45+ leukocytes in all cells is 4.89%.

[0226] Embodiment 14

[0227] A method for quantitative detection of semen leukocytes using the kit described in Example 11 comprises the following steps:

[0228] 1. Take 0.2 mL of semen sample into a 1.5 mL centrifuge tube, add 0.2 mL of sample treatment solution, invert 10 times to mix, and incubate at room temperature for 8 minutes.

[0229] 2. Add 0.8 mL of sample diluent and mix by inverting 10 times.

[0230] 3. Aspirate 200μL of sample separation solution, extend the pipette tip to the bottom of the solution and slowly add the sample separation solution. Note that at this time, you can see that there is a boundary layer between the sample separation solution and the sample to ensure the separation effect; centrifuge at 1200×g for 30min; after centrifugation, observe obvious stratification, aspirate the middle layer cells (middle layer liquid) and add them to a new 1.5ml centrifuge tube with a volume of about 0.2~0.3mL. Be careful not to aspirate the cells at the bottom.

[0231] 4. Add 0.6 mL of washing solution, invert 10 times to mix, and centrifuge at 400 g for 10 min. After the sample is centrifuged, discard the supernatant to obtain the cell pellet.

[0232] During the centrifugation process, the staining antibody can be prepared: calculate the amount of antibody required for the experiment. Taking 10 samples as an example, take 0.42mL of sample diluent, add 10μL of antibody solution, and mix well to obtain the staining antibody solution. The staining amount is 40μL / tube.

[0233] 5. Resuspend the cell pellet obtained by the above treatment in 40 μL of antibody solution to prepare the sample staining tube; incubate all stained samples at room temperature in the dark for 25 minutes.

[0234] Note: During the cell resuspension process, be sure to thoroughly mix by pipetting or vortexing to ensure the staining effect.

[0235] 6. Resuspend the cells in 0.5 mL of sample fixative and fix for 20 min; centrifuge at 300 × g for 10 min and discard the supernatant.

[0236] At this time, the volume of the cell suspension is about 20 μL. Add 0.2 mL of sample diluent to resuspend the cells. At this time, the volume of the cell suspension is 240 μL.

[0237] 7. Vortex the absolute counting microspheres for 15 seconds to evenly disperse the microspheres. Take about 1.96×10 4 Add the absolute counting microspheres beads obtained in step 7 to the cell suspension, mix well by pipetting, and then perform detection on the machine (the detection can be completed within 24 hours).

[0238] The detection is performed using a flow cytometer, where the instrument is required to be able to detect PE and FITC channels.

[0239] 8. Analyze the flow cytometry results using Flow Jo software and calculate CD45 by absolute bead counting. + The number of cells, including CD45 +The cells are in the FITC channel; opening the FITC channel and the PerCP-Cy5.5 channel at the same time can set a gate for the beads.

[0240] CD45 + The cells are calculated as follows:

[0241] CD45 + Absolute number of cells (A) = (CD45 + Number of cells (X) / Number of flow cytometry beads (Y)) * (Concentration of beads used for detection (N) * Volume of beads used for detection (V))

[0242] The number of beads used for detection = beads concentration * volume of beads used for detection. If the operation steps are strictly followed, the number of beads used for detection = (9.84 × 10 5 particles / mL)×0.02mL=1.968×10 4 .

[0243] Embodiment 15

[0244] A method for quantitative detection of semen leukocytes using the kit described in Example 12 comprises the following steps:

[0245] 1. Take 0.2 mL of semen sample into a 1.5 mL centrifuge tube, add 0.2 mL of sample treatment solution, invert 10 times to mix, and incubate at room temperature for 10 minutes.

[0246] 2. Add 1.0 mL of sample diluent and mix by inverting 10 times.

[0247] 3. Aspirate 200μL of sample separation solution, extend the pipette tip to the bottom of the solution and slowly add the sample separation solution. Note that at this time, you can see that there is a boundary layer between the sample separation solution and the sample to ensure the separation effect; centrifuge at 1800×g for 20min; after centrifugation, observe obvious stratification, aspirate the middle layer cells (middle layer liquid) and add them to a new 1.5ml centrifuge tube with a volume of about 0.2~0.3mL. Be careful not to aspirate the cells at the bottom.

[0248] 4. Add 0.8 mL of washing solution, invert 10 times to mix, and centrifuge at 800 g for 5 min. After the sample is centrifuged, discard the supernatant to obtain the cell pellet.

[0249] During the centrifugation process, the staining antibody can be prepared: calculate the amount of antibody required for the experiment. Taking 10 samples as an example, take 0.62mL of sample diluent, add 10μL of antibody solution, and mix well to obtain the staining antibody solution. The staining amount is 60μL / tube.

[0250] 5. Resuspend the cell pellet obtained by the above treatment in 60 μL of antibody solution to prepare the sample staining tube; incubate all stained samples at room temperature in the dark for 30 minutes.

[0251] Note: During the cell resuspension process, be sure to thoroughly mix by pipetting or vortexing to ensure the staining effect.

[0252] 6. Resuspend the cells in 0.5 mL of sample fixative and fix for 20 min; centrifuge at 400 × g for 5 min and discard the supernatant.

[0253] At this time, the volume of the cell suspension is about 20 μL. Add 0.2 mL of sample diluent to resuspend the cells. At this time, the volume of the cell suspension is 240 μL.

[0254] 7. Vortex the absolute counting microspheres for 25 seconds to evenly disperse the microspheres. Take about 3.92×10 4 Add the absolute counting microspheres beads obtained in step 7 to the cell suspension, mix well by pipetting, and then perform detection on the machine (the detection can be completed within 24 hours).

[0255] The detection is performed using a flow cytometer, where the instrument is required to be able to detect PE and FITC channels.

[0256] 8. Analyze the flow cytometry results using Flow Jo software and calculate CD45 by absolute bead counting. + The number of cells, including CD45 + The cells are in the FITC channel; opening the FITC channel and the PerCP-Cy5.5 channel at the same time can set a gate for the beads.

[0257] CD45 + The cells are calculated as follows:

[0258] CD45 + Absolute number of cells (A) = (CD45 + Number of cells (X) / Number of flow cytometry beads (Y)) * (Concentration of beads used for detection (N) * Volume of beads used for detection (V))

[0259] The number of beads used for detection = beads concentration * volume of beads used for detection. If the operation steps are strictly followed, the number of beads used for detection = (9.84 × 10 5 particles / mL)×0.04mL=3.936×10 4 .

[0260] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A flow cytometry quantitative detection reagent for semen leukocyte population, characterized in that: The detection reagent comprises: 1) Sample treatment solution: a solution composed of Na2HPO4, KH2PO4 and NaCl, with concentrations of Na2HPO4, KH2PO4 and NaCl being 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L and 7 g / L-9 g / L respectively; 2) Sample diluent: a solution composed of Na2HPO4, KH2PO4, NaCl and KCl, with concentrations of Na2HPO4, KH2PO4, NaCl and KCl being 1.30 g / L-1.5 g / L, 0.19 g / L-0.39 g / L, 7 g / L-9 g / L and 0.1-0.3 g / L respectively; 3) Sample separation solution: a solution composed of polysucrose, diatrizoate and a hydrophilic polymer with non-glycosyl units, including, by mass percentage, 1-6% diatrizoate, 1-5% polysucrose, 0.3-5% hydrophilic polymer with non-glycosyl units, and the remainder is deionized water without a heat source; the hydrophilic polymer with non-glycosyl units is selected from at least one of polyethylene glycol, polyvinyl pyrrolidone and polystyrene sulfonic acid; 4) Antibody solution: a solution of fluorescently labeled anti-human CD45 IgG monoclonal antibody with an antibody concentration of 0.01 μg-0.05 μg / 10 6 cells; 5) Sample fixative: 0.5%-4% paraformaldehyde solution; The sample treatment solution, sample diluent, sample separation solution, antibody solution and sample fixative are all prepared from deionized water.

2. The detection reagent according to claim 1, characterized in that: The antibody solution further contains fetal bovine serum (FBS), and the antibody solution contains 0.01% of fetal bovine serum (FBS).

3. The detection reagent according to claim 1, characterized in that: The fluorescently labeled anti-human CD45 IgG monoclonal antibody is derived from rabbit; And / or, the fluorescent label is selected from any one of FITC, PE, APC, and PerCP-cy5.

5.

4. A kit for flow cytometry quantification of semen leukocyte population comprising the detection reagent according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that: The kit further comprises: 6) washing solution; 7) absolute counting fluorescent microsphere beads.

6. The kit according to claim 5, characterized in that: The washing solution is a PBS solution containing 0.01% Tween-20; And / or, the absolute counting fluorescent microsphere beads are absolute counting fluorescent microspheres for flow cytometry.

7. A method for quantitatively detecting semen leukocyte population for purposes other than disease diagnosis or treatment, characterized in that: Using the detection reagent according to any one of claims 1 to 3 and / or the detection kit according to any one of claims 4 to 6, the method comprises the following steps: (1) Treating the semen sample with a sample processing solution; (2) diluting the semen sample treated with the sample treatment solution with a sample diluent; (3) Add sample separation solution, centrifuge, observe the stratification, and aspirate the intermediate layer liquid; (4) Add the washing solution to the middle layer liquid, mix well, centrifuge, and discard the supernatant; (5) Resuspend the cell pellet with antibody solution and incubate for staining; (6) Fixing the cell-antibody complex in the solution obtained in step (5) with a sample fixative, centrifuging and discarding the supernatant; (7) Add sample diluent to resuspend the cells, then add absolute counting fluorescent microspheres, and finally detect using a flow cytometer to record and calculate the results.

8. The detection method according to claim 7, characterized in that: In step (1), the amount of sample processing solution used is the same as that of the semen sample; and / or, in step (1), treating the semen sample with the sample treatment solution at room temperature for 5-10 minutes; And / or, in step (2), the dilution factor is 1-3 times; and / or, in step (3), the centrifugation conditions are: 1200-1800×g, 20-30 min; and / or, in step (4), adding 1-3 mL of washing solution to each mL of the intermediate layer liquid; And / or, in step (4), the centrifugation conditions are: 400-800×g, centrifugation for 5-10 min; and / or, in step (5), the amount of antibody solution required to resuspend each milliliter of cell pellet is 40-60 μL; And / or, in step (5), incubating in a dark environment for 20-30 min; and / or, in step (6), adding 0.1-0.5 mL of fixative solution per mL of the solution obtained in step (5); and / or, in step (6), the fixing time is 10-20 min; and / or, in step (6), the centrifugation conditions are: 300-400×g, 5-10 min; And / or, in step (7), the flow cytometry results are analyzed using flow cytometry data analysis software, and CD45 + The number of cells, including CD45 + The cells are in the FITC channel; open the FITC channel and the PerCP-Cy5.5 channel at the same time and set the gate for the beads.

9. The detection method according to claim 8, characterized in that: The calculation formula of CD45+ cells is: A=(X / Y)*(N*V), where A is CD45 + Absolute number of cells, X is CD45 detected by flow cytometry + The number of cells, Y is the number of flow cytometry beads, N is the concentration of beads used for detection, and V is the volume of beads used for detection; The number of beads used for detection = beads concentration * beads volume used for detection.

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