Quality control substance for Treg cell analysis and preparation method thereof

Abstract

The invention relates to the technical field of quality control products in medical immunology, in particular to a quality control substance for Treg cell analysis and a preparation method of the quality control substance. Comprising a negative quality control substance of CD45 and a positive quality control substance of CD45, a negative quality control substance of CD3 and a positive quality control substance of CD3, a negative quality control substance of CD4 and a positive quality control substance of CD4, a negative quality control substance of CD25 and a positive quality control substance of CD25, a negative quality control substance of CD127 and a positive quality control substance of CD127, and can be used for different test schemes of clinical Treg cell analysis. Compared with the prior art, the quality control substance for Treg cell analysis and the preparation method of the quality control substance have the advantages that the quality control substance capable of simultaneously performing negative and positive detection in one test is provided, and the method is simple to operate, higher in yield, better in stability and small in batch difference; the method can be applied to quality control in the field of quality control substance detection for Treg cell analysis in flow cytometry, and is beneficial to wider application.

Description

[Technical field]

[0001] The present invention relates to the technical field of quality control products in medical immunology, and in particular to a quality control material for Treg cell analysis and a preparation method thereof. [Background technology]

[0002] Regulatory T cells (Tregs) are a type of T cell subset that controls the body's autoimmune reactivity, accounting for about 5%-10% of the total number of CD4+T lymphocytes in peripheral blood. This type of cell participates in the regulation of immune response and immune tolerance in an "active" manner. It not only participates in the regulation of autoimmune tolerance, but also plays an important role in tumor immunity and transplantation immunity.

[0003] Traditionally, Treg cells are identified mainly through labeling with CD4 and CD25. As research progresses, two more markers, FoxP3 and CD127, have been discovered one by one. Since FoxP3 is expressed in Treg cells, the labeling requires membrane permeation and fixation, and only dead cells can be obtained after labeling, while CD127 labeling can obtain live cells. Since the CD45 antigen is expressed on all white blood cells, it is also called the common leukocyte antigen. Therefore, CD45, CD4, CD25, and CD127 are often used in clinical practice to detect and screen Treg cells, or CD45, CD3, CD4, CD25, and CD127 are often used to detect and screen Treg cells.

[0004] There is no quality control material for Treg cell analysis in the prior art, and there is no biological control for clinical application detection. At the same time, CD127 is weakly expressed or negative, and quality control materials are needed to evaluate the stability and accuracy of the detection system, which seriously affects clinical application. [Summary of the invention]

[0005] In order to overcome the above problems, the present invention proposes a quality control substance for Treg cell analysis and a preparation method thereof which can effectively solve the above problems.

[0006] A technical solution provided by the present invention to solve the above technical problems is: to provide a quality control substance for Treg cell analysis and a preparation method thereof, including a negative quality control substance for CD45 and a positive quality control substance for CD45, a negative quality control substance for CD3 and a positive quality control substance for CD3, a negative quality control substance for CD4 and a positive quality control substance for CD4, a negative quality control substance for CD25 and a positive quality control substance for CD25, a negative quality control substance for CD127 and a positive quality control substance for CD127, and a preservation solution.

[0007] Preferably, the CD45 positive quality control substance is obtained by coupling CD45 antigen to the surface of microspheres; if the microspheres are not coupled to CD45 antigen, the CD45 negative quality control substance is obtained.

[0008] Preferably, the CD3 positive quality control substance is obtained by coupling CD3 antigen to the surface of microspheres; if the microspheres are not coupled to CD3 antigen, the CD3 negative quality control substance is obtained.

[0009] Preferably, the CD4 positive quality control substance is obtained by coupling CD4 antigen to the surface of microspheres; if the microspheres are not coupled to CD4 antigen, the CD4 negative quality control substance is obtained.

[0010] Preferably, the CD25 positive quality control substance is obtained by coupling CD25 antigen to the surface of microspheres; if the microspheres are not coupled to CD25 antigen, the CD25 negative quality control substance is obtained.

[0011] Preferably, the CD127 positive quality control material is obtained by coupling CD127 antigen to the surface of the microspheres; if the microspheres are not coupled to the CD127 antigen, the CD127 negative quality control material is obtained.

[0012] Preferably, the preservation solution comprises a buffer, a dispersant, an antibacterial agent and a protein protectant, the concentration of the buffer is 10-100 mmol / L, the concentration of the dispersant is 0.005%-5%, and the concentration of the antibacterial agent is 0.005%-5%.

[0013] Preferably, the microspheres are microspheres with a particle size of 3-8 μm, surface carboxyl or amino activated groups, and magnetic or non-magnetic microspheres.

[0014] Preferably, the pH value of the preservation solution is controlled within the range of 6.0-8.0.

[0015] A method for preparing a quality control substance for Treg cell analysis comprises the following steps:

[0016] Step S1, taking antigen, diluting with MES buffer to an antigen concentration of 1 mg / mL, and mixing for later use;

[0017] Step S2, taking the microspheres, magnetically separating them, discarding the supernatant, and adding MES buffer to resuspend them to form a microsphere suspension;

[0018] Step S3, taking EDC and Sulfo-NHS, and preparing EDC solution and Sulfo-NHS solution respectively with MES buffer;

[0019] Step S4, adding EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mixing, and reacting at 25° C. for 30 minutes;

[0020] Step S5, magnetically separate the microspheres and discard the supernatant, resuspend them in MES buffer, mix them, and magnetically separate them again and discard the supernatant;

[0021] Step S6, adding antigen to the microspheres of step S5, adding MES buffer, mixing, and reacting at 25° C. for 3-12 hours;

[0022] Step S7, magnetic separation of microspheres, discarding the supernatant, adding blocking solution to the microspheres, mixing and blocking reaction at 25°C for 4-18 hours;

[0023] Step S8, the microspheres are magnetically separated, the supernatant is discarded, the microspheres are added to the preservation solution and resuspended, and stored at 4°C.

[0024] Compared with the prior art, the quality control material for Treg cell analysis of the present invention and the preparation method thereof provide a quality control material that can perform both negative and positive detection in one test. The method is simple to operate, has higher yield, better stability, and small batch difference. It can be applied to quality control in the field of quality control material detection for Treg cell analysis using flow cytometry, and is conducive to wider application.

Brief Description of the Drawings

[0025] Figure 1 This is a flow chart of the method for preparing quality control material for Treg cell analysis of the present invention;

[0026] Figure 2 The front scatter and side scatter distribution diagrams of the quality control material for Treg cell analysis obtained in Example 1 of the present invention;

[0027] Figure 3 This is the distribution diagram of CD45- and CD45+ of the quality control material for Treg cell analysis obtained in Example 1 of the present invention;

[0028] Figure 4 This is the distribution diagram of CD3- and CD3+ of the quality control material for Treg cell analysis obtained in Example 1 of the present invention;

[0029] Figure 5 This is the distribution diagram of CD4- and CD4+ of the quality control material for Treg cell analysis obtained in Example 1 of the present invention;

[0030] Figure 6 This is the distribution diagram of CD25- and CD25+ of the quality control material for Treg cell analysis obtained in Example 1 of the present invention;

[0031] Figure 7 This is the distribution diagram of CD127- and CD127+ of the quality control material for Treg cell analysis obtained in Example 1 of the present invention;

[0032] Figure 8 This is the front scatter and side scatter distribution diagram of the quality control material for Treg cell analysis obtained in Example 2 of the present invention. [Specific implementation method]

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] It should be noted that in the embodiments of the present invention, all directional indications (such as up, down, left, right, front, back, etc.) are limited to relative positions on the specified view, rather than absolute positions.

[0035] In addition, in the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0036] See also Figures 1 to 8 The quality control material for Treg cell analysis of the present invention, based on flow cytometry technology, includes negative quality control material of CD45 and positive quality control material of CD45, negative quality control material of CD3 and positive quality control material of CD3, negative quality control material of CD4 and positive quality control material of CD4, negative quality control material of CD25 and positive quality control material of CD25, negative quality control material of CD127 and positive quality control material of CD127, and includes preservation solution. The quality control material for Treg cell analysis of the present invention can be used for different test schemes of Treg cell analysis in clinical practice.

[0037] CD45, CD3, CD4, CD25, and CD127 are important cell surface antigens in immunology, and they play key roles in the recognition, activation, and function of different immune cells.

[0038] The CD45 positive quality control material is obtained by coupling the CD45 antigen on the surface of the microspheres. If the microspheres are not coupled with the CD45 antigen, the CD45 negative quality control material is obtained.

[0039] The CD3 positive quality control material is obtained by coupling CD3 antigen on the surface of the microsphere. If the microsphere is not coupled with CD3 antigen, the CD3 negative quality control material is obtained.

[0040] The CD4 positive quality control material is obtained by coupling CD4 antigen on the surface of the microsphere. If the microsphere is not coupled with CD4 antigen, the CD4 negative quality control material is obtained.

[0041] The CD25 positive quality control material is obtained by coupling the CD25 antigen on the surface of the microsphere. If the microsphere is not coupled with the CD25 antigen, the CD25 negative quality control material is obtained.

[0042] The positive quality control material of CD127 is obtained by coupling CD127 antigen on the surface of microspheres. If the microspheres are not coupled with CD127 antigen, the negative quality control material of CD127 is obtained.

[0043] The microspheres are microspheres with a particle size of 3-8 μm, surface carboxyl or amino activated groups, and magnetic or non-magnetic microspheres.

[0044] The surfaces of the microspheres are coupled with CD45 antigen, CD3 antigen, CD4 antigen, CD25 antigen, and CD127 antigen respectively; or coupled with CD45 antigen, CD4 antigen, CD25 antigen, and CD127 antigen respectively; or coupled with CD25 antigen and CD127 antigen respectively.

[0045] The coupling method of the microspheres and the antigen is independently selected from any of the following:

[0046] Streptavidin and biotin reactions;

[0047] or -NH2 (primary amine) and -NHS (N-hydroxysuccinimide ester) reaction;

[0048] or -NH2 and -NCS (isothiocyanate) reaction;

[0049] or -SH2 reacts with maleimide groups;

[0050] Or -NH2 reacts with epoxy groups;

[0051] Or -NH2 reacts with tosyl group.

[0052] The preservation solution comprises a buffer, a dispersant, an antibacterial agent and a protein protectant. The concentration of the buffer is 10-100 mmol / L, the concentration of the dispersant is 0.005%-5%, and the concentration of the antibacterial agent is 0.005%-5%.

[0053] The buffer is one of phosphate buffer, HEPES buffer, Tris buffer or glycine buffer.

[0054] The dispersant is a surfactant, and the surfactant can be one of Tween-20, Tween-40, Tween-80, Triton-100 or poloxamer.

[0055] The antibacterial agent is one of sodium azide, Prclin-300, BND-10 or P rclin-950.

[0056] The protein protective agent is gelatin BSA (0.01-0.5% w / v), sucrose BSA (0.1-0.5% w / v), lactose BSA (0.1-1% w / v), trehalose (0.1-1% w / v) or one of the commercially available protein protective agents.

[0057] The pH value of the preservation solution is controlled within the range of 6.0-8.0.

[0058] The method for preparing a quality control material for Treg cell analysis of the present invention comprises the following steps:

[0059] Step S1, antigen preparation: take the antigen, dilute it to an antigen concentration of 1 mg / mL with MES buffer, and mix well for later use.

[0060] Step S2, taking the microspheres, magnetically separating them, discarding the supernatant, and adding MES buffer to resuspend them to form a microsphere suspension.

[0061] Step S3, take EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and Sulfo-NHS (N-hydroxysuccinimide-4-sulfonic acid butyl ester), and prepare EDC solution and Sulfo-NHS solution respectively using MES buffer.

[0062] Step S4, add EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25° C. for 30 minutes.

[0063] Step S5, the microspheres are magnetically separated and the supernatant is discarded, and the microspheres are resuspended with MES buffer, mixed and magnetically separated again and the supernatant is discarded.

[0064] Step S6, adding antigen to the microspheres of step S5, adding MES buffer, mixing, and reacting at 25° C. for 3-12 hours.

[0065] Step S7, magnetically separate the microspheres, discard the supernatant, add blocking solution to the microspheres, mix well, and perform blocking reaction at 25°C for 4-18 hours.

[0066] Step S8, the microspheres are magnetically separated, the supernatant is discarded, the microspheres are added to the preservation solution and resuspended, and stored at 4°C.

[0067] The following is an explanation of the method for preparing the quality control material for Treg cell analysis of the present invention through specific examples.

[0068] Embodiment 1

[0069] Composition of quality control materials

[0070]

[0071] Step X1, Antigen Preparation:

[0072] Antigen A: Take 10-100 μg of CD45 antigen, dilute to 1 mg / mL with MES buffer, mix well and set aside;

[0073] Antigen B: Take 10-100 μg of CD3 antigen, dilute to 1 mg / mL with MES buffer, mix well and set aside;

[0074] C antigen: Take 10-100 μg of CD4 antigen, dilute to 1 mg / mL with MES buffer, mix well and set aside;

[0075] D antigen: Take 10-100 μg of CD25 antigen, dilute to an antigen concentration of 1 mg / mL with MES buffer, mix well and set aside;

[0076] E antigen: Take 10-100 μg of CD127 antigen, dilute to an antigen concentration of 1 mg / mL with MES buffer, mix well and set aside;

[0077] Step X2, take 5 mL of microspheres, separate by magnetic separation, discard the supernatant, and add 1 mL of MES buffer to resuspend;

[0078] Step X3, weigh 5 and 50 mg of EDC and Sulfo-NHS respectively, and prepare a 50 mg / mL solution with MES buffer;

[0079] Step X4, add 0.1 mL of EDC solution and 0.1 mL of Sulfo-NHS solution to the microsphere suspension in sequence, mix well, and react at 25° C. for 30 minutes;

[0080] Step X5, magnetically separate the microspheres and discard the supernatant, resuspend with 1 mL of MES buffer, mix and evenly divide into 5 tubes marked A, B, C, D, and E, 0.2 mL per tube, and magnetically separate again and discard the supernatant;

[0081] Step X6, add corresponding antigens to the 5 tubes of microspheres in step X5, which are the above-mentioned antigen A, antigen B, antigen C, antigen D and antigen E, add 10-50 μL of each, add MES buffer to 0.2 mL, mix well, and react at 25° C. for 3-12 hours;

[0082] Step X7, magnetic separation of microspheres, discarding the supernatant, adding 0.4 mL of blocking solution to each microsphere, mixing and blocking reaction at 25°C for 4-18 hours;

[0083] Step X8, magnetically separate the microspheres, discard the supernatant, add 10-100 mL of the preservation solution in this protocol to each microsphere, resuspend, and store at 4°C.

[0084] The Treg cell analysis quality control material obtained in Example 1 was tested on a flow cytometer. Figure 2-7 shown.

[0085] The quality control material for Treg cell analysis obtained in Example 1 was tested experimentally and found to be sealed and stored at 2-8°C for 12 months with a decay rate of ≤15%, meeting clinical requirements. The stability and grouping of the quality control material for Treg cell analysis obtained in Example 1 of the present invention are illustrated by experimental data, and the data are as follows:

[0086]

[0087] Embodiment 2

[0088] Composition of quality control materials

[0089]

[0090]

[0091] Step Y1, antigen preparation: antigen preparation is the same as in Example 1.

[0092] Step Y2, microspheres: select microspheres of 5 particle sizes. In this embodiment, magnetic microspheres with diameters of about 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are selected;

[0093] Step Y3, preparation:

[0094] CD45 coupled to 3 μm magnetic microspheres;

[0095] CD3 coupled to 4 μm magnetic microspheres;

[0096] CD4 coupled to 5 μm magnetic microspheres;

[0097] CD25 coupled to 6 μm magnetic microspheres;

[0098] CD127 coupled to 7 μm magnetic microspheres;

[0099] The coupling scheme of each antigen is the same as that of Example 1.

[0100] The Treg cell analysis quality control material obtained in Example 2 was tested on a flow cytometer. Figure 8 shown.

[0101] Compared with the prior art, the quality control material for Treg cell analysis and the preparation method thereof of the present invention solve the gap of the current market lack of quality control material for Treg cell analysis, and have the following beneficial effects:

[0102] 1. It has the advantages of stable performance, low production cost, mass production, small batch difference, wider clinical application, etc., which can better control the quality of Treg cell analysis system.

[0103] 2. It can be flexibly combined and used for quality control of different Treg cell testing schemes.

[0104] 3. It can be widely used in inter-laboratory quality assessment.

[0105] 4. Calibration objects that can be used for debugging different instruments.

[0106] 5. One test can perform negative and positive quality control at the same time, saving reagents and time, and performing controls at the same time to avoid differences in results due to operational deviations.

[0107] 6. The quality control concentration can be adjusted and can be used to evaluate the sensitivity of the reagent.

[0108] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any modifications, equivalent substitutions and improvements made within the concept of the present invention should be included in the patent protection scope of the present invention.

Claims

1. A quality control substance for Treg cell analysis, characterized in that: Including negative control substance and positive control substance of CD45, negative control substance and positive control substance of CD3, negative control substance and positive control substance of CD4, negative control substance and positive control substance of CD25, negative control substance and positive control substance of CD25, negative control substance and positive control substance of CD127, and preservation solution.

2. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The CD45 positive quality control material is obtained by coupling the CD45 antigen on the surface of the microsphere; the CD45 negative quality control material is obtained when the microsphere is not coupled with the CD45 antigen.

3. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The CD3 positive quality control material is obtained by coupling CD3 antigen on the surface of the microsphere; the CD3 negative quality control material is obtained when the microsphere is not coupled with the CD3 antigen.

4. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The CD4 positive quality control material is obtained by coupling CD4 antigen on the surface of the microsphere; the CD4 negative quality control material is obtained when the microsphere is not coupled with the CD4 antigen.

5. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The CD25 positive quality control material is obtained by coupling the CD25 antigen on the surface of the microsphere; the CD25 negative quality control material is obtained when the microsphere is not coupled with the CD25 antigen.

6. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The CD127 positive quality control material is obtained by coupling the CD127 antigen to the surface of the microsphere; the CD127 negative quality control material is obtained when the microsphere is not coupled to the CD127 antigen.

7. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The preservation solution comprises a buffer, a dispersant, an antibacterial agent and a protein protectant. The concentration of the buffer is 10-100 mmol / L, the concentration of the dispersant is 0.005%-5%, and the concentration of the antibacterial agent is 0.005%-5%.

8. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The microspheres are microspheres with a particle size of 3-8 μm, surface carboxyl or amino activated groups, and magnetic or non-magnetic microspheres.

9. The quality control substance for Treg cell analysis according to claim 1, characterized in that: The pH value of the preservation solution is controlled within the range of 6.0-8.

0.

10. A method for preparing a quality control substance for Treg cell analysis, characterized in that: The steps include: Step S1, taking antigen, diluting with MES buffer to an antigen concentration of 1 mg / mL, and mixing for later use; Step S2, taking the microspheres, magnetically separating them, discarding the supernatant, and adding MES buffer to resuspend them to form a microsphere suspension; Step S3, taking EDC and Sulfo-NHS, and preparing EDC solution and Sulfo-NHS solution respectively with MES buffer; Step S4, adding EDC solution and Sulfo-NHS solution to the microsphere suspension in sequence, mixing, and reacting at 25° C. for 30 minutes; Step S5, magnetically separate the microspheres and discard the supernatant, resuspend them in MES buffer, mix them, and magnetically separate them again and discard the supernatant; Step S6, adding antigen to the microspheres of step S5, adding MES buffer, mixing, and reacting at 25° C. for 3-12 hours; Step S7, magnetic separation of microspheres, discarding the supernatant, adding blocking solution to the microspheres, mixing and blocking reaction at 25°C for 4-18 hours; Step S8, the microspheres are magnetically separated, the supernatant is discarded, the microspheres are added to the preservation solution and resuspended, and stored at 4°C.

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