Freeze-drying protective agent, flow cytometry detection reagent, and preparation method and application of freeze-drying protective agent and flow cytometry detection reagent

Abstract

The invention discloses a freeze-drying protective agent, a flow cytometry detection reagent as well as a preparation method and application of the freeze-drying protective agent and the flow cytometry detection reagent, and belongs to the technical field of biology. The freeze-drying protective agent is prepared from the following components: 5%-30% w / v of trehalose, 1%-15% w / v of mannitol, 1% w / v of glucose, 0.01%-1% w / v of animal serum albumin, 0.01%-1% v / v of a surfactant, 0.03%-0.5% v / v of a preservative and 1%-5% w / v of polyethylene glycol. And the balance is a buffer solution. The freeze-drying protective agent component in the flow cytometry detection reagent is improved, and the freeze-drying reagent and antibody concentration are adaptively adjusted, so that the detection accuracy is effectively improved, and the application prospect is excellent.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a freeze-dried protective agent, a flow cytometry detection reagent, and a preparation method and use thereof. Background Art

[0002] Flow cytometry (FCM) is a highly sensitive, high-throughput, and rapid bioanalysis technique that can determine the proportion and number of target cells in a sample by analyzing specific markers on the cell surface. The advantage of flow cytometry is that it can analyze thousands of individual cells in a short period of time and can provide detailed information about cell size, granularity, DNA content, cell cycle, etc. It can also be used for cell sorting, that is, to separate specific cells from a population based on fluorescent signals.

[0003] In the existing flow cytometry fluorescent antibody detection technology, antibodies are usually coupled with different fluorescent dyes and stored separately in a buffer solution to avoid light. When used for multi-label detection, antibodies with different fluorescent dyes are combined to detect specific antigen molecules of cells. The disadvantage of this method is that different antibodies are stored separately in a buffer solution, and when used, a small amount of reagent is transferred from the reagent bottle to the reaction tube (usually 5 microliters to 20 microliters). The operation is time-consuming and labor-intensive, and there is a risk of reagent cross-over caused by continuous addition of samples, and repeated opening of the bottle causes contamination of the reagent.

[0004] On the other hand, the fluorescence intensity of the existing flow fluorescence antibody detection technology is not high enough, resulting in inaccurate test results, which needs further improvement. Patent application CN114264811A discloses a flow fluorescence quantitative detection freeze-dried reagent and kit, but the detection technology has poor discrimination between positive and negative cell populations and low positive cell ratio detection rate. Summary of the invention

[0005] The purpose of the present invention is to provide a lyophilized protective agent, a flow cytometry detection reagent, and a preparation method and use thereof.

[0006] The present invention provides a freeze-drying protective agent, which consists of the following components:

[0007] Trehalose 5%-30% w / v, mannitol 1%-15% w / v, glucose 1% w / v, animal serum albumin 0.01%-1% w / v, surfactant 0.01%-1% v / v, preservative 0.03%-0.5% v / v, polyethylene glycol 1%-5% w / v; the rest is buffer.

[0008] Furthermore, the lyophilization protective agent consists of the following components:

[0009] Trehalose 10% w / v, mannitol 5% w / v, glucose 1% w / v, animal serum albumin 0.5% w / v, surfactant 0.5% v / v, preservative 0.05% v / v, polyethylene glycol 3% w / v; the rest is buffer.

[0010] Furthermore, the buffer solution is a 0.3-0.7 mol / L phosphate buffer solution; the pH of the buffer solution is 6.0-8.0.

[0011] Furthermore, the buffer solution is a 0.5 mol / L phosphate buffer solution; the pH of the buffer solution is 7.0±0.5.

[0012] Further, the animal serum albumin is selected from bovine serum albumin, sheep serum albumin, donkey serum albumin, horse serum albumin, rabbit serum albumin, porcine serum albumin or chicken serum albumin;

[0013] The surfactant is Tween 20;

[0014] The preservative is sodium azide or Proclin 300.

[0015] Furthermore, the animal serum is bovine serum albumin.

[0016] The present invention also provides a flow cytometry detection reagent, which is prepared from the above-mentioned freeze-dried protective agent, counting microspheres and detection antibodies.

[0017] Furthermore, the counting microspheres are microspheres with fluorescent dyes; the fluorescent dyes are selected from FITC, PE, APC, PerCP (PerCP-Cy5.5), PE-Cy7, APC-Cy7, or dyes with an emission wavelength of 500 to 800 nm;

[0018] The detection antibody is selected from one or a mixture of two or more of a monoclonal antibody, a polyclonal antibody or an antigen.

[0019] Furthermore, the number of the counting microspheres is 5000 to 100000 per test, the content of the detection antibody is 0.0125 ug to 0.2 ug per test; the detection antibody is selected from one or a mixture of two or more of CD45, CD3, CD4, CD8, CD16, CD19, CD25, CD56, and CD127.

[0020] Further, the number of the counting microspheres is 20,000 to 50,000 per test;

[0021] The content of CD45 is 0.1μg / test, the content of CD3 is 0.1μg / test, the content of CD4 is 0.05μg / test, the content of CD8 is 0.1μg / test, the content of CD16 is 0.05μg / test, the content of CD19 is 0.0125μg / test, the content of CD25 is 0.1μg / test, the content of CD56 is 0.05μg / test, and the content of CD127 is 0.05μg / test.

[0022] The present invention also provides a method for preparing the above-mentioned flow cytometry detection reagent, which comprises the following steps: mixing the above-mentioned freeze-dried protective agent with the counting microspheres and the detection antibody, and freeze-drying to obtain the above-mentioned flow cytometry detection reagent.

[0023] Furthermore, the freeze-drying comprises the following stages:

[0024] Pre-freezing: temperature -70~-30℃, vacuum pressure 0~20Pa, time 100~500min;

[0025] Sublimation drying: temperature -30~10℃, vacuum pressure 0~40Pa, time 1300~1700min;

[0026] Analytical drying: temperature 10-50°C, vacuum pressure 0-10Pa, time 100-300min.

[0027] Furthermore, the freeze-drying comprises the following stages:

[0028] Pre-freezing: temperature -50°C, vacuum pressure 10Pa, time 300min;

[0029] Sublimation drying: temperature -10°C, vacuum pressure 20Pa, time 1500min;

[0030] Analytical drying: temperature 30°C, vacuum pressure 5Pa, time 180min.

[0031] The present invention also provides use of the flow cytometry detection reagent in antibody quantitative detection.

[0032] The present invention also provides use of the flow cytometry detection reagent in flow cytometry multi-factor detection.

[0033] Furthermore, when the above-mentioned flow cytometry detection reagent is used for antibody quantitative detection or flow cytometry multifactor detection, the total amount of cells in each test sample is 1×10 ^5 ~1×10 ^6 cells.

[0034] The present invention has achieved the following beneficial effects:

[0035] The present invention provides a new system for detecting cell CD45 and other markers. By adapting and adjusting the freeze-dried reagents and antibody concentrations in the system, efficient and accurate detection is achieved, overcoming many problems of the prior art:

[0036] 1. Avoid the systematic errors caused by traditional methods in the antibody loading process;

[0037] 2. Multiple fluorescent antibodies are mixed and freeze-dried, which reduces the pipetting steps during use, saves time and improves efficiency;

[0038] 3. The flow cytometry fluorescent antibody is stored in a solid form by the freeze-dried buffer, and the reagent can be stored at room temperature, avoiding the increase in costs during low temperature and cold chain storage and transportation and the impact of temperature fluctuations on the reagent;

[0039] 4. Pre-loading the reagents into the flow sample tube can achieve rapid detection, greatly saving working time;

[0040] 5. Provides a faster testing solution for projects that require timely testing;

[0041] 6. Compared with patent application CN114264811A, the flow cytometric detection system provided by the present invention has significantly improved the differentiation between positive and negative cell populations and the detection rate of positive cell ratio.

[0042] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0043] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Results of CD45 antibody content screening.

[0045] Figure 2 Results of CD3 antibody content screening.

[0046] Figure 3 Results of screening for CD4 antibody content.

[0047] Figure 4 Results of CD8 antibody content screening.

[0048] Figure 5 Results of CD16 antibody content screening.

[0049] Figure 6 Results of CD19 antibody content screening.

[0050] Figure 7 Results of CD25 antibody assay screening

[0051] Figure 8 Results of CD56 antibody content screening.

[0052] Fig. 9 The results are for the CD127 antibody content screening.

[0053] Fig.10 The flow cytometry detection reagents of (a) Example 2, (b) Control Example 1, (c) Control Example 2, (d) Control Example 3, and (e) Control Example 4 were tested in the CD3-CD56 antibody combination.

[0054] Fig.11 The freeze-dried reagent (a) and antibody stock solution (b) of Example 1 were used to detect the percentage of TBNK cells.

[0055] Fig.12 The antibody stock solution (a) and lyophilized reagent (b) of Example 3 were used to detect the percentage of Treg cells. DETAILED DESCRIPTION

[0056] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0057] In the present invention, w / v means mass-to-volume ratio, and the unit is g / mL. For example, 10% w / v means 10 g / 100 mL.

[0058] Example 1: Preparation of flow cytometry detection reagents

[0059] Preparation method:

[0060] 1. Take the TBNK detection antibody combination + absolute counting microspheres, add the lyophilized protective agent and mix evenly to obtain the TBNK detection antibody combination + absolute counting microsphere solution.

[0061] Among them, the freeze-drying protective agent is composed of: trehalose (10% w / v), mannitol (5% w / v), glucose (1% w / v), bovine serum albumin (0.5% w / v), surfactant (Tween20 0.5% v / v), Proclin300 (0.05% v / v), polyethylene glycol (PEG) (3% w / v), and the rest is 0.5 mol / L phosphate buffer; pH is 7.0±0.5.

[0062] The preparation ratio of TBNK detection antibody combination + absolute counting microsphere solution is as follows:

[0063] 25ul of lyophilized protective agent contains:

[0064] CD45 (0.1ug), CD3 (0.1ug), CD4 (0.05ug), CD8 (0.1ug), CD19 (0.05ug), CD16 (0.05ug) and CD56 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0065] 2. Drop 5-100 μl of TBNK detection antibody combination + absolute counting microsphere solution into liquid nitrogen to form an ice ball, which is then transferred to a freeze dryer to run the set freeze-drying program. The microspheres obtained after freeze-drying are flow cytometry detection reagents with a volume of 0.003 cubic centimeters to 0.1 cubic centimeters and a diameter of 1 mm to 5 mm.

[0066] The freeze-drying procedure is as follows:

[0067]

[0068] Directions:

[0069] When used, the microspheres are directly added to serum, plasma and other biological samples (10-200 μl). The total number of cells in each test sample is 1×10 ^5 ~1×10 ^6 , the microspheres dissolve to release the antibodies, thus completing the automatic incubation process, and then the antibody titer test is performed.

[0070] Example 2: Preparation of flow cytometry detection reagents

[0071] Preparation method:

[0072] 1. Take CD3-CD56 antibody combination + absolute counting microspheres, add the lyophilized protective agent of Example 1 and mix evenly to obtain a CD3-CD56 antibody combination + absolute counting microsphere solution.

[0073] The preparation ratio of CD3-CD56 antibody combination + absolute counting microsphere solution is as follows:

[0074] 25ul of lyophilized protective agent contains:

[0075] CD3 (0.1ug) and CD56 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0076] 2. Referring to the method of step 2 of Example 1, the microspheres obtained by freeze-drying are flow cytometry detection reagents.

[0077] Usage: Same as Example 1.

[0078] Example 3: Preparation of flow cytometry detection reagents

[0079] Preparation method:

[0080] 1. Take Treg detection antibody combination + absolute counting microspheres, add the lyophilized protective agent of Example 1 and mix evenly to obtain a Treg detection antibody combination + absolute counting microsphere solution.

[0081] The preparation ratio of Treg detection antibody combination + absolute counting microsphere solution is as follows:

[0082] 25ul of lyophilized protective agent solution contains:

[0083] CD45 (0.1ug) / CD3 (0.1ug) / CD4 (0.05ug) / CD25 (0.1ug) / CD127 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0084] 2. Referring to the method of step 2 of Example 1, the microspheres obtained by freeze-drying are flow cytometry detection reagents.

[0085] Usage: Same as Example 1.

[0086] The following is a control sample preparation.

[0087] Control Example 1: Preparation of flow cytometry detection reagents

[0088] Preparation method:

[0089] 1. Take CD3-CD56 antibody combination + absolute counting microspheres, add lyophilized protective agent and mix evenly to obtain CD3-CD56 detection antibody combination + absolute counting microsphere solution.

[0090] Among them, the freeze-drying protective agent is composed of: trehalose (10% w / v), mannitol (5% w / v), glucose (3% w / v), bovine serum albumin (0.5% w / v), surfactant (Tween20 0.5% v / v), Proclin300 (0.05% v / v), polyethylene glycol (PEG) (3% w / v), 0.5 mol / L phosphate buffer, pH 7.0±0.5.

[0091] The preparation ratio of CD3-CD56 detection antibody combination + absolute counting microsphere solution is as follows:

[0092] 25ul of lyophilized protective agent contains:

[0093] CD3 (0.1ug) and CD56 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0094] 2. Referring to the method of step 2 of Example 1, the microspheres obtained by freeze-drying are flow cytometry detection reagents.

[0095] Usage: Same as Example 1.

[0096] Control Example 2: Preparation of flow cytometry detection reagents

[0097] Preparation method:

[0098] 1. Take CD3-CD56 antibody combination + absolute counting microspheres, add lyophilized protective agent and mix evenly to obtain CD3-CD56 detection antibody combination + absolute counting microsphere solution.

[0099] Among them, the freeze-drying protective agent is composed of: trehalose (10% w / v), mannitol (5% w / v), glucose (0.5% w / v), bovine serum albumin (0.5% w / v), surfactant (Tween20 0.5% v / v), Proclin300 (0.05% v / v), polyethylene glycol (PEG) (3% w / v), 0.5 mol / L phosphate buffer, pH 7.0±0.5.

[0100] The preparation ratio of CD3-CD56 detection antibody combination + absolute counting microsphere solution is as follows:

[0101] 25ul of lyophilized protective agent contains:

[0102] CD3 (0.1ug) and CD56 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0103] 2. Referring to the method of step 2 of Example 1, the microspheres obtained by freeze-drying are flow cytometry detection reagents.

[0104] Usage: Same as Example 1.

[0105] Control Example 3: Preparation of flow cytometry detection reagents

[0106] Preparation method:

[0107] 1. Take CD3-CD56 antibody combination + absolute counting microspheres, add lyophilized protective agent and mix evenly to obtain CD3-CD56 detection antibody combination + absolute counting microsphere solution.

[0108] The freeze-drying protective agent is composed of: trehalose (10% w / v), mannitol (5% w / v), bovine serum albumin (1% w / v), surfactant (Tween20 0.5% v / v), Proclin300 (0.05% v / v), 0.1 mol / L phosphate buffer, pH 7.0.

[0109] The preparation ratio of CD3-CD56 antibody combination + absolute counting microsphere solution is as follows:

[0110] 25ul of lyophilized protective agent contains:

[0111] CD3 (0.1ug) and CD56 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0112] 2. Referring to the method of step 2 of Example 1, the microspheres obtained by freeze-drying are flow cytometry detection reagents.

[0113] Usage: Same as Example 1.

[0114] Control Example 4: Preparation of flow cytometry detection reagents

[0115] Preparation method:

[0116] 1. Take CD3-CD56 antibody combination + absolute counting microspheres, add lyophilized protective agent and mix evenly to obtain CD3-CD56 detection antibody combination + absolute counting microsphere solution.

[0117] Among them, the freeze-drying protective agent is composed of: trehalose (10% w / v), mannitol (5% w / v), glucose (1% w / v), bovine serum albumin (1% w / v), surfactant (Tween20 0.5% v / v), Proclin300 (0.05% v / v), polyethylene glycol (PEG) (3% w / v), 0.1 mol / L phosphate buffer, pH 7.0.

[0118] The preparation ratio of CD3-CD56 detection antibody combination + absolute counting microsphere solution is as follows:

[0119] 25ul of lyophilized protective agent contains:

[0120] CD3 (0.1ug) and CD56 (0.05ug); the number of microspheres counted was 20,000 / tube.

[0121] 2. Referring to the method of step 2 of Example 1, the microspheres obtained by freeze-drying are flow cytometry detection reagents.

[0122] Usage: Same as Example 1.

[0123] The difference between the freeze-dried protective agent formulations of reference examples 1 to 4 and the freeze-dried protective agent formulation of the present invention is that:

[0124] Compared with the freeze-dried protective agent formulation of Example 1, the freeze-dried protective agent formulation of Control Example 1 increases the content of glucose from 1% w / v to 3% w / v;

[0125] Compared with the freeze-dried protective agent formulation of Example 1, the freeze-dried protective agent formulation of Control Example 2 reduces the content of glucose from 1% w / v to 0.5% w / v;

[0126] The freeze-dried protective agent formula of comparative example 3 is the freeze-dried protective agent formula in patent application CN114264811A;

[0127] The freeze-dried protective agent formula of comparative example 4 is prepared by adding 1% w / v glucose and 3% w / v polyethylene glycol of the present invention to the freeze-dried protective agent of patent application CN114264811A.

[0128] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0129] Experimental Example 1: Antibody Titer Screening

[0130] 1. Experimental methods

[0131] Different amounts (0.0125 ug to 0.2 ug) of monoclonal antibodies coupled with different fluorescent dye molecules (CD45, CD3, CD4, CD8, CD16, CD19, CD25, CD56, CD127) were taken, and the lyophilized protective agent in Example 1 was added respectively and mixed evenly to obtain different detection antibody combinations + absolute counting microsphere solutions.

[0132] Refer to the method of Example 1, freeze-dry, and then perform antibody titer test.

[0133] 2. Experimental results

[0134] The antibody content screening results of CD45, CD3, CD4, CD8, CD16, CD19, CD25, CD56 and CD127 are shown in Tables 1-9, Figure 1-9 shown.

[0135] Among them, rSD represents the relative standard deviation; -MFI and +MFI represent the mean fluorescence intensity of negative and positive cell populations, respectively.

[0136] SI stands for Stain Index, and the calculation formula is:

[0137]

[0138] Table 1 CD45 antibody content screening results

[0139]

[0140] Table 2 CD3 antibody content screening results

[0141]

[0142] Table 3 CD4 antibody content screening results

[0143]

[0144] Table 4 CD8 antibody content screening results

[0145]

[0146] Table 5 CD16 antibody content screening results

[0147]

[0148] Table 6 CD19 antibody content screening results

[0149]

[0150]

[0151] Table 7 CD25 antibody content screening results

[0152]

[0153] Table 8 CD56 antibody content screening results

[0154]

[0155] Table 9 Antibody content screening results of CD127

[0156]

[0157] Screening showed that the optimal contents of CD45, CD3, CD4, CD8, CD16, CD19, CD25, CD56 and CD127 in each test were 0.1ug, 0.1ug, 0.05ug, 0.1ug, 0.05ug, 0.0125ug, 0.1ug, 0.05ug and 0.05ug respectively.

[0158] Experimental Example 2: Test results of different flow cytometry detection reagents

[0159] The effect of grouping the positive and negative cell populations was tested, and the effects of the flow cytometry detection reagents in Example 2 and Control Examples 1-4 were compared.

[0160] like Fig.10 As shown, the freeze-dried protective agent formula of Example 1 ( Fig.10 a) Compared with control example 1 ( Fig.10 b) and Comparative Example 2 ( Fig.10c) The grouping is more obvious, the differentiation between positive and negative cell groups is higher, and the detection rate of positive cell ratio is higher. The freeze-dried protective agent formula of Example 1 ( Fig.10 a) Compared with control example 3 ( Fig.10 The lyophilized protective agent formula of Example 1 (d) has a more obvious grouping, and the differentiation between positive and negative cell populations is higher. Fig.10 a) Compared with control example 4 ( Fig.10 The freeze-dried protective agent formula e) has a higher degree of differentiation between positive and negative cell populations and a better detection rate of positive cell ratio.

[0161] Experimental Example 3: Performance test of antibody stock solution before and after freeze-drying

[0162] 1. Experimental methods

[0163] The antibody stock solutions and lyophilized reagents of test examples 1 and 3 were tested for the lymphocyte percentage (Lym%), CD3 positive cell percentage (CD3+%), CD3 and CD4 positive cell percentage (CD3+CD4%), CD3 and CD8 positive cell percentage (CD3+CD8%), B cell percentage (B cell%) and NK cell percentage (NK cell%) in lymphocyte subpopulations.

[0164] 2. Experimental results

[0165] Table 10 Fig.11 The figure shows the proportion of TBNK cells between the lyophilized reagent (a) and the antibody stock solution (b) of Example 1. It can be seen that before and after the antibody stock solution is lyophilized to form the lyophilized reagent, the relative deviations of Lym%, CD3+%, CD3+CD8%, B cell% and NK cell% are between 0.3% and 3.77%.

[0166] Table 10 Detection of TBNK cell ratio in Example 1

[0167] Lym% CD3+% CD3+CD4+% CD3+CD8+% B Cell % NK cells% Lyophilized reagents 9.89 75.85 39.58 31.48 14.36 9.25 Antibody stock solution 9.68 75.62 41.13 31.13 13.95 9.17 Relative deviation 2.12％ 0.3％ 3.77％ 1.11％ 2.86％ 0.8％

[0168] Table 11 Fig.12 The figure shows the proportion of Treg cells between the lyophilized reagent (a) and the antibody stock solution (b) of Example 3. It can be seen that before and after the antibody stock solution is lyophilized to form the lyophilized reagent, the relative deviations of Lym%, CD4+% and CD25+CD127LOW% are between 2.19% and 3.53%.

[0169] Table 11 Detection of Treg cell ratio in Example 3

[0170] Lym% CD4% CD25+CD127Low% Lyophilized reagents 28.12 38.05 11.46 Antibody stock solution 28.75 37.11 11.90 Relative deviation 2.19％ 2.47％ 3.53％

[0171] The above results show that the performance of the lyophilized reagent after reconstitution is very close to that of the antibody stock solution, which means that the lyophilization process effectively maintains the biological activity and immunological properties of the antibody, ensures the stability, consistency and reliability of the antibody under different conditions. The lyophilized reagent is stored in solid form and can be stored at room temperature, avoiding the increase in costs during low temperature and cold chain storage and transportation and the impact of temperature fluctuations on the reagent.

[0172] In summary, the present invention discloses a lyophilized protective agent, a flow cytometry detection reagent, and a preparation method and use thereof. The lyophilized protective agent is composed of the following components: trehalose 5% to 30% w / v, mannitol 1% to 15% w / v, glucose 1% w / v, animal serum albumin 0.01% to 1% w / v, surfactant 0.01% to 1% v / v, preservative 0.03% to 0.5% v / v, polyethylene glycol 1% to 5% w / v; the rest is a buffer. The present invention improves the lyophilized protective agent component in the flow cytometry detection reagent, adapts and adjusts the lyophilized reagent and antibody concentration, effectively improves the detection accuracy, and has excellent application prospects.

Claims

1. A freeze-drying protective agent, characterized in that It consists of the following components: Trehalose 5%-30% w / v, mannitol 1%-15% w / v, glucose 1% w / v, animal serum albumin 0.01%-1% w / v, surfactant 0.01%-1% v / v, preservative 0.03%-0.5% v / v, polyethylene glycol 1%-5% w / v; the rest is buffer.

2. The lyoprotectant according to claim 1, characterized in that The freeze-drying protective agent consists of the following components: Trehalose 10% w / v, mannitol 5% w / v, glucose 1% w / v, animal serum albumin 0.5% w / v, surfactant 0.5% v / v, preservative 0.05% v / v, polyethylene glycol 3% w / v; the rest is buffer.

3. The lyoprotectant according to claim 1 or 2, characterized in that The animal serum albumin is selected from bovine serum albumin, sheep serum albumin, donkey serum albumin, horse serum albumin, rabbit serum albumin, porcine serum albumin or chicken serum albumin; The surfactant is Tween 20; The preservative is sodium azide or Proclin 300.

4. A flow cytometry detection reagent, characterized in that: The method is prepared from the freeze-dried protective agent described in any one of claims 1 to 3, counting microspheres and detection antibodies.

5. The flow cytometry detection reagent according to claim 4, characterized in that: The counting microspheres are microspheres with fluorescent dyes; the fluorescent dyes are selected from FITC, PE, APC, PerCP (PerCP-Cy5.5), PE-Cy7, APC-Cy7, or dyes with an emission wavelength of 500 to 800 nm; The detection antibody is selected from one or a mixture of two or more of a monoclonal antibody, a polyclonal antibody or an antigen.

6. The flow cytometry detection reagent according to claim 5, characterized in that: The number of the counting microspheres is 5000 to 100000 per test, and the content of the detection antibody is 0.0125 ug to 0.2 ug per test; the detection antibody is selected from one or a mixture of two or more of CD45, CD3, CD4, CD8, CD16, CD19, CD25, CD56, and CD127.

7. The flow cytometry detection reagent according to claim 6, characterized in that: The number of the counting microspheres is 20,000 to 50,000 per test; The content of CD45 is 0.1μg / test, the content of CD3 is 0.1μg / test, the content of CD4 is 0.05μg / test, the content of CD8 is 0.1μg / test, the content of CD16 is 0.05μg / test, the content of CD19 is 0.0125μg / test, the content of CD25 is 0.1μg / test, the content of CD56 is 0.05μg / test, and the content of CD127 is 0.05μg / test.

8. A method for preparing the flow cytometry detection reagent according to any one of claims 4 to 7, characterized in that: The method comprises the following steps: mixing the lyophilized protective agent according to any one of claims 1 to 3 with counting microspheres and detection antibodies, and freeze-drying to obtain the lyophilized protective agent.

9. The method according to claim 8, characterized in that The freeze-drying process includes the following stages: Pre-freezing: temperature -70~-30℃, vacuum pressure 0~20Pa, time 100~500min; Sublimation drying: temperature -30~10℃, vacuum pressure 0~40Pa, time 1300~1700min; analytical drying: temperature 10~50℃, vacuum pressure 0~10Pa, time 100~300min.

10. Use of the flow cytometry detection reagent according to any one of claims 4 to 7 in antibody quantitative detection.

Images