A six-color lymphocyte subset absolute counting freeze-dried pellet and its preparation method and application

Abstract

The present invention provides a six-color lymphatic subpopulation absolute counting freeze-dried pellet and a preparation method and application thereof. The freeze-dried pellet is prepared by mixing a fluorescent microsphere solution, a fluorescent antibody solution and a freeze-dried protective solution with the simplest ingredients. The freeze-dried protective solution contains a blocking agent, a polymer, an antioxidant, a saccharide, a polyol, an amino acid, a surfactant and a buffer, and each component is screened in large quantities to finally prepare a six-color lymphatic subpopulation absolute counting freeze-dried pellet, which can be stored for a long time without adding a preservative, can ensure the stability of each component in the freeze-dried pellet, and can ensure the accuracy of the six-color lymphatic subpopulation absolute counting after long-term storage, and has the advantages of good stability, long shelf life, and convenient use.

Description

Technical Field

[0001] The invention relates to the field of freeze-drying technology, in particular to a method for preparing freeze-dried beads, and in particular to a six-color lymphoid subset absolute counting freeze-dried bead and a preparation method and application thereof. Background Art

[0002] Peripheral blood lymphocytes are important immune cells in the body, mainly including T lymphocytes, B lymphocytes, and natural killer cells (NK). The percentage of peripheral blood lymphocyte subpopulations and absolute cell counts play an important role in medical research and clinical testing. They help understand the immune function status in the body under different conditions, assist in clinical disease diagnosis, explore the pathogenesis of diseases, progression of the disease, prognosis, and monitor and guide clinical treatment plans. Especially in immunology research, the percentage and number of specific cells are indicators for evaluating immunological effects. Clinically, lymphocyte subpopulations are mainly analyzed by flow cytometry. In the detection of lymphocyte subpopulations, flow cytometry (FCM) uses a variety of different fluorescently labeled leukocyte surface marker monoclonal antibodies to fluorescently stain leukocytes, and then uses flow cytometry to distinguish the different leukocyte marker fluorescence positive patterns on each leukocyte one by one, so that leukocytes can be carefully classified and the content of various leukocyte subtypes can be obtained simply and quickly.

[0003] However, liquid antibodies are currently mostly used on the market for lymphoid subpopulation detection, and all of them are liquid six-color antibodies. Liquid antibodies have problems such as liquid collection deviation, which will affect the accuracy of the detection. In addition, liquid antibodies used for flow cytometry detection usually require fluorescent microsphere solutions, fluorescent antibody solutions, buffer solutions, etc. to be packaged separately, and then mixed and incubated when used. The detection process is cumbersome. At the same time, liquid antibodies also have problems such as high requirements for storage and transportation conditions and a short shelf life.

[0004] In order to facilitate storage, transportation and easier operation during the detection process, it is hoped that the liquid antibody detection reagent can be prepared into a freeze-dried pellet and directly used for the detection of flow cytometer antigens. In the prior art, 10% sucrose is used to freeze-dry anti-human liver cancer monoclonal antibody fragment antibodies. The freeze-dried product is a white cake with a fine sand surface and a loose and porous microstructure. However, the antibody freeze-dried into a white cake has the problem of adhesion to the cup wall and incomplete re-dissolution. Since there are many links that can be changed in the freeze-drying process, such as multi-component protective agents, pre-freezing cooling rate, vacuum degree, etc., which will affect the freeze-drying effect, a freeze-drying process with better effects is extremely needed in this field.

[0005] Invention patents CN117159480B (a recombinant human IFN-γ protein freeze-dried pellet and its preparation method and application) and CN118225545B (a recombinant human TNF-α protein freeze-dried pellet and its preparation method and application) respectively provide methods for preparing IFN-γ protein and TNF-α protein into freeze-dried pellets, which can be directly used as detection standards, and maintain high uniformity between different freeze-dried pellets, with a shelf life of more than 5 years. However, both freeze-dried pellets are prepared with only one protein and can only be used for standard products, not for cell detection, let alone for absolute counting of six-color lymphoid subsets.

[0006] Compared with standard proteins, the detection reagents for absolute counting of six-color lymphoid subsets by flow cytometry usually need to include multiple solutions such as fluorescent microsphere solution, fluorescent antibody solution, buffer solution, etc. After being prepared into freeze-dried beads, the long-term storage stability of all components must be guaranteed, not only the activity of the antibodies, but also the binding stability of the fluorescent markers with the microspheres and antibodies must be guaranteed, and the stability of various components in the buffer solution must be guaranteed. It is also necessary to provide a suitable detection environment when used for detection, and there must be no cross-influence between each other, so as to accurately achieve absolute counting of six-color lymphoid subsets.

[0007] CN118091117B (A protective agent for preparing freeze-dried beads and its application) discloses a freeze-dried bead that can achieve absolute counting of six-color lymphoid subsets, but the freeze-dried beads prepared by the method cannot be stored for a long time, and it is difficult to ensure the stability of the absolute counting of six-color lymphoid subsets after long-term storage.

[0008] Therefore, it is urgent to find a six-color lymphocyte subset absolute counting freeze-dried pellet to solve the currently difficult to overcome stability problem, enable long-term storage, and ensure the accuracy, sensitivity and specificity of detection after long-term storage. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a six-color lymphatic subpopulation absolute counting freeze-dried bead and its preparation method and application. The freeze-dried bead is prepared by mixing a fluorescent microsphere solution, a fluorescent antibody solution and a freeze-dried protective solution with simple ingredients. The freeze-dried protective solution contains a blocking agent, a polymer, an antioxidant, a sugar, a polyol, an amino acid, a surfactant and a buffer, and each component is screened in large quantities to finally prepare a six-color lymphatic subpopulation absolute counting freeze-dried bead, which can be stored for a long time without adding a preservative, and can ensure the stability of each component in the freeze-dried bead, and can ensure the accuracy of the six-color lymphatic subpopulation absolute counting after long-term storage, and has the advantages of good stability, long shelf life, and convenient use.

[0010] On the one hand, the present invention provides a six-color lymphoid subset absolute counting freeze-dried pellet, wherein the freeze-dried pellet is prepared by mixing a fluorescent microsphere solution, a fluorescent antibody solution and a freeze-dried protective solution into a freeze-dried stock solution, and then freeze-dried into a pellet; the freeze-dried protective solution comprises a blocking agent, a sugar, a polyol, a polymer, an antioxidant, a surfactant and a buffer; the blocking agent comprises skim milk powder and arginine; the antioxidant comprises sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate; the antibodies in the fluorescent antibody solution comprise CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody.

[0011] The six-color lymphatic subpopulation absolute counting freeze-dried beads prepared by the present invention contain all the detection reagents for flow cytometry detection. The prepared freeze-dried beads are separately packed into flow tubes and packaged separately. When used for detection, it is only necessary to directly add samples to the flow tube containing the freeze-dried beads to perform six-color lymphatic subpopulation absolute counting by flow cytometer. Moreover, the freeze-dried beads can not only ensure the stability and detection activity of each reagent component after freeze-drying, but also provide a suitable detection environment for sample detection, provide guarantee for accurate detection of samples, eliminate batch differences caused by the transfer process of multiple liquid reagents, and the most important thing is that they can be stored for a long time, with a shelf life of more than 5 years, and can be used as needed, which greatly simplifies the detection process, and the detection process is very convenient and efficient.

[0012] Existing freeze-dried beads containing fluorescent microspheres and fluorescent antibodies usually need to add preservatives to improve their stability, but the addition of preservatives has a certain impact on the detection accuracy after long-term storage, resulting in the prepared freeze-dried beads being difficult to store for a long time, which has serious defects. The six-color lymphoid subset absolute counting freeze-dried beads prepared by the present invention adopt a suitable combination of blocking agent and antioxidant, so that no preservatives need to be added, eliminating the impact of preservatives on later detection.

[0013] There are significant differences in the antioxidant effects of different antioxidants on freeze-dried beads. When the antioxidant is prepared by combining L-sodium ascorbate and disodium ethylenediaminetetraacetic acid dihydrate, it is more versatile and has a simple formula. The oxidation effect has a synergistic effect and can be used for the combined detection of multiple antibodies with better results. There is no need to add preservatives, which will not affect the accuracy of the detection.

[0014] The amino acids in the freeze-dried pellets can not only act as a blocking agent to ensure the accuracy and specificity of the detection, but also maintain the change of pH value during freeze-drying. The amino acids include one or more of proline, glycine, glutamic acid, histidine, arginine, 4-hydroxyproline, L-serine, β-alanine, lysine hydrochloride, lysine, and sarcosine, among which the most preferred amino acid is arginine.

[0015] In some embodiments, the polymer includes one or more of dextran, polysucrose, polyethylene glycol, hydroxyethyl starch, gum arabic, gelatin, polyvinyl pyrrolidone, cellulose, β-cyclodextrin, methylcellulose, maltodextrin, and cross-linked dextran; the sugar includes one or more of sucrose, glucose, trehalose, fructose, galactose, lactose, maltose, chitosan, mannose, ribose, xylose, and raffinose; the polyol includes one or more of sorbitol, glycerol, mannitol, xylitol, and inositol; the surfactant includes one or more of Tween 80, Tween 20, sodium dodecyl sulfate, hydroxypropyl-β-cyclodextrin, Triton X-100, sucrose fatty acid ester, and fatty alcohol polyoxyethylene ether; the buffer includes one or more of citrate buffer, Tris-HCL buffer, and phosphate buffer.

[0016] In the six-color lymphoid subset absolute counting freeze-dried beads prepared by the present invention, the addition of polymers can significantly accelerate the freezing speed of the solution, thereby significantly improving the detection activity of all detection reagents after freeze-drying, and improving the detection accuracy and sensitivity; sugars help to protect the protein structure; surfactants can prevent the fluorescent microspheres from aggregating and adhering; polyols mainly play the role of fillers and also help to improve the stability of proteins.

[0017] The buffer in the freeze-dried pellet should not only provide a stable freeze-drying environment for the components in the freeze-dried pellet, but also provide a suitable detection environment for all detection reagent combinations when used for detection, thereby ensuring the smooth progress of the flow cytometer detection process.

[0018] Furthermore, the polymer includes polysucrose; the sugar includes trehalose and ribose; the antioxidant includes sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate; the polyol includes xylitol; the surfactant includes hydroxypropyl-β-cyclodextrin; and the buffer includes phosphate buffer.

[0019] Different polymers, sugars, antioxidants, polyols, surfactants and buffers have significantly different effects on the preparation of freeze-dried pellets. The present invention obtains the simplest and most effective formula of polymers, sugars, antioxidants, polyols, amino acids, surfactants and buffers through a large number of screenings.

[0020] Furthermore, in terms of mass percentage, the freeze-dried protective solution includes 0.1-2% skim milk powder, 1-5% polysucrose, 0.1-1% L-sodium ascorbate, 0.1-1% ethylenediamineacetic acid disodium salt dihydrate, 1-10% trehalose, 0.5-5% ribose, 2-8% xylitol, 0.5-3% arginine, 1-5% polysucrose, and 0.005-1 mol / L phosphate buffer.

[0021] In some embodiments, calculated by mass percentage, the freeze-dried protective solution includes 1% skimmed milk powder, 3% polysucrose, 0.5% sodium L-ascorbate, 0.5% disodium ethylenediamineacetic acid dihydrate, 5% trehalose, 1% ribose, 1% xylitol, 1% arginine, 0.5% hydroxypropyl-β-cyclodextrin, and 0.01 mol / L phosphate buffer.

[0022] Furthermore, the volume ratio of the fluorescent microsphere solution, the fluorescent antibody solution and the freeze-dried protective solution is (1-5):(2-10):(2-10).

[0023] In some embodiments, the volume ratio of the fluorescent microsphere solution, the fluorescent antibody solution and the lyophilization protection solution is 1:2:2.

[0024] Furthermore, the pH of the freeze-dried stock solution is 7.0-7.4.

[0025] The pH of the lyophilized stock solution also has a critical influence on the lyophilized pellets for antigen detection provided by the present invention, because it is not only related to the effect of lyophilization stability, but also to the accuracy of sample detection. The most appropriate pH value must be selected to ensure the stability and detection activity of each reagent component after lyophilization, and to provide a suitable detection environment for sample detection.

[0026] Furthermore, the antibodies in the fluorescent antibody solution include: PerCP-cy5.5-CD45 monoclonal antibody with clone number 2D1, FITC-CD3 monoclonal antibody with clone number SK7, PE-cy7-CD4 monoclonal antibody with clone number SK3, APC-cy7-CD8 monoclonal antibody with clone number SK1, APC-CD19 monoclonal antibody with clone number SJ25-C1, PE-CD16 monoclonal antibody with clone number B73.1, and PE-CD56 monoclonal antibody with clone number 5.1H11.

[0027] Monoclonal antibodies with different clone numbers were evaluated by antibody titration for their performance in flow cytometry, and it was found that there were obvious differences in the staining index, the degree of grouping was different, and the ability to bind to antigens on the cell surface was also different. Therefore, the present invention screened monoclonal antibodies with different clone numbers, and selected the monoclonal antibody with the best staining effect to prepare the microsphere solution of the coupled antibody and the antibody solution with the label, and prepared them into freeze-dried beads.

[0028] Furthermore, the fluorescent microspheres are fluorescent counting microspheres produced by Suzhou Weidu Company.

[0029] In another aspect, the present invention provides a method for preparing a freeze-dried pellet for detecting antigen content, the method comprising the following steps:

[0030] (1) preparing a fluorescent antibody solution, wherein the antibodies include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody;

[0031] (2) Preparation of freeze-drying protective solution: skim milk powder, arginine, polysucrose, trehalose, ribose, sodium L-ascorbate, disodium ethylenediaminetetraacetic acid dihydrate, xylitol, hydroxypropyl-β-cyclodextrin and phosphate buffer to prepare a freeze-drying protective solution;

[0032] (3) Mix the freeze-dried protective solution, fluorescent microsphere solution and fluorescent antibody solution to form a freeze-dried stock solution;

[0033] (4) Dropping the lyophilized stock solution into liquid nitrogen through a bead dropping device to freeze it into spherical frozen beads;

[0034] (5) The frozen beads are prepared into freeze-dried pellets for detecting antigen content through a freeze-drying process.

[0035] Furthermore, the antibodies in the fluorescent antibody solution of step (1) include: the CD45 monoclonal antibody is the PerCP-cy5.5-CD45 monoclonal antibody with a clone number of 2D1; the CD3 monoclonal antibody is the FITC-CD3 monoclonal antibody with a clone number of SK7; the CD4 monoclonal antibody is the PE-cy7-CD4 monoclonal antibody with a clone number of SK3; the CD8 monoclonal antibody is the APC-cy7-CD8 monoclonal antibody with a clone number of SK1; the CD19 monoclonal antibody is the APC-CD19 monoclonal antibody with a clone number of SJ25-C1; the CD16 monoclonal antibody is the PE-CD16 monoclonal antibody with a clone number of B73.1; and the CD56 monoclonal antibody is the PE-CD56 monoclonal antibody with a clone number of 5.1H11.

[0036] Furthermore, the freeze-drying process in step (5) includes the following steps:

[0037] b-1: Set the temperature of the shelf to -50°C and maintain this temperature for 2 hours;

[0038] b-2: Set the temperature of the shelf to -50°C, evacuate the freeze dryer (vacuum degree less than 20 Pa) and maintain this temperature for 4.5 hours;

[0039] b-3: Set the temperature of the shelf to -40°C and maintain this temperature for 10 hours;

[0040] b-4: Set the temperature of the shelf to -30°C and maintain this temperature for 10 hours;

[0041] b-5: Set the temperature of the shelf to -20°C and maintain this temperature for 10 hours;

[0042] b-6: Set the temperature of the shelf to -10°C and maintain this temperature for 1 hour;

[0043] b-7: Set the temperature of the shelf to 0°C and maintain this temperature for 1 hour;

[0044] b-8: Set the temperature of the shelf to 10°C and maintain this temperature for 1 hour;

[0045] b-9: Set the temperature of the shelf to 25°C and maintain this temperature for 2-8 hours.

[0046] Studies have shown that freeze-drying using this procedure can reduce the water content of the six-color lymphoid subset absolute counting freeze-dried pellets, improve the appearance, and improve the grouping effect, which is beneficial to improving the accuracy of the six-color lymphoid subset absolute counting.

[0047] In yet another aspect, the present invention provides a six-color lymphocyte subset absolute counting method, wherein the method uses the freeze-dried beads as described above for detection.

[0048] On the other hand, the present invention provides a freeze-dried protective solution, which includes a blocking agent, a sugar, a polyol, a polymer, an antioxidant, a surfactant and a buffer; wherein the blocking agent includes skimmed milk powder and arginine, the polymer includes polysucrose, the sugar includes trehalose and ribose, the antioxidant includes sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate, the polyol includes xylitol, the amino acid includes arginine, the surfactant includes hydroxypropyl-β-cyclodextrin, and the buffer includes phosphate buffer.

[0049] On the other hand, the present invention provides a freeze-dried protective solution for preparing a reagent for improving the detection stability of six-color lymphocyte subset absolute counting freeze-dried beads, wherein the freeze-dried protective solution includes a blocking agent, a sugar, a polyol, a polymer, an antioxidant, a surfactant and a buffer; wherein the blocking agent includes skimmed milk powder and arginine, the polymer includes polysucrose, the sugar includes trehalose and ribose, the antioxidant includes sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate, the polyol includes xylitol, the amino acid includes arginine, the surfactant includes hydroxypropyl-β-cyclodextrin, and the buffer includes phosphate buffer.

[0050] On the other hand, the present invention provides a freeze-dried protective solution for preparing a reagent for improving the detection accuracy and repeatability of six-color lymphoid subset absolute counting freeze-dried beads, wherein the six-color lymphoid subset absolute counting detection includes the detection of CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody; the freeze-dried protective solution includes a blocking agent, a sugar, a polyol, a polymer, an antioxidant, a surfactant and a buffer; wherein the blocking agent includes skimmed milk powder and arginine, the polymer includes polysucrose, the sugar includes trehalose and ribose, the antioxidant includes sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate, the polyol includes xylitol, the amino acid includes arginine, the surfactant includes hydroxypropyl-β-cyclodextrin, and the buffer includes phosphate buffer.

[0051] On the other hand, the present invention provides a use of a freeze-dried protective solution for preparing six-color lymphoid subset absolute counting freeze-dried beads without the need for preservatives, wherein the six-color lymphoid subset absolute counting freeze-dried beads include a fluorescent microsphere solution, a fluorescent antibody solution and a freeze-dried protective solution; the freeze-dried protective solution includes a blocking agent, a sugar, a polyol, a polymer, an antioxidant, a surfactant and a buffer; wherein the blocking agent includes skimmed milk powder and arginine, the polymer includes polysucrose, the sugar includes trehalose and ribose, the antioxidant includes sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate, the polyol includes xylitol, the amino acid includes arginine, the surfactant includes hydroxypropyl-β-cyclodextrin, and the buffer includes phosphate buffer.

[0052] Compared with existing products, the present invention has the following beneficial effects:

[0053] (1) Provided is a six-color lymphocyte subset absolute counting freeze-dried pellet, which contains fluorescent antibodies and fluorescent microspheres, which can ensure the stability of each component in the freeze-dried pellet and ensure accurate detection. The components are simple, easy to prepare, easy to use, and can be stored for a long time. After long-term storage, the accuracy and batch-to-batch precision of the six-color lymphocyte subset absolute counting can still be guaranteed;

[0054] (2) Through component selection and concentration optimization, the appearance of the prepared freeze-dried beads is smooth and flat; when used to detect the percentage of each component and cell concentration of lymphoid subpopulations in blood samples, the clustering effect is better, and the accuracy and precision are high;

[0055] (3) It is used for the absolute counting of the percentage of six-color lymphocyte subset cells and cell concentration, etc. It has the advantages of no need to add preservatives, good stability, long shelf life, and easy use;

[0056] (4) The prepared freeze-dried pellets are easy to transport and store, while maintaining stable biological properties, providing an ideal storage solution;

[0057] (5) The use of the protective agent of the present invention to prepare freeze-dried beads containing fluorescent antibodies and fluorescent microspheres can avoid errors in liquid extraction, which may lead to deviations in test results. In addition, the beads will not adhere to the inner wall of the container, thus avoiding incomplete reconstitution of powdered freeze-dried products and uneven mixing.

[0058] (6) The protective agent of the present invention is used to prepare freeze-dried beads containing fluorescent antibodies and fluorescent microspheres, which can quickly and easily detect the percentage of lymphoid subpopulations and the number of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is an example of the detection result of the absolute count of six-color lymphoid subsets in Example 1;

[0060] Figure 2 This is a cell cluster diagram with the highest staining index of different FITC-CD3 monoclonal antibodies in Example 12;

[0061] Figure 3 This is a comparative cell clustering diagram of FITC-CD3 monoclonal antibodies of different brands of SK7 clone numbers in Example 12;

[0062] Figure 4 This is a cell cluster diagram with the highest staining index of different PE-cy7-CD4 monoclonal antibodies in Example 12;

[0063] Figure 5 This is a comparative cell clustering diagram of different brands of SK3 clone number PE-cy7-CD4 monoclonal antibodies in Example 12;

[0064] Figure 6 This is a cell cluster diagram with the highest staining index of different APC-cy7-CD8 monoclonal antibodies in Example 12;

[0065] Figure 7 This is a comparative cell clustering diagram of different brands of SK1 clone number APC-cy7-CD8 monoclonal antibodies in Example 12;

[0066] Figure 8 This is a cell cluster diagram with the highest staining index of different PE-CD16 monoclonal antibodies in Example 12;

[0067] Fig. 9 This is a comparative cell clustering diagram of different brands of B73.1 clone PE-CD16 monoclonal antibodies in Example 12;

[0068] Fig.10 This is a cell cluster diagram with the highest staining index of different APC-CD19 monoclonal antibodies in Example 12;

[0069] Fig.11This is a comparative cell clustering diagram of different brands of SJ25-C1 clone number APC-CD19 monoclonal antibodies in Example 12;

[0070] Fig.12 This is a cell cluster diagram with the highest staining index of different PE-CD56 monoclonal antibodies in Example 12;

[0071] Fig.13 This is a cell cluster diagram with the highest staining index of different PerCP-cy5.5-CD45 monoclonal antibodies in Example 12;

[0072] Fig.14 This is a comparative cell clustering diagram of different brands of 2D1 clone number PerCP-cy5.5-CD4 monoclonal antibodies in Example 12;

[0073] Fig.15 This is a clustering diagram of different fluorescent microsphere flow cytometry detection in Example 13;

[0074] Fig.16 This is the appearance of the freeze-dried pellets prepared by different freeze-drying procedures in Example 14. DETAILED DESCRIPTION

[0075] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be understood by people with general skills in the field to which the present invention belongs. The reagents used in the present embodiment, unless otherwise stated, are all known products, obtained by purchasing commercially available products.

[0076] Example 1: Preparation of six-color lymphocyte subset absolute counting freeze-dried beads

[0077] 1. The preparation method of the six-color lymphocyte subset absolute counting freeze-dried beads provided in this embodiment is as follows:

[0078] (1) Prepare fluorescent antibody solution;

[0079] CD45-Percp-Cy5.5 (6.25 μg / mL) (clone 2D1, purchased from biolegend, catalog number: 368504), CD3-FITC (25 μg / mL) (clone SK7, purchased from biolegend, catalog number: 344804), CD4-PE-Cy7 (3.125 μg / mL) (clone SK3, purchased from biolegend, catalog number: 344612), CD8-APC-Cy (12.5 μg / mL) (clone SK1, Equal volumes of CD45-PE (purchased from biolegend, catalog number: 344714), CD19-APC (6.25 μg / mL) (clone number SJ25-C1, purchased from biolegend, catalog number: 363006), CD16-PE (25 μg / mL) (clone number B73.1, purchased from biolegend, catalog number: 360704), and CD56-PE (10 μg / mL) (clone number 5.1H11, purchased from biolegend, catalog number: 362508) were mixed to prepare a fluorescent antibody solution.

[0080] (2) Preparation of freeze-dried protective solution:

[0081] First, according to the phosphate buffer formula (as shown in Table 1), accurately weigh sodium chloride (purchased from Sinopharm, item number: 10019318), potassium chloride (purchased from Sinopharm, item number: 10016318), disodium hydrogen phosphate (purchased from Sinopharm, item number: 20040618), and potassium dihydrogen phosphate (purchased from Sinopharm, item number: 10017608) and add them to 1L of purified water, and mix thoroughly to obtain a clear, transparent, colorless liquid.

[0082] Then, according to the formula of protective agent #1 (as shown in Table 2), sodium L-ascorbate (purchased from Sinopharm, item number: TA0539500G), disodium ethylenediaminetetraacetic acid dihydrate (purchased from Sinopharm, item number: 10009717), hydroxypropyl-β-cyclodextrin (purchased from Sinopharm, item number: C297565000), skimmed milk powder (purchased from Yuanye Biotechnology, item number: R21306), trehalose (purchased from McLean, item number: D81847) were accurately taken. 4), ribose (purchased from McLean, item number: L817253), xylitol (purchased from Sinopharm, item number: 63011983), arginine (purchased from McLean, item number: L800637), polysucrose (purchased from Sinopharm, item number: XW0126873858012) in a beaker, and add phosphate buffer to the beaker, fully dissolve, stir and mix, and filter with a 0.22μm filter membrane to obtain a light yellow, clear and transparent solution. And use 0.01mol / L hydrochloric acid solution or 0.01mol / L sodium hydroxide solution to adjust the pH value to 7.2±0.2 (preferably 7.2 in this embodiment) to prepare a freeze-dried protective solution, which is placed in a refrigerator at 2-8°C for storage.

[0083] Table 1. Phosphate buffer formula

[0084]

[0085] Table 2, formula of protective agent #1

[0086]

[0087] Among them, sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate are antioxidants for anti-oxidation, which can make the freeze-dried reagent have a longer stability; skim milk powder and arginine are blocking agents for blocking non-specific binding sites, and arginine can maintain the overall pH value without a significant change when the water gradually sublimates during the freeze-drying process; hydroxypropyl-β-cyclodextrin is used as a surfactant to prevent the aggregation of fluorescent microspheres in the solution; trehalose and ribose are used as cryoprotectants to form a special protective film on the surface of the freeze-dried product, which can prevent the freeze-dried material from changing its structure and becoming inactive in a low-temperature environment, and significantly improve the stability after freeze-drying; xylitol is used as a filler to support the molecular structure; polysucrose is used as a polymer protective agent to increase the viscosity of the solution and the glass transition during freeze-drying, which can effectively prevent the product from becoming inactive during the freeze-drying process, and polysucrose and the above-mentioned xylitol have the function of supporting the structure and imparting properties, which can make the freeze-dried beads form normally during the freeze-drying process; phosphate buffer is used as a buffer to maintain an appropriate pH value to prevent the antibody from being denatured and inactivated when the pH changes.

[0088] (3) First, mix the freeze-dried protective solution with the fluorescent antibody solution, and then add the fluorescent microsphere solution (2.5×104 The six-color lymphocyte subset absolute counting reagent freeze-dried stock solution was prepared by mixing fluorescent microsphere solution, fluorescent antibody solution and freeze-dried protective solution in a volume ratio of 1:2:2, and the six-color lymphocyte subset absolute counting reagent freeze-dried stock solution was fully oscillated and mixed before dropping beads.

[0089] (4) Dropping the lyophilized stock solution into liquid nitrogen through a bead dropping device to freeze it into spherical frozen beads;

[0090] Adjust the parameters of the bead dropper so that the volume of each drop is within the range of 10μL±0.5μL, place the freeze-dried stock solution on the oscillator and keep oscillating. Use a protective agent to rinse the catheter, empty the liquid in the catheter, and inhale the prepared solution. After the solution fills the catheter, pour about four-fifths of the volume of liquid nitrogen into the thermos cup of the liquid separation device. Start the automatic function of the liquid separation device, and the instrument begins to separate the liquid. After the separation is completed, transfer the frozen beads to a single-layer stainless steel cup prepared separately for the next step. The frozen beads must be in a liquid nitrogen environment during the transfer process to avoid melting.

[0091] (5) The frozen beads are prepared into freeze-dried pellets for detecting antigen content through a freeze-drying process.

[0092] The temperature at which the lyophilized stock solution is completely frozen (including all kinds of chemical reagents in it) is around -40°C, which is called the eutectic point. The temperature of the first section must be set below the eutectic point temperature. Secondly, in a low-temperature environment, most of the frozen water in the sample exists in the form of free water. The temperature rise interval cannot be too large, which will affect the activity of the protein. The general interval is 5-10 minutes. The first section is the pre-freezing stage to completely freeze the sample. Sections 2 to 7 are sublimation drying. The purpose of this section is mainly to directly sublimate the free water in the sample. At this time, the remaining frozen water in the sample is attached to the surface of the protein structure in the form of bound water. At this time, the next stage of analytical drying, i.e., sections 8-9, is carried out. At this time, because the free water has been sublimated, the temperature can quickly rise to room temperature to remove the remaining bound water.

[0093] The freeze dryer needs to be started at least one hour in advance to ensure that the ambient temperature meets the equipment operation requirements. After precooling to the precooling temperature, the pellets are placed in the freeze dryer for freeze drying. The specific steps are as follows:

[0094] a: Turn on the freeze dryer, precool the freeze dryer to the precooling temperature (less than -20°C), and then place the frozen pellets in the freeze dryer;

[0095] b: Set the temperature of the shelves in the freeze dryer for the first stage of drying. The specific steps are:

[0096] b-1: Set the temperature of the shelf to -50°C and maintain this temperature for 2 hours;

[0097] b-2: Set the temperature of the shelf to -50°C, evacuate the freeze dryer (vacuum degree less than 20 Pa) and maintain this temperature for 4.5 hours;

[0098] b-3: Set the temperature of the shelf to -40°C and maintain this temperature for 10 hours;

[0099] b-4: Set the temperature of the shelf to -30°C and maintain this temperature for 10 hours;

[0100] b-5: Set the temperature of the shelf to -20°C and maintain this temperature for 10 hours;

[0101] b-6: Set the temperature of the shelf to -10°C and maintain this temperature for 1 hour;

[0102] b-7: Set the temperature of the shelf to 0°C and maintain this temperature for 1 hour;

[0103] b-8: Set the temperature of the shelf to 10°C and maintain this temperature for 1 hour;

[0104] b-9: Set the temperature of the shelf to 25°C and maintain this temperature for 2-8 hours;

[0105] c: After the instrument b-9 step has been running for at least 2 hours, restore the freeze dryer to normal atmospheric pressure and then remove the freeze-dried pellets from the instrument.

[0106] (6) Storage of freeze-dried pellets

[0107] Turn on the dehumidifier in the room and wait until the humidity in the room drops below 40% before proceeding to the next step. Turn off the freeze dryer. Dispense the freeze-dried pellets into vials as needed, then add stoppers, cover with aluminum caps and press with a capping machine to obtain freeze-dried pellets #1, which are stored in an environment of 2-8°C away from light.

[0108] 2. Detection and analysis

[0109] (1) Method for absolute counting of six-color lymphocyte subsets using freeze-dried beads

[0110] Detection principle: The prepared lyophilized beads are individually packaged into flow tubes, and 50μL of whole blood sample is added to the flow tube. The lyophilized beads in the tube are dissolved, releasing a known number of fluorescent microspheres. The fluorescent antibodies in the reagent specifically bind to the leukocyte surface antigens, and the fluorescent microspheres do not participate in any reaction. In the flow cytometer, blood cells are surrounded and constrained by the sheath fluid to form a single cell suspension that is ejected from the flow chamber nozzle at high speed. When irradiated by the excited light, forward scattered light (ESC), side scattered light (SSC) and fluorescent signals are generated. The forward angle scattered light is closely related to the square of the diameter of the measured cell, reflecting the size of the cell volume. The side angle scattered light is more sensitive to the refractive index of the cell membrane, cytoplasm and nuclear membrane, and can provide information about the fine structure and particle properties of the cell. By detecting and analyzing the fluorescent signal, the lymphocytes (CD45+ cells) in the peripheral blood are confirmed, and the percentage of the subpopulations of lymphocytes, T lymphocytes (CD3* cells), B lymphocytes (CD19- cells) and natural killer (NK) cells (CD16+CD56) cells) and CD4- and CD8+ subpopulations of I lymphocytes are determined. At the same time, by comparing the number of cells and the number of microspheres, the absolute count (individuals / u) of positive cells in the sample can be determined. The test results are as follows: Figure 1 As shown. Figure 1 The P2 gate in the upper left picture indicates that the fluorescent microspheres themselves do not participate in any reaction, and the other CD45+, CD3+, CD4+, CD8+, CD19+, and CD16 / 56+ gates are positive cells in the test sample that bind to the corresponding antibodies.

[0111] In the product, the total number of fluorescent microspheres is known. According to the number of fluorescent microspheres collected in the test results and the number of positive cells, the number of cells and concentration in the sample can be calculated by the following formula.

[0112] (Number of cells in the target area / Number of microspheres in the target area) × (Number of microspheres tested each time / Volume of sample to be tested (μL)) = Target cell concentration (cells / μL)

[0113] (2) Accuracy and repeatability test of freeze-dried pellets

[0114] The six-color lymphatic subpopulation absolute counting freeze-dried pellet reagent prepared in this embodiment has a smooth surface, uniform size, and no adhesion between the freeze-dried pellets. Compared with the reagents on the market that are mostly refrigerated for transportation, the six-color lymphatic subpopulation absolute counting freeze-dried pellet reagent can be transported at room temperature or high temperature; at the same time, the operation is more convenient, and the test process takes a short time; at the same time, the reagent dosage for each person is 1 freeze-dried pellet, which reduces the sampling error when sampling liquid reagents, and greatly reduces the experimental error.

[0115] 1) Positive percentage deviation test: Detect the fluorescence value of each positive cell in the sample and compare it with the deviation of the test sample of the commercially available reagent BDMultitest™ 6-color TBNK (Cat. No.: 662967) (a six-color liquid detection kit for absolute counting of lymphoid subpopulations). The relative deviation should be within the range of ±10%.

[0116] After testing, the positive percentage deviation test results of the six-color lymphoid subset absolute counting freeze-dried beads prepared in this embodiment are shown in Table 3 (because CD45 is used to screen lymphocytes, all lymphocytes express CD45, so the positive percentage of CD45 is 100%, so the deviation data of the CD45 positive percentage is not reflected in the subsequent tables).

[0117] Table 3. Positive percentage

[0118]

[0119] 2) Cell concentration deviation test: Detect the concentration of positive cells in each sample and compare it with the deviation of the test sample using the commercially available reagent BD counter tube (Cat. No.: 340334). The relative deviation of the positive cell concentration should be within the range of ±10%.

[0120] After testing, the cell concentration deviation test results of the six-color lymphoid subset absolute counting freeze-dried beads prepared in this example are shown in Table 4 (CD16 / 56 is the same type of cells used for testing).

[0121] Table 4. Cell concentration

[0122]

[0123] 3) Repeatability test: Use freeze-dried pellets to test the same sample 10 times and calculate its coefficient of variation (CV). The coefficient of variation (CV) = standard deviation (SD) / mean value. The coefficient of variation (CV) should be ≤8%.

[0124] After testing, the repeatability test results of the six-color lymphocyte subset absolute counting freeze-dried beads prepared in this example are shown in Table 5.

[0125] Table 5. Repeatability test

[0126]

[0127] 4) Accuracy test: Test quality control sample I (purchased from Beckman, catalog number: 6607098) and quality control sample II (purchased from Beckman, catalog number: 6607077), and their positive percentage and cell concentration should be within the target value range of quality control blood. The target value range of quality control blood is shown in Table 6. The results of the accuracy test are shown in Table 7 (the following table only considers the subpopulations of CD45 cells - CD3\CD4\CD8\CD16 / 56\CD19 analysis of positive percentage and cell concentration, and does not consider CD45 cells).

[0128] Table 6. Target value range of quality control samples

[0129]

[0130] Table 7. Accuracy test

[0131]

[0132] 5) Long-term stability: The freeze-dried pellets are stored at 2-8°C. The quality control samples are tested using freeze-dried pellets. After one year of storage, the positive percentage and cell concentration of quality control samples I and II should be within the target value range of the quality control blood.

[0133] After testing, the long-term stability test results of the six-color lymphocyte subset absolute counting freeze-dried beads prepared in this example are shown in Table 8.

[0134] Table 8. Long-term stability test

[0135]

[0136] According to Tables 3 to 8, the six-color lymphoid subset absolute counting freeze-dried pellet reagent prepared in this embodiment is compared with similar products on the market (BD Multitest™ 6-color TBNK (Cat. No.: 662967), D counting tube (Cat. No.: 340334), which is a liquid reagent). When the same sample is tested, the deviation of the results is within the range of ±10%, and the quality control samples are all within the target value range. It can be considered that the accuracy of the test results is high. At the same time, the same sample was tested repeatedly, and its variability (CV) was within 8%, which can be considered to have good repeatability of the test results. After 1 year of storage, the quality control sample was tested, and the result was still within the quality control target value range, indicating that the stability and detection activity of the freeze-dried pellets after 1 year of storage are very good.

[0137] Example 2: Comparison of similar products

[0138] This example uses the freeze-dried beads prepared in Example 1, the freeze-dried beads for absolute counting of six-color lymphoid subsets prepared by the method provided in Example 1 of CN118091117B in the prior art, and the liquid six-color reagent + counting microspheres for comparative testing.

[0139] 1. High temperature thermal acceleration stability comparison test

[0140] Comparison of high temperature thermal acceleration stability experiment: The freeze-dried beads prepared in Example 1, the freeze-dried beads for absolute counting of six-color lymphoid subsets prepared by the method provided in Example 1 of CN118091117B in the prior art, and the liquid six-color reagent + counting microspheres were placed in the corresponding environment for storage according to Table 9, and the same reagents were stored at 45°C for one month (equivalent to 2-8°C for one year), and the deviation of the test results (including cell concentration deviation and positive percentage deviation) compared with the same reagents stored at 2-8°C for one month. The design of the comparative experiment is shown in Table 9, and the results are shown in Tables 10 to 12.

[0141] Table 9, High temperature thermal acceleration stability

[0142]

[0143] Table 10. Comparison of thermal acceleration stability of six-color lymphoid subset absolute count freeze-dried pellets (Example 1)

[0144]

[0145] Table 11. Comparison of thermal acceleration stability of six-color lymphocyte subset absolute count freeze-dried pellets (CN118091117B)

[0146]

[0147] Table 12. Comparison of thermal acceleration stability of liquid six-color reagent + counting microspheres

[0148]

[0149] Comparing Table 10 and Table 12, it can be seen that the results of the absolute counting of the six-color lymphoid subsets of the freeze-dried beads prepared in Example 1 after being stored at 45°C for one month are basically the same as those stored at 2-8°C for one month, and are stable within a deviation of 10%. However, after the liquid reagent is thermally accelerated at high temperature, its test results are significantly lower than those stored at 2-8°C, and most samples are lower by more than 10%.

[0150] By comparing Table 10 and Table 11, it can be seen that the freeze-dried beads prepared by CN118091117B are not stable after high-temperature acceleration, and many sample counts are lower than 10%; the freeze-dried beads prepared in Example 1 have significantly improved high-temperature storage stability compared with the freeze-dried beads prepared by CN118091117B, and all deviations are within 5%, which proves that the six-color lymphoid subset absolute counting freeze-dried beads prepared in Example 1 have significantly better long-term storage stability than similar products, and the detection accuracy after long-term storage is significantly improved, and can be stably stored for at least one year.

[0151] 2. Comparison of experimental operation time

[0152] Experimental operation time comparison test method: The reference reagent liquid six-color reagent + counting microspheres and six-color antibody freeze-dried beads were used for experimental operation comparison, and 50 samples were tested at the same time, and the time required was recorded. Considering that the operation after staining and incubation is consistent, only the operation time before staining and incubation was recorded. The results are shown in Table 13.

[0153] Table 13. Comparison of experimental operation time

[0154]

[0155] As can be seen from Table 13, the freeze-dried pellet test takes less time and is more efficient.

[0156] 3. Repeatability test:

[0157] The reference reagent liquid six-color reagent + counting microspheres, the six-color antibody freeze-dried beads prepared in Example 1, and the six-color lymphoid subset absolute counting freeze-dried beads prepared in Example 1 of CN118091117B were used for experimental operation comparison. The same sample was tested using the above reagents, and 10 tubes were tested in parallel to evaluate the repeatability of the two reagents. The results are shown in Table 14.

[0158] Table 14. Repeatability

[0159]

[0160]

[0161] It can be seen from Table 14 that the overall repeatability of cell concentration in the liquid reagent is worse than that of the freeze-dried pellet, and the CV value is higher, generally greater than 10%. The repeatability of the freeze-dried pellet prepared by CN118091117B is also unsatisfactory, while the CV value of the freeze-dried pellet in Example 1 is basically less than 5%, indicating that the repeatability of the freeze-dried pellet is higher, which effectively reduces the experimental error and eliminates the batch difference.

[0162] Example 3: Effect of preservatives

[0163] In this example, freeze-dried beads were prepared according to the method provided in Example 1, wherein the effects of preservatives, sealants, and antioxidants on the high temperature accelerated stability of freeze-dried beads were compared, and freeze-dried beads of six-color lymphoid subset absolute counts prepared by the method provided in Example 1 of CN118091117B were compared. The specific experimental scheme is shown in Table 15. The deviation of the test results (including cell concentration deviation and positive percentage deviation) of the same reagent stored at 45°C for one month (equivalent to storage at 2-8°C for one year) compared with storage at 2-8°C for one month is shown in Table 15. Since the test results of each monoclonal antibody are basically the same, this example only uses the deviation of CD3 as typical data (average value is taken after 10 repetitions) for comparison.

[0164] Table 15. Experimental plan

[0165]

[0166] According to Table 15, it can be seen that in comparison with Group 1 and Group 2, the freeze-dried beads prepared in Example 1, after adding preservatives, not only did not improve their stability, but also had a downward trend. The reason may be that the addition of preservatives has a certain impact on the detection accuracy of the absolute count of the six-color lymphoid subsets. It can also be explained that the freeze-dried beads prepared in Example 1 do not need to add preservatives in the freeze-dried protection solution. In comparison with Group 3 and Group 4, the freeze-dried beads prepared by CN118091117B, when not containing preservatives, show a serious decline in high temperature accelerated stability, indicating that the freeze-dried beads of this formula must contain preservatives, otherwise the stability is poor. At the same time, it can also be explained that the freeze-dried beads prepared in accordance with Example 1 can be stored for a long time without adding preservatives, and the stability is significantly improved.

[0167] Example 4: Effect of buffer type and solution pH on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagent

[0168] Protein has the properties of an amphoteric electrolyte, which can react with both acid and base. When the solution is at a certain pH value, the positive charge and negative charge carried by the protein are exactly equal, and the protein is not electrically charged. The pH value of the solution at this time is called the isoelectric point of the protein. Proteins are more stable in an environment at their isoelectric point. However, in an extreme pH environment, high static charge causes strong intramolecular electrical repulsion, which causes the protein molecules to unfold. During the freezing process of the protein solution, the concentration of the solution gradually increases. At high concentrations, the pH value of the solution may change, and in severe cases, it may cause protein denaturation, thereby directly inactivating the biological product. Therefore, in the formulation of the lyophilization protectant for biological products, an appropriate amount of buffer is often added.

[0169] The main function of phosphate buffer and pH adjustment is to provide a suitable environment for antibodies and maintain the stability of antibodies and microspheres in a liquid environment. Different buffers have different protective effects on antibodies and fluorescent microspheres, which will cause the antibodies to degrade and become inactivated directly in a liquid environment without being freeze-dried.

[0170] 1) Effect of buffer type on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagent

[0171] In this example, starting from the buffer in the freeze-dried protective solution, a six-color lymphoid subpopulation absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of different buffers on the preparation of the six-color lymphoid subpopulation absolute counting freeze-dried bead reagent were investigated (see Table 16 for details). The stability of the protective agent prepared with different buffers for preparing the fluorescent antibody solution stored at 45°C for 4h (fluorescence value deviation before and after storage) is shown in Table 17, and the fluorescence value deviation shall not exceed 10%. Different freeze-dried beads were prepared and stored at 45°C in the dark for one month (equivalent to storage at room temperature for 1 year) and five months (equivalent to storage at room temperature for 5 years), and the values ​​were compared with those before storage, respectively, to investigate the effects of the cell concentration deviation and positive percentage deviation of the freeze-dried beads for the six-color lymphoid subpopulation absolute counting before and after long-term storage, and the results are shown in Tables 18 and 19.

[0172] Table 16. Screening of buffer solutions

[0173]

[0174] Table 17. Stability of protective agents prepared with different buffers for preparing fluorescent antibody solutions after 4 h storage

[0175]

[0176] Table 18. Comparison of the stability of six-color lymphocyte subset absolute count freeze-dried pellets after one-month thermal accelerated storage with different buffers

[0177]

[0178] Table 19. Comparison of the stability of six-color lymphocyte subset absolute count freeze-dried pellets after five-month thermal accelerated storage with different buffers

[0179]

[0180] It can be seen from Table 17 that before freeze-drying and storage at 45°C for 4 hours, the storage effects of different buffers are different, among which phosphate buffer has the best storage effect and the fluorescence value remains stable.

[0181] It can be seen from Tables 18-19 that different buffers have a significant effect on the shelf life of the six-color lymphoid subset absolute counting freeze-dried pellet reagent, among which the phosphate buffer has the best effect. After being stored at 45°C in the dark for one month (equivalent to storage at room temperature for one year) and five months (equivalent to storage at room temperature for five years), the cell concentration deviation and positive percentage deviation of the six-color lymphoid subset absolute counting were both within 5%. It can also be shown that the shelf life of the six-color lymphoid subset absolute counting freeze-dried pellet provided by the present invention can be as long as more than 5 years.

[0182] 2) Effect of solution pH on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagent

[0183] In this example, the final pH value of the prepared solution was confirmed, and a six-color lymphoid subset absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When preparing the freeze-dried stock solution, the effects of different pH values ​​of the solution on the preparation of the six-color lymphoid subset absolute counting freeze-dried beads were investigated (see Table 20 for details). The stability of freeze-dried beads prepared by freeze-dried stock solutions prepared with different pH values ​​at 45°C for 4 hours (fluorescence value deviation before and after storage) is shown in Table 21, and the fluorescence value deviation shall not exceed 10%. Different freeze-dried beads were prepared, and each group of freeze-dried beads was tested according to the method provided in Example 1. They were stored in the dark at 45°C for one month (equivalent to storage at room temperature for 1 year) and five months (equivalent to storage at room temperature for 5 years), and the values ​​were compared with those before storage, and the positive percentage deviation and cell concentration deviation were calculated. The results are shown in Tables 22-23 respectively; at the same time, in this example, except for the different pH values ​​of the solution, the other conditions are the optimal conditions in Example 1.

[0184] Table 20, pH value screening

[0185]

[0186] Table 21. Stability of fluorescent antibody solution prepared with different pH protective agents after 4 h storage

[0187]

[0188] Table 22. Comparison of the stability of six-color lymphocyte subset absolute count freeze-dried pellets after heat accelerated storage for one month at different pH values

[0189]

[0190] Table 23. Comparison of the stability of six-color lymphocyte subset absolute count freeze-dried pellets after five-month thermal accelerated storage at different pH values

[0191]

[0192] According to Table 21, before freeze-drying and storage at 45°C for 4 hours, different pH values ​​have different effects on the preservation. The best preservation effect is achieved when the pH value is 7.0-7.4, and the fluorescence value remains stable.

[0193] According to Tables 22-23, it can be seen that different solution pH values ​​have different effects on the preparation of six-color lymphoid subset absolute count freeze-dried pellet reagents, and the effect on the shelf life is very obvious. The best effect is when the pH value is 7.0-7.4. The cell concentration deviation and positive percentage deviation of the six-color lymphoid subset absolute count are within 5% when stored at 45°C in the dark for one month (equivalent to 1 year at room temperature) and five months (equivalent to 5 years at room temperature). This example further verifies the repeatability, proving that the repeatability is also within 5%.

[0194] Example 5: Effect of the type of antioxidant on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagent

[0195] This example starts with the antioxidant in the freeze-dried protective solution, and prepares the six-color lymphoid subset absolute counting freeze-dried bead reagent according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of different antioxidants on the preparation of the six-color lymphoid subset absolute counting freeze-dried beads were investigated (see Table 24 for details), thereby preparing different freeze-dried beads, which were stored in the dark at 45°C for one month (equivalent to 1 year of storage at room temperature), and compared with the values ​​before storage, the deviations were calculated, and the effects of the cell concentration deviation and the positive percentage deviation of the six-color lymphoid subset absolute counting of the freeze-dried beads before and after long-term storage were investigated. Since the detection results of each monoclonal antibody were basically the same, this example only used the deviation of CD3 as typical data (repeated 10 times and averaged), and the results are shown in Table 24.

[0196] Table 24. Screening of antioxidants

[0197]

[0198] According to Table 24, it can be seen that different antioxidants have different effects on the detection activity of the six-color lymphoid subset absolute counting freeze-dried pellet reagent after long-term stability storage. Among them, the combination of L-sodium ascorbate and disodium ethylenediaminetetraacetic acid dihydrate has a better effect, which can make the freeze-dried pellet have better high-temperature storage stability, and still have good detection activity after long-term storage, and the cell concentration deviation and positive percentage deviation are minimal.

[0199] Example 6: Effect of the type of surfactant on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagent

[0200] In the whole freeze-drying process of biological products, surfactants can not only reduce the freezing and dehydration denaturation caused by the ice-water interfacial tension during freezing and dehydration, but also play the role of wetting agent and re-wrinkling agent for active components during rehydration. In this example, starting from the surfactant in the freeze-dried protection solution, the six-color lymphatic subpopulation absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When preparing the freeze-dried protection solution, the effects of different surfactants on the preparation of the six-color lymphatic subpopulation absolute counting freeze-dried bead test were investigated (see Table 25 for details), so that different freeze-dried beads were prepared, stored in the dark at 45°C for one month (equivalent to 1 year of storage at room temperature), and compared with the values ​​before storage, and the deviation was calculated to investigate the influence of cell concentration deviation and positive percentage deviation on the six-color lymphatic subpopulation absolute counting of freeze-dried beads before and after long-term storage. Since the detection results of each monoclonal antibody are basically the same, this example only uses the deviation of CD3 as typical data (repeated 10 times and averaged) for comparison, and the results are shown in Table 25.

[0201] Table 25. Screening of surfactants (0.5%)

[0202]

[0203] According to Table 25, different surfactants have different effects on the detection activity of the six-color lymphoid subset absolute counting freeze-dried pellet reagent after high-temperature stability storage. Among them, the hydroxypropyl-β-cyclodextrin used has a better effect and can still be used for accurate detection after long-term storage, with the smallest cell concentration deviation and positive percentage deviation.

[0204] Example 7: Effect of blocking agent on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagent

[0205] In this example, starting from the blocking agent in the freeze-dried protective solution, a six-color lymphoid subset absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of different blocking agents on the preparation of the six-color lymphoid subset absolute counting freeze-dried bead test were investigated (see Table 26 for details), thereby preparing different freeze-dried beads, which were stored in the dark at 45°C for one month (equivalent to 1 year of storage at room temperature), and compared with the values ​​before storage, the deviations were calculated, and the effects of the cell concentration deviation and the positive percentage deviation of the six-color lymphoid subset absolute counting of the freeze-dried beads before and after long-term storage were investigated. Since the detection results of each monoclonal antibody were basically the same, this example only used the deviation of CD3 as typical data (repeated 10 times and averaged), and the results are shown in Table 26.

[0206] Table 26. Screening of blocking agents

[0207]

[0208] According to Table 26, different blocking agents have different effects on the detection activity of the six-color lymphoid subset absolute counting freeze-dried beads after high-temperature stability storage. Among them, skimmed milk powder and arginine used in the blocking agent have better effects and can still be used for accurate detection after long-term storage, with the smallest cell concentration deviation and positive percentage deviation.

[0209] Example 8: Effect of carbohydrates on the preparation of six-color lymphocyte subset absolute counting freeze-dried pellet reagents

[0210] As far as the freeze-drying process is concerned, both reducing disaccharides and non-reducing disaccharides have a good protective effect. However, during the storage process of biological products after freeze-drying, the presence of reducing disaccharides will cause the freeze-dried products to undergo Maillard reaction (protein browning reaction), which will eventually lead to the deterioration of the freeze-dried products. Therefore, in the freeze-drying formula of food, medicine and organisms, sucrose and trehalose are the two most commonly used protective agents. However, which specific sugar is more suitable for preparing six-color lymphoid subset absolute counting freeze-dried beads still needs to be studied to find out. In this example, starting from the sugars in the freeze-dried protective solution, six-color lymphoid subset absolute count freeze-dried beads were prepared according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of different sugars on the preparation of the six-color lymphoid subset absolute count freeze-dried beads were investigated (see Table 27 for details), thereby preparing different freeze-dried beads, which were stored in the dark at 45°C for one month (equivalent to 1 year of storage at room temperature), and compared with the values ​​before storage, the deviations were calculated, and the effects of the cell concentration deviation and positive percentage deviation of the six-color lymphoid subset absolute count of the freeze-dried beads before and after long-term storage were investigated. Since the detection results of each monoclonal antibody were basically the same, this example only used the deviation of CD3 as typical data (repeated 10 times and averaged), and the results are shown in Table 27.

[0211] Table 27. Screening of sugars

[0212]

[0213] According to Table 27, different sugars have different effects on the detection activity of the six-color lymphoid subset absolute counting freeze-dried pellet reagent after high-temperature stability storage. The combination of trehalose and ribose has a better effect and can still be used for accurate detection after long-term storage, with the smallest cell concentration deviation and positive percentage deviation.

[0214] Example 9: Effect of polyols on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagents

[0215] In this example, starting from the polyols in the freeze-dried protective solution, a six-color lymphoid subset absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of different polyols on the preparation of the six-color lymphoid subset absolute counting freeze-dried bead reagent were investigated (see Table 28 for details), thereby preparing different freeze-dried beads, which were stored in the dark at 45°C for one month (equivalent to 1 year of storage at room temperature), and compared with the values ​​before storage, the deviations were calculated, and the effects of the cell concentration deviation and the positive percentage deviation of the six-color lymphoid subset absolute counting of the freeze-dried beads before and after long-term storage were investigated. Since the detection results of each monoclonal antibody were basically the same, this example only used the deviation of CD3 as typical data (average value was taken after 10 repetitions) for comparison, and the results are shown in Table 28.

[0216] Table 28. Screening of polyols (5%)

[0217]

[0218] It can be seen from Table 28 that different polyols have different effects on the preparation of six-color lymphocyte subset absolute counting freeze-dried pellet reagents, among which xylitol has a better effect.

[0219] Example 10: Effect of polymers on the preparation of six-color lymphocyte subset absolute counting lyophilized pellet reagents

[0220] In this example, starting from the polymers in the freeze-dried protective solution, a six-color lymphoid subset absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of different polymers on the preparation of the six-color lymphoid subset absolute counting freeze-dried bead reagent were investigated (see Table 29 for details), thereby preparing different freeze-dried beads, storing them in the dark at 45°C for one month (equivalent to 1 year of storage at room temperature), and comparing them with those before storage, calculating the deviation, and investigating the influence of the cell concentration deviation and positive percentage deviation of the six-color lymphoid subset absolute counting of the freeze-dried beads before and after long-term storage. Since the detection results of each monoclonal antibody are basically the same, this example only uses the deviation of CD3 as typical data (average value is taken after 10 repetitions) for comparison, and the results are shown in Table 29.

[0221] Table 29. Screening of polymers (3%)

[0222]

[0223] According to Table 29, different polymers have different effects on the detection activity of the six-color lymphoid subset absolute counting freeze-dried bead reagent after high-temperature stability storage. Among them, the polysucrose used has a better effect and can still be used for accurate detection after long-term storage, with the smallest cell concentration deviation and positive percentage deviation.

[0224] Example 11: Effect of the content of polymers and polyols on the formation of freeze-dried pellets

[0225] In this example, a six-color lymphoid subset absolute count freeze-dried bead reagent is prepared according to the method provided in Example 1. When preparing the freeze-dried protective solution, the effects of the dosage of xylitol and polysucrose on the preparation of the six-color lymphoid subset absolute count freeze-dried bead reagent are investigated (see Table 30 for details), thereby preparing different freeze-dried beads, and evaluating their appearance. The freeze-dried beads should be smooth and round without obvious wrinkles. At the same time, the positive percentage deviation, cell concentration deviation and repeatability results of each group of freeze-dried beads are detected according to the method provided in Example 1. Since the detection results of each monoclonal antibody are basically the same, this example only uses the deviation of CD3 as a typical data (average value is taken after 10 repetitions), and the results are shown in Table 30; at the same time, in this example, except for the different contents of xylitol and polysucrose, the other conditions are the optimal conditions in Example 1.

[0226] Table 30. Screening of polymer and polyol content

[0227]

[0228] According to Table 30, it can be seen that different contents of xylitol and polysucrose have an impact on the appearance and performance of the preparation of the six-color lymphocyte subset absolute counting freeze-dried pellet reagent. The optimal amounts of xylitol and polysucrose are 5% and 3%, respectively.

[0229] Example 12: Screening of antibodies

[0230] For antibody screening, this embodiment uses titration of antibodies to calculate the staining index to efficiently and accurately screen antibodies suitable for flow cytometry. The core of antibody titration is to quantitatively evaluate the performance of antibodies in flow cytometry applications by combining experiments and mathematical models. The following is a further detailed description of the above steps:

[0231] Antibody titration:

[0232] a. Serially dilute the antibody stock solution to be screened, for example, by 2-fold, 3-fold or 10-fold serial dilution to obtain a series of antibody solutions with different concentrations.

[0233] b. Add different concentrations of antibodies to the sample and react for a certain period of time under appropriate conditions to allow the antibodies to bind to antigens on the cell surface.

[0234] c. Set up a control group, that is, cells treated with diluent only without adding antibodies, to exclude the influence of nonspecific binding.

[0235] d. Use flow cytometry to detect the stained cells and record the fluorescence intensity of each sample.

[0236] e. Based on the fluorescence intensity data detected by the flow cytometer, subtract the background noise (i.e., the average fluorescence intensity of the control group) to obtain the corrected fluorescence intensity value.

[0237] f. For the antibodies with different concentrations of CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody, the staining index SI = (mean fluorescence intensity of positive cells - mean fluorescence brightness of negative cells) / 2 times the rSD value of negative cells was calculated. For CD45 monoclonal antibody, the ratio of the fluorescence value of lymphocytes to the fluorescence value of neutrophils was calculated.

[0238] g. The higher the staining index, the better the staining effect of the antibody at this concentration. The higher the ratio of lymphocyte fluorescence value to neutrophil fluorescence value, the more obvious the grouping and the better the staining effect.

[0239] The monoclonal antibodies needed for the six-color antibody raw materials include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody. The above scheme is used to screen out the best monoclonal antibody as the raw material for subsequent freeze-drying and other experiments.

[0240] 1) FITC-CD3 monoclonal antibody screening:

[0241] In this example, FITC-CD3 monoclonal antibodies with different clone numbers are used. The cell grouping diagrams with the highest staining index of different FITC-CD3 monoclonal antibodies are shown in FIG. Figure 2 , where P2 is a CD3-positive cell and P3 is a CD3-negative cell; from left to right and from top to bottom, the clone numbers of the antibodies are OKT3, HIT3a, SK7, UCHT1, CD3-12, 145-2C11, and 7D6. The staining index results are shown in Table 31.

[0242] Table 31. Highest staining index values ​​of different clone numbers of FITC-CD3 monoclonal antibodies

[0243]

[0244] from Figure 2 It can be seen that each clone number of the antibody has a good classification of CD3 negative and positive cells, and it is impossible to directly distinguish the difference between each antibody from the classification. However, it can be seen from Table 31 that the FITC-CD3 antibody with clone number SK7 has the highest staining index, which indicates that this clone number has the best staining effect.

[0245] At the same time, we compared the FITC-CD3 monoclonal antibody of other brands with SK7 clone number. See the cell clustering diagram for comparison. Figure 3 , from left to right the brands are BioLegend and BD. The staining index results are shown in Table 32.

[0246] Table 32. Highest staining index values ​​of different brands of FITC-CD3 monoclonal antibody SK7 clone

[0247]

[0248] from Figure 3 As can be seen from Table 32, the FITC-CD3 results of the two brands of SK7 clones are basically the same, which shows that the antibodies with the same clone number from different manufacturers have the same effect. Based on the comprehensive results, the clone number of the FITC-CD3 monoclonal antibody is SK7.

[0249] 2) PE-cy7-CD4 monoclonal antibody screening:

[0250] The cell clustering diagrams with the highest staining index of different PE-cy7-CD4 monoclonal antibodies are shown in Figure 4 ,in Figure 4 P2 is a CD4 positive cell, and P3 is a CD4 negative cell. The clone numbers of the antibodies from left to right and from top to bottom are RPA-T4, SK3, L200, A161A1, OKT4, MEM241, RM4-5, and S3.5. The staining index results are shown in Table 33.

[0251] Table 33. Highest staining index values ​​of different clone numbers of PE-cy7-CD4 monoclonal antibody

[0252]

[0253] from Figure 4 It can be seen that the antibodies of clone numbers RPA-T4, L200, A161A1, and OKT4 have a long tail on CD4-negative cells, and the effect is not good. However, it can be seen from Table 33 that the PE-cy7-CD4 antibody of clone number SK3 has the highest staining index, which indicates that this clone has the best staining effect.

[0254] At the same time, we compared the PE-cy7-CD4 monoclonal antibody of other brands of SK3 clones. The cell clustering diagram is shown in Figure 5 , from left to right the brands are BioLegend, BD, and Abcam. The staining index results are shown in Table 34.

[0255] Table 34. Highest staining index values ​​of different brands of PE-cy7-CD4 monoclonal antibody SK7 clone

[0256]

[0257] from Figure 5As can be seen from Table 34, the results of SK3 clone number PE-cy7-CD4 of the three brands are basically the same, which shows that the antibodies with the same clone number from different manufacturers have the same effect. Based on the results, the clone number of PE-cy7-CD4 monoclonal antibody is selected as SK3.

[0258] 3) APC-cy7-CD8 screening:

[0259] The cell clustering diagrams with the highest staining index of different APC-cy7-CD8 monoclonal antibodies are shown in Figure 6 , where P4 is a CD8 positive cell and P5 is a CD8 negative cell; from left to right and from top to bottom, the clone numbers of the antibodies are RPA-T8, SK1, HIT8a, MEM-31, 53-6.7, and RFT8. The staining index results are shown in Table 35.

[0260] Table 35. Maximum staining index values ​​of different clone numbers of APC-cy7-CD8 monoclonal antibodies

[0261]

[0262] from Figure 6 It can be seen that the CD8 negative cells of the antibody with clone number RPA-T8 have a long tail and the effect is not good. However, it can be seen from Table 35 that the APC-cy7-CD8 antibody with clone number SK1 has the highest staining index, which indicates that this clone has the best staining effect.

[0263] At the same time, we compared the APC-cy7-CD8 monoclonal antibody of other brands of SK1 clones. See the cell clustering diagram for comparison. Figure 7 , from left to right the brands are BioLegend and BD. The staining index results are shown in Table 36.

[0264] Table 36. Highest staining index values ​​of different brands of APC-cy7-CD8 monoclonal antibody SK1 clone

[0265]

[0266] from Figure 7 As can be seen from Table 36, the results of SK1 clone number APC-cy7-CD8 of the two brands are basically the same, which shows that the antibodies with the same clone number from different manufacturers have the same effect. Based on the results, the clone number of APC-cy7-CD8 monoclonal antibody is SK1.

[0267] 4) PE-CD16 screening:

[0268] The cell clustering diagrams with the highest staining index of different PE-CD16 monoclonal antibodies are shown in Figure 8 ,in Figure 8P2 is a CD16 positive cell, and P3 is a CD16 negative cell. The clone numbers of the antibodies from left to right and from top to bottom are CLB-gran11.5, B73.1, 3G8, QA18A13, EPR22409-124, SP175, J5511, and MEM-154. The staining index results are shown in Table 37.

[0269] Table 37. Highest staining index values ​​of different clone numbers of PE-CD16 monoclonal antibodies

[0270]

[0271] from Figure 8 As can be seen from Table 37, clone B73.1 has the highest PE-CD16 antibody staining index and better positive and negative cell grouping, which indicates that this clone has the best staining effect.

[0272] At the same time, we compared the PE-CD16 monoclonal antibody of other brands with clone number B73.1. The cell clustering diagram is shown in Fig. 9 , from left to right the brands are BD and BioLegend. The staining index results are shown in Table 38.

[0273] Table 38. Highest staining index values ​​of different brands of PE-CD16 monoclonal antibody B73.1 clone

[0274]

[0275] from Fig. 9 As can be seen from Table 38, the results of the two brands of PE-CD16 with clone number B73.1 are basically the same, which shows that antibodies with the same clone number from different manufacturers have the same effect. Based on the results, the clone number of PE-CD16 monoclonal antibody is selected as B73.1.

[0276] 5) APC-CD19 screening:

[0277] The cell clustering diagrams with the highest staining index of different APC-CD19 monoclonal antibodies are shown in Fig.10 , where P2 is a CD19 positive cell and P3 is a CD19 negative cell; from left to right and from top to bottom, the clone numbers of the antibodies are HIB19, QA18A75, 4G7, EPR5906, LT19, 6D5, and SJ25-C1. The staining index results are shown in Table 39.

[0278] Table 39. Maximum staining index values ​​of different clone numbers of APC-CD19 monoclonal antibodies

[0279]

[0280] from Fig.10As can be seen from Table 39, the APC-CD19 antibody staining index of clone SJ25-C1 is the highest, and its positive and negative cell clustering is also better, which indicates that this clone has the best staining effect.

[0281] At the same time, we compared the APC-CD19 monoclonal antibody of another brand, SJ25-C1 clone number, and the cell clustering diagram is shown in Fig.11 , from left to right the brands are eBioscience, BD, and BioLegend. The staining index results are shown in Table 40.

[0282] Table 40. Highest staining index values ​​of different brands of APC-CD19 monoclonal antibody SJ25-C1 clone number

[0283]

[0284] from Fig.11 As can be seen from Table 40, the results of APC-CD19 with clone number SJ25-C1 of three brands are basically the same, which shows that the antibodies with the same clone number from different manufacturers have the same effect. Based on the comprehensive results, the clone number of APC-CD19 monoclonal antibody is selected as SJ25-C1.

[0285] 6) PE-CD56 screening:

[0286] The cell clustering diagrams with the highest staining index of different PE-CD56 monoclonal antibodies are shown in Fig.12 , where P4 is a CD56-positive cell and P5 is a CD56-negative cell; the clone numbers of the antibodies from left to right and from top to bottom are MY31, B159, R19-760, QA18A21, QA17A16, 5.1H11, MEM-188, EPR21827, LT56, EP2567Y, CMSSB, TULY56, and C5.9. The staining index results are shown in Table 41.

[0287] Table 41. Maximum staining index values ​​of different clone numbers of PE-CD56 monoclonal antibody

[0288] from Fig.12 As can be seen from Table 41, some antibodies have poor clustering and the staining index cannot be calculated. Secondly, the PE-CD56 antibody with clone number 5.1H11 has the highest staining index and its positive and negative cell clustering is also good, which shows that this clone has the best staining effect. Because no PE-CD56 antibody of the same clone number from other brands was found, there is no comparison result. The clone number of PE-CD56 monoclonal antibody is 5.1H11.

[0289] 7) PerCP-cy5.5-CD45 screening:

[0290] The cell clustering diagrams with the highest staining index of different PerCP-cy5.5-CD45 monoclonal antibodies are shown in Fig.13 , where P1 is lymphocyte and P2 is monocyte; from left to right and from top to bottom, the clone numbers of the antibodies are QA21A24, QA17A19, MEM-28, 104, 2D1, and HI30. The staining index results are shown in Table 42.

[0291] Table 42. Fluorescence value ratios of lymphocytes and monocytes of different clone numbers of PerCP-cy5.5-CD45 monoclonal antibody

[0292]

[0293] from Fig.13 As can be seen from Table 42, the PerCP-cy5.5-CD45 antibody of clone 2D1 has the highest ratio of fluorescence values ​​of lymphocytes to monocytes, and its cell clustering is also good, which indicates that this clone has the best staining effect. At the same time, the PerCP-cy5.5-CD4 monoclonal antibody of other brands with clone 2D1 was compared, and the cell clustering diagram is shown in Fig.14 , from left to right the brands are Abcam, BD, and BioLegend. The staining index results are shown in Table 43.

[0294] Table 43. Highest staining index values ​​of different brands of PerCP-cy5.5-CD45 monoclonal antibody SJ25-C1 clone

[0295]

[0296] from Fig.14 As can be seen from Table 43, the results of the 2D1 clone number PerCP-cy5.5-CD45 of the three brands are basically the same, which shows that the antibodies with the same clone number from different manufacturers have the same effect. Based on the comprehensive results, the clone number of PerCP-cy5.5-CD45 monoclonal antibody is 2D1. Based on the above results, the antibody raw materials for the six-color lymphoid subsets are FITC-CD3 (clone number: SK7), PE-cy7-CD4 (clone number: SK3), APC-cy7-CD8 (clone number: SK1), PE-CD16 (clone number: B73.1), APC-CD19 (clone number: SJ25-C1), PerCP-cy5.5-CD45 (clone number: 2D1), PE-CD16 (clone number: 5.1H11), and the same clone number antibodies from different brands have consistent results.

[0297] Example 13: Screening of fluorescent microspheres

[0298] As a microsphere for absolute cell counting, fluorescent microspheres do not participate in the binding of antibodies and antigens, so it is important that they do not affect the cell gate on the instrument, cell clustering, and are in an appropriate position.

[0299] The suitability is evaluated by the ratio of fluorescence value to lymphocytes. The ratio of fluorescence value of fluorescent microspheres to lymphocytes in the PerCP channel should be within the range of 2-30, so that the microspheres are not close to cells when the fluorescence value is low, and do not exceed the boundary of the instrument software diagram when the fluorescence value is high. At the same time, the ratio of fluorescence value of fluorescent microspheres to neutrophils in the SSC channel should be within the range of 0.5-1, so that the fluorescent microspheres are not close to cells when the fluorescence value is low, and do not exceed the boundary of the instrument software diagram when the fluorescence value is high.

[0300] At the same time, the microspheres should not aggregate, which is reflected in the flow graph as another cluster of microspheres appearing in the upper right corner of the position of the microspheres themselves.

[0301] The screening results are as follows:

[0302] The PerCP-cy5.5-CD45 monoclonal antibody with clone number 2D1 selected above was used to react with the sample, and different fluorescent microspheres were added. The clustering situation and the fluorescence values ​​of the fluorescent microspheres and lymphocytes were detected by flow cytometry. The clustering diagram is shown in Fig.15 , from left to right are YCT300, YCT500, and YCT800. In the figure, P1 is lymphocyte, P2 is neutrophil, and P3 is fluorescent microsphere. The fluorescence ratio of each microsphere is shown in Table 44.

[0303] Table 44. Fluorescence ratio

[0304]

[0305] Among them, YCT300 meets the requirements, so the YCT300 fluorescent counting microsphere produced by Suzhou Weidu Company was selected.

[0306] Example 14: Effect of freeze-drying procedure on preparation of six-color lymphocyte subset absolute counting freeze-dried pellet reagent

[0307] Freeze drying is to cool down the material containing a large amount of water and freeze it into a solid in advance. Then, under vacuum conditions, water vapor is sublimated directly from the solid, while the material itself remains in the frozen ice shelf, so that the dried product does not lose its original solid skeleton structure, maintains the original shape of the material, and the product has excellent rehydration properties. Then, ice crystal sublimation drying is carried out at an appropriate temperature and vacuum degree, and desorption drying is carried out after the sublimation is completed to remove part of the bound water to obtain a dry product. Therefore, the appropriate freeze-drying procedure has a great influence on the amount of moisture and appearance of the freeze-dried product.

[0308] In this example, starting from the freeze-drying procedure, a six-color lymphoid subset absolute counting freeze-dried bead reagent was prepared according to the method provided in Example 1. When setting the freeze-drying procedure, the effects of different freeze-drying procedures on the preparation of the six-color lymphoid subset absolute counting freeze-dried bead reagent were investigated (see Table 45 for details), thereby preparing different freeze-dried beads. The water content of the freeze-dried beads prepared by different procedures was tested, and the water content requirement should meet <3%. The results are shown in Table 46. The appearance of the freeze-dried beads was compared, and the appearance should be smooth and round. The results are shown in Table 46. Fig.16 . At the same time, the clustering on the flow cytometer when testing the sample was compared. The clustering effect detection method was to use freeze-dried beads of different schemes to detect the same sample, and the staining index (SI) of CD3, CD4, CD8, CD19, CD16 / 56 and the ratio of CD45 lymphocyte fluorescence value to neutrophil fluorescence value in each scheme result were calculated according to the calculation method provided in the antibody titration step f in the above Example 12. The higher the staining index, the higher the fluorescence ratio, and the better the clustering effect. The results are shown in Table 46. At the same time, in this embodiment, except for the different freeze-drying procedures, the other conditions are the optimal conditions in Example 3.

[0309] Table 45. Screening of freeze drying procedures

[0310]

[0311] Table 46. Water content and grouping effect

[0312]

[0313] According to Table 46, different freeze-drying procedures have different effects on the water content of the samples. The water content of procedures 3 and 4 is less than 3%, and that of procedure 4 is even lower. Fig.16 It can be seen that the appearance of the samples corresponding to procedure 4 is poor, which may be due to the longer time resulting in the failure to effectively maintain the original appearance. It can also be seen from Table 46 that the grouping effect of procedure 3 is better. Therefore, the most preferred freeze-drying procedure is procedure 3.

[0314] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A six-color lymphocyte subset absolute counting freeze-dried pellet, characterized in that: The fluorescent microsphere solution, the fluorescent antibody solution and the freeze-drying protection solution are mixed into a freeze-dried stock solution, and then freeze-dried into a sphere; The freeze-dried protective solution is composed of a blocking agent, sugars, polyols, polymers, antioxidants, surfactants and buffer; the blocking agent is skim milk powder and arginine; the antioxidant is sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate; the antibodies in the fluorescent antibody solution include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody; the polymer is polysucrose; the sugars are trehalose and ribose; the polysucrose is 1% hydroxyethyl ester ... The monoalcohol is xylitol; the surfactant is hydroxypropyl-β-cyclodextrin; the buffer is a phosphate buffer; and the freeze-dried protective solution is, by mass percentage, 0.1-2% skim milk powder, 1-5% polysucrose, 0.1-1% L-sodium ascorbate, 0.1-1% ethylenediamineacetic acid disodium salt dihydrate, 1-10% trehalose, 0.5-5% ribose, 2-8% xylitol, 0.5-3% arginine, 1-5% polysucrose, and 0.005-1 mol / L phosphate buffer.

2. The freeze-dried pellet according to claim 1, characterized in that The antibodies in the fluorescent antibody solution include: PerCP-cy5.5-CD45 monoclonal antibody with clone number 2D1, FITC-CD3 monoclonal antibody with clone number SK7, PE-cy7-CD4 monoclonal antibody with clone number SK3, APC-cy7-CD8 monoclonal antibody with clone number SK1, APC-CD19 monoclonal antibody with clone number SJ25-C1, PE-CD16 monoclonal antibody with clone number B73.1, and PE-CD56 monoclonal antibody with clone number 5.1H11.

3. A method for preparing freeze-dried beads for detecting antigen content, characterized in that: The following steps are involved: (1) preparing a fluorescent antibody solution, wherein the antibodies include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody; (2) Preparing a freeze-drying protective solution: taking skim milk powder, arginine, polysucrose, trehalose, ribose, sodium L-ascorbate, disodium ethylenediaminetetraacetic acid dihydrate, xylitol, hydroxypropyl-β-cyclodextrin and phosphate buffer to prepare a freeze-drying protective solution; in terms of mass percentage, the freeze-drying protective solution includes 0.1-2% skim milk powder, 1-5% polysucrose, 0.1-1% sodium L-ascorbate, 0.1-1% disodium ethylenediaminetetraacetic acid dihydrate, 1-10% trehalose, 0.5-5% ribose, 2-8% xylitol, 0.5-3% arginine, 1-5% polysucrose and 0.005-1 mol / L phosphate buffer; (3) Mix the freeze-dried protective solution, fluorescent microsphere solution and fluorescent antibody solution to form a freeze-dried stock solution; (4) Dropping the lyophilized stock solution into liquid nitrogen through a bead dropping device to freeze it into spherical frozen beads; (5) The frozen beads are prepared into freeze-dried pellets for detecting antigen content through a freeze-drying process.

4. A freeze-dried protective solution, characterized in that: The invention is composed of a blocking agent, a sugar, a polyol, a polymer, an antioxidant, a surfactant and a buffer; wherein the blocking agent is skimmed milk powder and arginine, the polymer is polysucrose, the sugar is trehalose and ribose, the antioxidant is sodium L-ascorbate and disodium ethylenediaminetetraacetic acid dihydrate, the polyol is xylitol, the surfactant is hydroxypropyl-β-cyclodextrin, and the buffer is a phosphate buffer; and in terms of mass percentage, the freeze-dried protective solution is 0.1-2% skimmed milk powder, 1-5% polysucrose, 0.1-1% sodium L-ascorbate, 0.1-1% disodium ethylenediaminetetraacetic acid dihydrate, 1-10% trehalose, 0.5-5% ribose, 2-8% xylitol, 0.5-3% arginine, 1-5% polysucrose, and 0.005-1 mol / L phosphate buffer.

5. A use of the freeze-dried protective solution as claimed in claim 4 for preparing a reagent for improving the detection stability of six-color lymphocyte subset absolute counting freeze-dried beads, characterized in that: The six-color lymphoid subset absolute counting freeze-dried beads are prepared by mixing a fluorescent microsphere solution, a fluorescent antibody solution and a freeze-dried protective solution into a freeze-dried stock solution, and then freeze-dried into a sphere; the antibodies in the fluorescent antibody solution include CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody.

6. A use of the freeze-dried protective solution as claimed in claim 4 for preparing a reagent for improving the detection accuracy and repeatability of six-color lymphocyte subset absolute counting freeze-dried beads, characterized in that: The six-color lymphoid subset absolute count detection includes the detection of CD45 monoclonal antibody, CD3 monoclonal antibody, CD4 monoclonal antibody, CD8 monoclonal antibody, CD19 monoclonal antibody, CD16 monoclonal antibody and CD56 monoclonal antibody.

7. A use of the freeze-dried protective solution as claimed in claim 4 for preparing six-color lymphocyte subset absolute counting freeze-dried beads without preservatives, characterized in that: The six-color lymphoid subset absolute counting freeze-dried beads include a fluorescent microsphere solution, a fluorescent antibody solution and a freeze-dried protective solution.

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