Freeze-drying protective agent, antibody freeze-drying microsphere stock solution, antibody freeze-drying microsphere and application of antibody freeze-drying microsphere stock solution

Abstract

The invention belongs to the technical field of medical detection, and particularly relates to a freeze-drying protective agent, an antibody freeze-dried microsphere stock solution, an antibody freeze-dried microsphere and application thereof. The freeze-drying protective agent comprises trehalose, mannitol, polyethylene glycol 8000, a protein protective agent, a surfactant, a preservative and a PBS (Phosphate Buffer Solution). The antibody freeze-dried microsphere based on the freeze-drying protective agent has excellent stability, and still has stable performance after being stored for 90 days at room temperature; however, the currently obtained lymphocyte subpopulation detection reagents need to be stored at 4 DEG C; in addition, the consistency of the detection result of the lymphocyte subpopulation detection reagent and the currently obtained lymphocyte subpopulation detection reagent is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and particularly relates to a lyoprotectant, a stock solution of antibody lyophilized microspheres, antibody lyophilized microspheres and their uses. Background Art

[0002] Flow cytometry, as the gold standard for lymphocyte subset detection, depends corely on the precise combination and reaction of multi-color fluorescently labeled antibodies. Currently, the widely used flow cytometry TBNK lymphocyte subset detection kits in clinics adopt the form of liquid mixed antibodies, which contain antibody components such as CD3, CD4, CD8, CD19, CD16 / 56, etc. In actual operation, the detection personnel need to repeatedly perform pipetting operations to accurately distribute the liquid antibodies into the detection tubes, then add the samples for incubation and labeling, and finally perform upper machine analysis. This process is not only time-consuming and laborious, but also has risks such as cross-contamination and dosage errors due to the manual pipetting steps, which may lead to deviation of the detection results, especially when the operation standardization in primary medical institutions is insufficient.

[0003] Furthermore, the existing liquid reagents are limited by the stability of bioactive substances and need to be transported and stored at a constant cold chain (2-8°C) throughout the process, which significantly increases the reagent distribution cost in remote areas and is prone to antibody titer attenuation due to temperature fluctuations. Although some studies have tried to improve the stability of antibodies through the freeze-drying process, traditional freeze-drying technologies mostly adopt plate freeze-drying methods, and the formed blocky freeze-dried bodies need to be redissolved and sub-packaged, which fails to truly achieve the "ready-to-use" design. In addition, when multi-antibody mixed freeze-drying is carried out, problems such as uneven component distribution and different reconstitution efficiencies are prone to occur, which restricts its application in flow cytometry multiplex detection.

[0004] Therefore, developing a new type of reagent form that can be stored and transported at room temperature, does not require pipetting operation, and can maintain the synchronous stability of multi-antibody activity has become a technical bottleneck that urgently needs to be broken through in the field of flow cytometry detection. Summary of the Invention

[0005] The present invention has developed an antibody lyophilized microsphere that can be used for lymphocyte subset detection. The antibody lyophilized microsphere can be stored at room temperature; and when performing lymphocyte subset detection, it does not require reconstitution and pipetting, and has high detection accuracy.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a lyoprotectant, which includes trehalose, mannitol, polyethylene glycol 8000, a protein protectant, a surfactant, a preservative and a PBS solution.

[0008] Preferably, among the above freeze-drying protectants, the protein protectant is selected from bovine serum albumin and / or casein; and / or the surfactant is selected from one or more of Tween-20, hydroxypropyl-β-cyclodextrin, and Tween-80; and / or the preservative is selected from ProClin300 and / or sodium azide.

[0009] Preferably, in the above freeze-drying protectant, the mass fraction of trehalose is 10% - 14%; and / or the mass fraction of mannitol is 10% - 14%; and / or the mass fraction of polyethylene glycol 8000 is 10% - 14%; and / or the mass fraction of the protein protectant is 1% - 5%; and / or the mass fraction of the surfactant is 0.1% - 0.3%; and / or the mass fraction of the preservative is 0.01% - 0.1%.

[0010] On the other hand, the present invention provides a freeze-dried antibody microsphere stock solution, which includes the freeze-drying protectant in the present invention and a fluorescent antibody solution, and the fluorescent antibody solution contains a specific labeled antibody against the surface antigen of lymphocyte subsets.

[0011] Preferably, in the above freeze-dried antibody microsphere stock solution, the fluorescent antibody solution includes one or more of the following antibodies: CD3FITC, CD16PE, CD56PE, CD45PerCP-Cy5.5, CD4PE-Cy7, CD19APC, or CD8APC-Cy7.

[0012] More preferably, in the fluorescent antibody solution of the above freeze-dried antibody microsphere stock solution, the concentration of CD3FITC is 2 μg / mL - 3 μg / mL; and / or the concentration of CD16PE is 1 μg / mL - 2 μg / mL; and / or the concentration of CD56PE is 1 μg / mL - 2 μg / mL; and / or the concentration of CD45PerCP-Cy5.5 is 5.5 μg / mL - 6.5 μg / mL; and / or the concentration of CD4PE-Cy7 is 1 μg / mL - 2 μg / mL; and / or the concentration of CD19APC is 2 μg / mL - 3 μg / mL; and / or the concentration of CD8APC-Cy7 is 6 μg / mL - 7 μg / mL.

[0013] Preferably, in the above freeze-dried antibody microsphere stock solution, the volume ratio of the fluorescent antibody solution to the freeze-drying protectant is 1:(2 - 4).

[0014] On the other hand, the present invention provides a freeze-dried antibody microsphere for detecting lymphocyte subsets, which is obtained by freeze-drying the freeze-dried antibody microsphere stock solution in the present invention.

[0015] Preferably, in the above freeze-dried antibody microsphere, the freeze-drying parameters include: the vacuum degree ≤ 20 Pa; and / or the cold trap temperature ≤ -55°C.

[0016] On the other hand, the present invention provides a product for detecting lymphocyte subsets, which comprises the antibody freeze-dried microspheres in the present invention.

[0017] The beneficial effects of the present invention include:

[0018] (1) The antibody freeze-dried microspheres provided by the present invention have excellent stability and remain stable in performance after 90 days of storage at room temperature; while the currently approved lymphocyte subset detection reagents all need to be stored at 4°C.

[0019] (2) When detecting lymphocyte subsets with the detection reagent based on the antibody freeze-dried microspheres provided by the present invention, pipetting is not required, which can reduce the operation time of the operator and avoid the errors caused by pipetting.

[0020] (3) The detection of lymphocyte subsets with the detection reagent based on the antibody freeze-dried microspheres provided by the present invention has high accuracy. After verification, the detection results have high consistency with those of the currently approved lymphocyte subset detection reagents.

[0021] (4) When detecting lymphocyte subsets with the detection reagent based on the antibody freeze-dried microspheres provided by the present invention, it has high compatibility with the absolute counting microspheres and does not affect the performance of the absolute counting microspheres.

[0022] (5) When detecting lymphocyte subsets with the detection reagent based on the antibody freeze-dried microspheres provided by the present invention, reconstitution is not required and direct labeling detection can be carried out, which improves the detection efficiency. Description of the Drawings

[0023] Figure 1A It is a schematic structural diagram of a flow tube; wherein, 1: flow tube cap; 2: flow tube body; 3: stainless steel mesh; 4: antibody freeze-dried microspheres;

[0024] Figure 1B It is a physical picture of a flow tube;

[0025] Figure 1C It is the appearance of the antibody freeze-dried microsphere 0# prepared in Example 1;

[0026] Figure 2 It is the appearance of the antibody freeze-dried microspheres prepared from different raw materials;

[0027] Figure 3 It is the detection of lymphocyte subsets in the peripheral blood of healthy people with the single-person portion lymphocyte subset freeze-dried microsphere detection reagent;

[0028] Figure 4 It is the detection of lymphocyte subsets in the peripheral blood of healthy people with the BD lymphocyte subset liquid detection reagent;

[0029] Figure 5This is the Bland-Altman plot (difference analysis plot) of the proportion of CD3+ cells in the detection results of the detection reagent and BD detection reagent in the present invention;

[0030] Figure 6 It is the stability detection of the single-person lymphocyte subset freeze-dried microsphere detection reagent at different temperatures. Detailed implementation manners

[0031] The embodiments cited are for better illustration of the present invention, but the content of the present invention is not limited only to the cited embodiments. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners according to the above invention content still fall within the protection scope of the present invention.

[0032] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having significantly different meanings in the context, the singular form of the expression includes the plural form of the expression. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility of the existence or addition of one or more other features, numbers, operations, components, parts, elements, materials or their combinations. As used herein, depending on the situation, " / " can be interpreted as "and" or "or".

[0033] The embodiment of the present invention provides a lyoprotectant, and the lyoprotectant includes trehalose, mannitol, polyethylene glycol 8000, protein protectant, surfactant, preservative and PBS solution.

[0034] It should be noted that in the lyoprotectant of the present invention, polyethylene glycol 8000 has an important influence on the formation of freeze-dried microspheres. If polyethylene glycol 8000 is not added in the present invention, the prepared antibody freeze-dried microspheres are significantly non-spherical. Even if spherical antibody freeze-dried microspheres are prepared by adjusting the proportion of other substances on this basis, in the subsequent flow cytometry detection, it will affect the hemolysis of hemolysin, resulting in insufficient hemolysis. After the specified incubation time, it is still turbid (after sufficient hemolysis of normal whole blood, it should be clear).

[0035] In some specific examples, in the above lyoprotectant, the protein protectant is selected from bovine serum albumin and / or casein; and / or the surfactant is selected from one or more of Tween-20, hydroxypropyl-β-cyclodextrin, and Tween-80; and / or the preservative is selected from ProClin300 and / or sodium azide.

[0036] It should be noted that the protein protectant, surfactant and preservative are all well-known in the art. For example, the protein protectant can be selected from bovine serum albumin and / or casein, the surfactant can be selected from one or more of Tween-20, hydroxypropyl-β-cyclodextrin, and Tween-80, and the preservative can be selected from ProClin300 and / or sodium azide. In addition, when Tween-80 is used as the surfactant, the prepared antibody lyophilized microspheres are slightly non-spherical. Therefore, Tween-20 and / or hydroxypropyl-β-cyclodextrin can be preferably used as the surfactant in the present invention.

[0037] In some specific examples, in the above-mentioned lyoprotectant, the mass fraction of trehalose is 10% - 14%; and / or the mass fraction of mannitol is 10% - 14%; and / or the mass fraction of polyethylene glycol 8000 is 10% - 14%; and / or the mass fraction of the protein protectant is 1% - 5%; and / or the mass fraction of the surfactant is 0.1% - 0.3%; and / or the mass fraction of the preservative is 0.01% - 0.1%.

[0038] It should be noted that the mass fractions of the components in the lyoprotectant of the present invention have an impact on the detection effect of the prepared antibody lyophilized microspheres. In the present invention, the mass fraction of trehalose can be 10% - 14%, such as 11%, 12% or 13%, etc.; and / or the mass fraction of mannitol is 10% - 14%, such as 11%, 12% or 13%, etc.; and / or the mass fraction of polyethylene glycol 8000 is 10% - 14%, such as 11%, 12% or 13%, etc.; and / or the mass fraction of the protein protectant is 1% - 5%, such as 2%, 3% or 4%, etc.; and / or the mass fraction of the surfactant is 0.1% - 0.3%, such as 0.15%, 0.2% or 0.25%, etc.; and / or the mass fraction of the preservative is 0.01% - 0.1%, such as 0.02%, 0.05% or 0.08%, etc.

[0039] In some specific examples, in the above-mentioned lyoprotectant, the mass fraction of trehalose is 12%; and / or the mass fraction of mannitol is 12%; and / or the mass fraction of polyethylene glycol 8000 is 12%; and / or the mass fraction of the protein protectant is 3%; and / or the mass fraction of the surfactant is 0.15%; and / or the mass fraction of the preservative is 0.05%.

[0040] It should be noted that the mass fractions of the above components are preferably used in the present invention. The antibody lyophilized microspheres prepared with this mass fraction not only have a good appearance shape, but also have more accurate detection results and higher detection stability when performing lymphocyte subset detection.

[0041] An embodiment of the present invention also provides an antibody freeze-dried microsphere stock solution, which includes the cryoprotectant in the present invention and a fluorescent antibody solution, and the fluorescent antibody solution contains specific labeled antibodies against lymphocyte subset surface antigens.

[0042] In some specific examples, in the above antibody freeze-dried microsphere stock solution, the fluorescent antibody solution includes one or more of the following antibodies: CD3FITC, CD16PE, CD56PE, CD45PerCP-Cy5.5, CD4PE-Cy7, CD19APC or CD8APC-Cy7.

[0043] In some specific examples, in the fluorescent antibody solution of the above antibody freeze-dried microsphere stock solution,

[0044] the concentration of CD3FITC can be 2 μg / mL to 3 μg / mL, such as 2.3 μg / mL, 2.5 μg / mL or 2.8 μg / mL, etc.; and / or

[0045] the concentration of CD16PE is 1 μg / mL to 2 μg / mL, such as 1.3 μg / mL, 1.5 μg / mL or 1.8 μg / mL, etc.; and / or

[0046] the concentration of CD56PE is 1 μg / mL to 2 μg / mL, such as 1.3 μg / mL, 1.5 μg / mL or 1.8 μg / mL, etc.; and / or

[0047] the concentration of CD45PerCP-Cy5.5 is 5.5 μg / mL to 6.5 μg / mL, such as 5.8 μg / mL, 6 μg / mL or 6.3 μg / mL, etc.; and / or

[0048] the concentration of CD4PE-Cy7 is 1 μg / mL to 2 μg / mL, such as 1.3 μg / mL, 1.5 μg / mL or 1.8 μg / mL, etc.; and / or

[0049] the concentration of CD19APC is 2 μg / mL to 3 μg / mL, such as 2.3 μg / mL, 2.5 μg / mL or 2.8 μg / mL, etc.; and / or

[0050] the concentration of CD8APC-Cy7 is 6 μg / mL to 7 μg / mL, such as 6.3 μg / mL, 6.5 μg / mL or 6.8 μg / mL, etc.

[0051] In some specific examples, the fluorescence antibodies in the fluorescence antibody solution of the above-mentioned antibody freeze-dried microsphere stock solution may preferably have the following concentrations: the concentration of CD3 FITC is 2.3 μg / mL; and / or the concentration of CD16 PE is 1.65 μg / mL; and / or the concentration of CD56 PE is 1.1 μg / mL; and / or the concentration of CD45 PerCP-Cy5.5 is 6.0 μg / mL; and / or the concentration of CD4 PE-Cy7 is 1.5 μg / mL; and / or the concentration of CD19 APC is 2.3 μg / mL; and / or the concentration of CD8 APC-Cy7 is 6.3 μg / mL.

[0052] In some specific examples, in the above-mentioned antibody freeze-dried microsphere stock solution, the volume ratio of the fluorescence antibody solution to the lyoprotectant is 1:(2 - 4), for example, 1:2.5, 1:3 or 1:3.5, etc.

[0053] The embodiment of the present invention also provides an antibody freeze-dried microsphere for detecting lymphocyte subsets, which is obtained by freeze-drying the antibody freeze-dried microsphere stock solution in the present invention.

[0054] It should be noted that freeze-drying the above-mentioned antibody freeze-dried microsphere stock solution into an antibody freeze-dried microsphere is more convenient for storage, transportation and use. For example, during use, pipetting is not required, which can reduce the operation time of the operator and avoid errors caused by pipetting.

[0055] In some specific examples, in the above-mentioned antibody freeze-dried microsphere, the freeze-drying parameters include: the vacuum degree ≤ 20 Pa; and / or the cold trap temperature ≤ -55°C.

[0056] It should be noted that the freeze-drying method of freeze-drying the antibody freeze-dried microsphere stock solution into an antibody freeze-dried microsphere is well-known in the art. In the present invention, the vacuum degree ≤ 20 Pa and / or the cold trap temperature ≤ -55°C are preferably used. The antibody freeze-dried microspheres prepared under these parameters have a better appearance and better stability.

[0057] The embodiment of the present invention also provides a product for detecting lymphocyte subsets, which includes the antibody freeze-dried microsphere in the present invention.

[0058] It should be noted that the antibody freeze-dried microsphere in the present invention can be prepared into a detection reagent in the Figure 1A way to prevent breakage during transportation, etc. It is packed into an aluminum foil bag, and a desiccant is put in and sealed to obtain a detection kit.

[0059] To better understand the present invention, the content of the present invention will be further clarified below in combination with specific examples, but the content of the present invention is not limited to the following examples only.

[0060] I. Screening of antibody freeze-dried microsphere components

[0061] In the following examples, the raw material information of the lyoprotectant used is shown in Table 1 below.

[0062] Table 1 Raw material information of the lyoprotectant

[0063] Raw material Manufacturer Article number Trehalose Sangon Biotech A600966-0025 Mannitol Sangon Biotech A100122-0100 Polyethylene glycol 8000 Sangon Biotech A600433-0500 Bovine serum albumin Proliant 69100 Tween-20 Beyotime ST825-100mL ProClin300 Sigma-Aldrich 48912-U PBS Biosharp BL601A

[0064] In the following examples, the antibody information in the fluorescent antibody solution used is shown in Table 2 below.

[0065] Table 2 Antibody information in the fluorescent antibody solution

[0066] Antibody Clone number CD3FITC SK7 CD16PE B73.1 CD56PE NCAM16.2 CD45PerCP-Cy5.5 2D1 CD4PE-Cy7 SK3 CD19APC SJ25C1 CD8APC-Cy7 SK1

[0067] Example

[0068] Example 1

[0069] (1) Preparation of the lyoprotectant

[0070] Weigh accurately trehalose, mannitol, polyethylene glycol 8000, bovine serum albumin, Tween-20, and ProClin 300 according to the ingredients in Table 3, and dissolve them thoroughly with phosphate buffer solution (PBS solution, weigh 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4, dissolve them in 800 mL of distilled water, adjust the solution to pH 7.4 with HCl, and finally make up to 1 L with distilled water to obtain 0.01 M PBS buffer solution, the same below), then adjust the pH to 7.3, filter with a 0.22 μm filter membrane, and store it in a refrigerator at 4°C for later use.

[0071] Table 3 Components of the lyoprotectant in Example 1

[0072] Component name Dosage Content (%) Trehalose 12g 12 Mannitol 12g 12 Polyethylene glycol 8000 12g 12 Bovine serum albumin 3g 3 Tween-20 0.15g 0.15 ProClin300 0.05g 0.05 PBS solution 100mL /

[0073] (2) Preparation of the fluorescent antibody solution

[0074] Prepare the fluorescent antibody solution according to the ingredients in Table 4.

[0075] Table 4 Components of the fluorescent antibody solution in Example 1

[0076] Antibody Addition amount Concentration (ug / mL) CD3FITC 2.3 2.3 CD16PE 1.65 1.65 CD56PE 1.1 1.1 CD45PerCP-Cy5.5 6.0 6.0 CD4PE-Cy7 1.5 1.5 CD19APC 2.3 2.3 CD8APC-Cy7 6.3 6.3 PBS solution 1mL -

[0077] (3) Preparation of the lyophilized stock solution

[0078] Mix the fluorescent antibody solution and the lyoprotectant in a volume ratio of 1:3 to prepare the lyophilized stock solution.

[0079] (4) Preparation of antibody lyophilized microspheres

[0080] Use a pipette to drop 10 μL of the freeze-dried stock solution into liquid nitrogen to quickly freeze and solidify it into spherical preparations; place the spherical preparations in a stainless-steel tray, put it into a freeze-dryer, and set the freeze-drying parameters: vacuum degree 0.1 mbar, cold trap temperature -55 °C, the temperature of the partition plate rises slowly, and the freeze-drying time is 24 h; after freeze-drying, obtain antibody freeze-dried microspheres 0#, and wait to be sub-packed and stored.

[0081] Perform operations on the drying table to prevent the antibody freeze-dried microspheres from absorbing moisture and denaturing; sub-pack the antibody freeze-dried microspheres 0# into flow tubes, and add a stainless-steel wire mesh to press the microspheres so that they are always at the bottom of the bottle to prevent breakage during transportation, etc.; and tightly cover the cap of the flow tube to obtain the detection reagent of the present invention; then pack 50 portions into an aluminum foil bag, put in a desiccant and seal it for storage; the structure of the detection reagent is as Figure 1A shown, including a flow tube cap, a flow tube body, a stainless-steel mesh, and antibody freeze-dried microspheres; the physical diagram is as Figure 1B shown.

[0082] The appearance diagram of the antibody freeze-dried microspheres 0# is as Figure 1C shown, and the antibody freeze-dried microspheres 0# are in a uniform white spherical shape with a smooth surface.

[0083] Example 2

[0084] The difference between Comparative Example 5 and Example 1 is that the formula of the freeze-drying protective liquid is different. In Example 2, bovine serum albumin in Example 1 is replaced with casein (Macklin, S829595-100 g), and the others are the same as in Example 1, and antibody freeze-dried microspheres 5# are prepared.

[0085] In Example 2, when bovine serum albumin in Example 1 is replaced with casein, the appearance of the prepared antibody freeze-dried microspheres 5# has no obvious change compared with the antibody freeze-dried microspheres 0# (see Figure 2 ).

[0086] Example 3

[0087] The difference between Example 3 and Example 1 is that the formula of the freeze-drying protective liquid is different. In Example 3, Tween-20 in Example 1 is replaced with 2-hydroxypropyl-β-cyclodextrin (Macklin, H871853-5 g), and the others are the same as in Example 1, and antibody freeze-dried microspheres 7# are prepared.

[0088] In Example 3, when Tween-20 in Example 1 is replaced with 2-hydroxypropyl-β-cyclodextrin, the appearance of the prepared antibody freeze-dried microspheres 7# has no obvious change compared with the antibody freeze-dried microspheres 0# (see Figure 2 ).

[0089] Example 4

[0090] The difference between Example 4 and Example 1 is that the formula of the lyophilization protective solution is different. In Example 4, Tween-20 in Example 1 is replaced with Tween 80 (Sangon Biotech, A600562-0500), and the others are the same as in Example 1, and antibody lyophilized microspheres 6# are prepared.

[0091] In Example 4, Tween-20 was replaced with Tween-80, and the appearance of the prepared antibody lyophilized microspheres 6# showed slight non-spherical shape (see Figure 2 ).

[0092] Comparative Example

[0093] Comparative Example 1

[0094] The difference between Comparative Example 1 and Example 1 is that the formula of the lyophilization protective solution is different. Comparative Example 1 is a conventional lyophilization formula, specifically as follows: 5% trehalose, 5% mannitol, 1% BSA, 0.05% Tween-20; the components and preparation method of the fluorescent antibody solution are the same as in Example 1, and antibody lyophilized microspheres 1# are prepared.

[0095] The appearance of the antibody lyophilized microspheres 1# prepared in Comparative Example 1 showed obvious non-spherical shape (see Figure 2 ).

[0096] Comparative Example 2

[0097] The difference between Comparative Example 2 and Example 1 is that the formula of the lyophilization protective solution is different. In Comparative Example 2, trehalose in Example 1 is replaced with sucrose (Shanghai Reagent, 10021463), and the others are the same as in Example 1, and antibody lyophilized microspheres 2# are prepared.

[0098] After replacing trehalose with sucrose in Comparative Example 2, the appearance of the prepared antibody lyophilized microspheres 2# showed slight bubble-like voids (see Figure 2 ).

[0099] Comparative Example 3

[0100] The difference between Comparative Example 3 and Example 1 is that the formula of the lyophilization protective solution is different. In Comparative Example 3, polyethylene glycol 8000 is not contained, and the others are the same as in Example 1, and antibody lyophilized microspheres 3# are prepared.

[0101] Without adding polyethylene glycol 8000 in Comparative Example 3, the appearance of the prepared antibody lyophilized microspheres 3# showed obvious non-spherical shape (see Figure 2 ).

[0102] Comparative Example 4

[0103] The difference between Comparative Example 4 and Example 1 is that the formula of the lyophilization protective solution is different. In Comparative Example 4, polyethylene glycol 8000 is not contained, and the proportion of trehalose is 10%, and the proportion of mannitol is 5%, and the others are the same as in Example 1, and antibody lyophilized microspheres 8# are prepared.

[0104] In Comparative Example 4, polyethylene glycol 8000 was not contained, the proportion of trehalose was 10%, and the proportion of mannitol was 5%. The obtained antibody lyophilized microspheres #8 had obvious flaking on the surface, were not spherical, and were hollow inside (see Figure 2 ).

[0105] Comparative Example 5

[0106] The difference between Comparative Example 5 and Example 1 was that the formulation of the lyophilization protective solution was different. In Comparative Example 5, polyethylene glycol 8000 was not contained, the proportion of trehalose was 20%, and the proportion of mannitol was 10%. Other conditions were the same as those in Example 1, and the obtained antibody lyophilized microspheres #9 were prepared.

[0107] In Comparative Example 5, polyethylene glycol 8000 was not contained, the proportion of trehalose was 20%, and the proportion of mannitol was 10%. The appearance of the obtained antibody lyophilized microspheres #9 had no obvious change compared with that of the antibody lyophilized microspheres #0 (see Figure 2 ); however, it was found in subsequent experiments that the antibody lyophilized microspheres #9 prepared in Comparative Example 9 affected the hemolysis of hemolysin during the flow cytometry detection operation, resulting in insufficient hemolysis. After reaching the specified incubation time, it was still turbid (after sufficient hemolysis of normal whole blood, it should be clear). The possible reason was that the alcohol component was too high, affecting the cell osmotic pressure and the effect of the hemolysis solution.

[0108] Comparative Example 6

[0109] The difference between Comparative Example 6 and Example 1 was that the formulation of the lyophilization protective solution was different. In Comparative Example 6, polyethylene glycol 8000 in Example 1 was replaced with gelatin (Shanghai Reagent, 10010326), and the concentration was adjusted to 4%. Other conditions were the same as those in Example 1, and the obtained antibody lyophilized microspheres #4 were prepared.

[0110] In Comparative Example 6, polyethylene glycol 8000 was replaced with gelatin, and the concentration ratio was 4%. The appearance of the obtained antibody lyophilized microspheres #4 had obvious wrinkles and was not smooth (see Figure 2 ).

[0111] Comparative Example 7

[0112] The difference between Comparative Example 7 and Example 1 was that the formulation of the lyophilization protective solution was different. In Comparative Example 7, polyethylene glycol 8000 in Example 1 was replaced with gelatin, and the proportion of gelatin in Comparative Example 7 was 1%, the proportion of trehalose was 10%, and the proportion of mannitol was 5%. Other conditions were the same as those in Example 1, and the obtained antibody lyophilized microspheres #10 were prepared.

[0113] In Comparative Example 7, polyethylene glycol 8000 was replaced with gelatin, the proportion of gelatin was 1%, the proportion of trehalose was 10%, and the proportion of mannitol was 5%. The appearance of the obtained antibody lyophilized microspheres #10 had obvious wrinkles and the surface was not smooth (see Figure 2 ).

[0114] Comparative Example 8

[0115] The difference between Comparative Example 8 and Example 1 is that the lyophilization protective liquid formulations are different. In Comparative Example 8, polyethylene glycol 8000 in Example 1 is replaced with gelatin, and the proportion of gelatin in Comparative Example 8 is 4%, the proportion of trehalose is 10%, and the proportion of mannitol is 5%. The rest is the same as in Example 1, and antibody lyophilized microspheres 11# are prepared.

[0116] In Comparative Example 8, polyethylene glycol 8000 is replaced with gelatin, with the proportion of gelatin being 4%, the proportion of trehalose being 10%, and the proportion of mannitol being 5%. The appearance of the prepared antibody lyophilized microspheres 11# shows slight wrinkles and the surface is not smooth (see Figure 2 ).

[0117] From the above examples and comparative examples, it can be known that when comparing different saccharides, trehalose affects the formation of antibody lyophilized microspheres, and adding other saccharides may affect the appearance; when comparing different surfactants, Tween-20 and hydroxypropyl-β-cyclodextrin have the best forming effect, and Tween-80 is slightly worse; protein protectants such as bovine serum albumin and casein have no obvious effect on the appearance of antibody lyophilized microspheres; polyethylene glycol 8000 has an important influence on the formation of antibody lyophilized microspheres. In the case of not adding polyethylene glycol, the concentrations of trehalose and mannitol must be increased; gelatin has a certain influence on the formation of antibody lyophilized microspheres, and the higher the concentration, the better the forming effect, but gelatin is not easily soluble in water, which is not conducive to later operations, so this scheme is discarded.

[0118] II. Optimization of the Concentration of Core Components

[0119] In the following experiments, the method for detecting lymphocyte subsets using the existing BD lymphocyte subset detection reagent (manufacturer: BD, product number: 662967, the same hereinafter, hereinafter referred to as BD detection reagent) (which can also be operated according to the instruction manual) includes: Transfer 20 μL of the lymphocyte subset detection reagent (flow cytometry method - 6 colors) to the bottom of the reagent tube, and then aspirate 50 μL of fully mixed anticoagulated whole blood (venous blood drawn from an anticoagulant tube) and place it at the bottom of the test tube; cover the test tube and gently rotate to mix; incubate in the dark at 25 °C for 15 min; then add 450 μL of BD FACS lysin with a concentration of 1x (manufacturer: BD, product number: 349202, the same hereinafter) to the tube; cover the test tube cap and gently shake until evenly mixed; then incubate in the dark at 25 °C for 15 min to obtain the test sample; analyze the test sample using a flow cytometer.

[0120] In the following experiments, the method for detecting lymphocyte subsets using the detection reagent based on the antibody freeze-dried microspheres of the present invention (hereinafter referred to as the detection reagent of the present invention) is as follows: Pipette 50 μL of well-mixed anticoagulated whole blood and place it at the bottom of a test tube; cover the test tube and gently rotate it to mix; incubate in the dark at 25 °C for 15 min; then add 450 μL of BD FACS lysing solution with a concentration of 1x to the tube; cover the test tube cap and gently shake until evenly mixed; then incubate in the dark at 25 °C for 15 min to obtain the sample to be detected; analyze the sample to be detected using a flow cytometer.

[0121] (I) Optimization of trehalose concentration

[0122] According to the formula of Example 1, adjust the trehalose concentration (see Table 5), and keep other conditions the same as in Example 1 to prepare different antibody freeze-dried microspheres and the detection reagent of the present invention; then use different detection reagents of the present invention and BD detection reagent to detect the same healthy human peripheral blood sample respectively, and compare the differences in the mean fluorescence intensity (MFI) of CD3+, CD3+CD8+, CD3+CD4+, CD19+, CD16+CD56+ (analyzed using FlowJo 10.8.1); the results are shown in Table 5 below.

[0123] Table 5 Influence of trehalose concentration on MFI values of different antigens

[0124]

[0125] It can be seen from Table 5 above (in Table 5, BD is the BD detection reagent, the same below) that the results detected by the detection reagent of the present invention prepared with 12% trehalose are the closest to those of the BD detection reagent. For 8% and 10% trehalose concentrations, the overall coefficient of variation (CV) is too large, and the average fluorescence intensity of the trehalose antigen with 14% and 16% trehalose concentrations is abnormal.

[0126] (II) Optimization of mannitol concentration

[0127] According to the formula of Example 1, adjust the mannitol concentration (see Table 6), and keep other conditions the same as in Example 1 to prepare different antibody freeze-dried microspheres and the detection reagent of the present invention; then use different detection reagents of the present invention and BD detection reagent to detect the same healthy human peripheral blood sample respectively, and compare the differences in the mean fluorescence intensity (MFI) of CD3+, CD3+CD8+, CD3+CD4+, CD19+, CD16+CD56+ (analyzed using FlowJo 10.8.1); the results are shown in Table 6 below.

[0128] Table 6 Influence of mannitol concentration on MFI values of different antigens

[0129]

[0130] As can be seen from Table 6 above, comparing the test results of the reagents of BD, the test results of the test reagent of the present invention prepared with mannitol at a concentration of 12% are the closest and most consistent; for mannitol at concentrations of 8% and 10%, the overall coefficient of variation is too large; for mannitol with trehalose concentrations of 14% and 16%, the average fluorescence intensity of the mannitol antigen is abnormal and the coefficient of variation is relatively large.

[0131] (III) Optimization of polyethylene glycol 8000 concentration

[0132] According to the formula of Example 1, adjust the concentration of polyethylene glycol 8000 (see Table 7), and the others are the same as in Example 1, to prepare different antibody lyophilized microspheres and the test reagent of the present invention; then use different test reagents of the present invention and BD test reagents to detect the same healthy human peripheral blood sample respectively, and compare the differences in the average fluorescence intensity (MFI) of CD3+, CD3+CD8+, CD3+CD4+, CD19+, CD16+CD56+ (analyzed by FlowJo 10.8.1); the results are shown in Table 7 below.

[0133] Table 7 Influence of polyethylene glycol 8000 concentration on MFI values of different antigens

[0134]

[0135]

[0136] As can be seen from Table 7 above, comparing the test results of the reagents of BD, the test results of the test reagent of the present invention prepared with polyethylene glycol 8000 at a concentration of 12% are the closest and most consistent; for mannitol with polyethylene glycol 8000 concentrations of 8% and 10%, the average fluorescence intensity of the mannitol antigen is abnormal and the overall coefficient of variation is too large; for mannitol with polyethylene glycol 8000 concentrations of 14% and 16%, the average fluorescence intensity of the mannitol antigen is abnormal and the coefficient of variation is relatively large.

[0137] III. Application test of antibody lyophilized microspheres

[0138] In the following tests, the method for detecting lymphocyte subsets using the existing BD lymphocyte subset detection reagent (manufacturer: BD, product number: 662967, the same hereinafter, hereinafter referred to as the BD detection reagent) (which can also be operated according to the instructions) is as follows: Transfer 20 uL of the lymphocyte subset detection reagent (flow cytometry method - 6-color) to the bottom of the reagent tube, and then aspirate 50 uL of fully mixed anticoagulated whole blood (venous blood drawn from an anticoagulant tube) and place it at the bottom of the test tube; cover the test tube and gently rotate it to mix; incubate in the dark at 25 °C for 15 min; then add 450 uL of BD FACS lysing solution with a concentration of 1x (manufacturer: BD, product number: 349202, the same hereinafter) to the tube; cover the test tube cap and gently shake until evenly mixed; then incubate in the dark at 25 °C for 15 min to obtain the sample to be tested; analyze the sample to be tested using a flow cytometer.

[0139] In the following tests, the method for detecting lymphocyte subsets using the detection reagent based on the antibody freeze-dried microsphere 0# in the present invention (hereinafter referred to as the detection reagent of the present invention) is as follows: Aspirate 50 uL of fully mixed anticoagulated whole blood and place it at the bottom of the test tube; cover the test tube and gently rotate it to mix; incubate in the dark at 25 °C for 15 min; then add 450 uL of BD FACS lysing solution with a concentration of 1x to the tube; cover the test tube cap and gently shake until evenly mixed; then incubate in the dark at 25 °C for 15 min to obtain the sample to be detected; analyze the sample to be detected using a flow cytometer.

[0140] (1) Comparative test of antibody freeze-dried microspheres and BD detection reagent in the present invention

[0141] (1) Comparative test for detecting lymphocyte subsets in peripheral blood of a single healthy person

[0142] Use the detection reagent of the present invention and the BD detection reagent to detect the lymphocyte subsets in the peripheral blood of the same healthy person according to the corresponding detection methods described above. The detection results are as Figure 3 and Figure 4 and shown in Table 8 and Table 9. Among them, Figure 3 and Table 8 show the detection of lymphocyte subsets in the peripheral blood of healthy people based on the detection reagent of the present invention, Figure 4 and Table 9 show the detection of lymphocyte subsets in the peripheral blood of healthy people by the BD detection reagent;

[0143] Table 8 Detection of lymphocyte subsets in the peripheral blood of healthy people by the detection reagent of the present invention

[0144] Parameter Percent Value / AbsCnt LymphEvents 2503 CD3+ 69.04 0 CD3+CD8+ 35.08 0 CD3+CD4+ 30.80 0 CD3+CD4+CD8+ 1.20 0 CD16+CD56+ 18.74 0 D19+ 9.67 0 CD45+ 0 4 / 8Ratio(CD4+ / CD8+) 0.88

[0145] Table 9 Detection of lymphocyte subsets in the peripheral blood of healthy people by the BD detection reagent

[0146] Parameter Percent Value / AbsCnt LymphEvents 2505 CD3+ 70.86 0 CD3+CD8+ 35.37 0 CD3+CD4+ 31.10 0 CD3+CD4+CD8+ 0.80 0 CD16+CD56+ 17.88 0 D19+ 9.46 0 CD45+ 0 4 / 8Ratio(CD4+ / CD8+) 0.88

[0147] From Figure 3 and Figure 4 as well as Tables 8 and 9, it can be seen that the detection results of the peripheral blood lymphocyte subsets of healthy people by the detection reagent of the present invention are basically the same as those of the BD detection reagent.

[0148] (2) Comparison of the detection of peripheral blood lymphocyte subsets in multiple healthy people

[0149] The detection reagent of the present invention and the BD detection reagent respectively detect the peripheral blood lymphocyte subsets of the same batch of 30 healthy people according to the corresponding detection methods described above, so as to compare the consistency between the detection reagent of the present invention and the detection reagent (BD lymphocyte subset detection reagent) currently used in clinical applications.

[0150] The results are as Figure 5 shown, Figure 5 shows the Bland-Altman plot (difference analysis plot) of the proportion of CD3+ cells in the detection results of the two detection reagents. It can be seen from this that the average value of all the detection result differences is -0.168, and the average value of the differences ± 1.96 times the standard deviation is 2.491 and -2.827 respectively, and most of the differences are within this range, indicating that the consistency of the two detection reagents is good.

[0151] (2) Antibody freeze-dried microsphere stability test

[0152] The detection reagent of the present invention is placed at 4°C, room temperature (25°C), and 37°C respectively; among them, the detection reagent of the present invention placed at 4°C and room temperature (25°C) is used to detect the peripheral blood lymphocyte subsets of the same healthy person on the 90th day respectively according to the corresponding detection methods described above; the detection reagent of the present invention placed at 37°C is used to detect the peripheral blood lymphocyte subsets of healthy people on the 7th day according to the above detection method; and the BD detection reagent within the validity period is used to detect the same sample respectively, and the consistency of different reagents in detecting the same sample is observed; the detection results are shown in Table 10 and Figure 6 shown.

[0153] Table 10 Comparison of detection results under different temperature conditions

[0154]

[0155] As shown in Table 10 above, the detection results of the detection reagent in the present invention after storage at 4°C for 90 days, at room temperature (25°C) for 90 days, and at 37°C for 7 days are similar to those of the BD detection reagent within the validity period. In addition, all the currently approved lymphocyte subset detection reagents need to be stored at 4°C, and the antibody freeze-dried microsphere detection reagent of the present invention is still stable in performance after storage at room temperature for 90 days.

[0156] In addition, as Figure 6The single - person lymphocyte subset antibody freeze - dried microsphere detection reagent of the present invention was stored at 4°C for 90 days, at room temperature (25°C) for 90 days, and at 37°C for 7 days. The comparison of the detection results shows that the cell subset distribution is similar to that of the BD lymphocyte subset detection reagent.

[0157] (III) Compatibility test of the detection reagent of the present invention with absolute counting microspheres

[0158] This test aims to test the situation of the lymphocyte subsets in the peripheral blood of the same healthy person detected by the detection reagent of the present invention and the BD detection reagent respectively together with absolute counting microspheres, and to test whether the detection reagent of the present invention affects the lymphocyte subset ratio and the detection results of lymphocyte numbers. The specific steps are as follows:

[0159] Among them, the operation steps of the detection reagent of the present invention are as follows: Take out the antibody freeze - dried microsphere #0 and put it into an absolute counting tube (manufacturer: BD, product number: 662995); then aspirate 50 μL of fully mixed anticoagulated whole blood, cover the test tube and gently rotate to mix; incubate in the dark at room temperature (25°C) for 15 min; add 450 μL of BD FACS lysin with a concentration of 1x to the tube; cover the test tube cap and gently shake until evenly mixed; incubate in the dark at room temperature (25°C) for 15 min to obtain the sample to be detected, and the sample to be detected is analyzed using a flow cytometer.

[0160] The operation steps of the BD detection reagent are as follows: Take 20 μl of the lymphocyte subset detection reagent (flow cytometer method - 6 - color) and transfer it to the bottom of an absolute counting tube (manufacturer: BD, product number: 662995), then aspirate 50 μL of fully mixed anticoagulated whole blood, cover the test tube and gently rotate to mix; incubate in the dark at room temperature (25°C) for 15 min; add 450 μL of BD FACS lysin with a concentration of 1x to the tube; cover the test tube cap and gently shake until evenly mixed; incubate in the dark at room temperature (25°C) for 15 min to obtain the sample to be detected, and the sample to be detected is analyzed using a flow cytometer.

[0161] Table 11 Comparison of detection results under different conditions

[0162]

[0163] The analysis results are shown in Table 11 above. Among them, YT+B represents the result of the combined detection of the BD detection reagent and the absolute counting tube (manufacturer: BD, product number: 662995), DG+B represents the result of the combined detection of the detection reagent of the present invention and the absolute counting tube (manufacturer: BD, product number: 662995), and YT represents the detection result of the BD detection reagent (the function of using the BD detection reagent alone for testing is to exclude the error in detecting the proportion of cell subsets after adding the absolute counting microspheres). The detection results of each group are similar, indicating that the detection reagent of the present invention does not affect the performance of the absolute counting microspheres.

[0164] 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 them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Lyophilization protectant, characterized in that The lyophilization protectant includes trehalose, mannitol, polyethylene glycol 8000, protein protectant, surfactant, preservative and PBS solution.

2. The lyophilization protectant according to claim 1, wherein the protein protectant is selected from bovine serum albumin and / or casein; and / or the surfactant is selected from one or more of Tween-20, hydroxypropyl-β-cyclodextrin, and Tween-80; and / or the preservative is selected from ProClin300 and / or sodium azide.

3. The lyophilization protectant according to claim 1, wherein the mass fraction of trehalose is 10% - 14%; and / or the mass fraction of mannitol is 10% - 14%; and / or the mass fraction of polyethylene glycol 8000 is 10% - 14%; and / or the mass fraction of the protein protectant is 1% - 5%; and / or the mass fraction of the surfactant is 0.1% - 0.3%; and / or the mass fraction of the preservative is 0.01% - 0.1%.

4. The antibody freeze-dried microsphere stock solution is characterized in that, It includes the lyophilization protectant according to any one of claims 1 to 3 and a fluorescent antibody solution, and the fluorescent antibody solution contains specific labeled antibodies against lymphocyte subset surface antigens.

5. The lyophilized microsphere stock solution of the antibody according to claim 4, wherein The fluorescent antibody solution includes one or more of the following antibodies: CD3FITC, CD16PE, CD56PE, CD45PerCP-Cy5.5, CD4PE-Cy7, CD19APC or CD8APC-Cy7.

6. The antibody lyophilized microsphere stock solution according to claim 5, characterized in that, In the fluorescent antibody solution, the concentration of CD3FITC is 2 μg / mL - 3 μg / mL; and / or the concentration of CD16PE is 1 μg / mL - 2 μg / mL; and / or the concentration of CD56PE is 1 μg / mL - 2 μg / mL; and / or the concentration of CD45PerCP-Cy5.5 is 5.5 μg / mL - 6.5 μg / mL; and / or the concentration of CD4PE-Cy7 is 1 μg / mL - 2 μg / mL; and / or the concentration of CD19APC is 2 μg / mL - 3 μg / mL; and / or the concentration of CD8APC-Cy7 is 6 μg / mL - 7 μg / mL.

7. The antibody lyophilized microsphere stock solution according to any one of claims 4 to 6, characterized in that, The volume ratio of the fluorescent antibody solution to the lyophilization protectant is 1:(2 - 4).

8. Antibody freeze-dried microspheres for lymphocyte subset detection, characterized in that, It is obtained by lyophilizing the antibody lyophilized microsphere stock solution according to any one of claims 4 to 6.

9. The antibody lyophilized microspheres according to claim 8, wherein, The lyophilization parameters include: the vacuum degree ≤ 20 Pa; and / or the cold trap temperature ≤ -55°C.

10. A product for detecting lymphocyte subsets, characterized in that, It includes the antibody lyophilized microspheres according to claim 8 or 9.

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