阅读说明：本技术 Pbmc的免疫系统重建能力的检测方法及应用 (Detection method for immune system reconstruction capability of PBMC (peripheral blood mononuclear cell) and application thereof ) 是由 李佐青 陈大为 康恺 刘津 于 2021-06-18 设计创作，主要内容包括：本发明涉及一种PBMC的免疫系统重建能力的检测方法及应用,其包括以下步骤：获取PBMC样本；获取所述PBMC样本中CD3阳性细胞的比例a,以及所述CD3阳性细胞中CD45RA阳性细胞的比例b,通过a×b计算得到所述PBMC样本中CD45RA阳性细胞的比例作为重建指数；所述重建指数数值越大,则所述PBMC样本的免疫系统重建能力越高,且所述重建指数的Cut-off值为0.2。本发明的检测方法可以脱离小鼠,通过体外的方式准确快速地判定Donor在小鼠体内重建状况好坏,极大方便了Donor的选取和实验时间规划,同时也降低了成本,能够既快又好地推进药物研发所用的人源化实验动物模型的建立,助力于药物开发。(The invention relates to a detection method and application of immune system reconstruction capability of PBMC, which comprises the following steps: obtaining a PBMC sample; obtaining the proportion a of CD3 positive cells in the PBMC sample and the proportion b of CD45RA positive cells in the CD3 positive cells, and obtaining the proportion of CD45RA positive cells in the PBMC sample as a reconstruction index through a x b calculation; the larger the reconstitution index value, the higher the immune system reconstitution capacity of the PBMC sample, and the Cut-off value of the reconstitution index is 0.2. The detection method can be separated from a mouse, the quality of the reconstruction condition of the Donor in the mouse can be accurately and quickly judged in an in-vitro mode, the Donor selection and the experiment time planning are greatly facilitated, the cost is reduced, the establishment of a humanized experiment animal model used for drug research and development can be quickly and well promoted, and the drug development is assisted.)

1. A method for detecting immune system reconstitution ability of PBMC, which is characterized by comprising the following steps:

obtaining a PBMC sample;

obtaining the proportion a of CD3 positive cells in the PBMC sample and the proportion b of CD45RA positive cells in the CD3 positive cells, and obtaining the proportion of CD45RA positive cells in the PBMC sample as a reconstruction index through a x b calculation;

the larger the reconstitution index value, the higher the immune system reconstitution capacity of the PBMC sample, and the Cut-off value of the reconstitution index is 0.2.

2. The detection method according to claim 1, wherein the method for obtaining the proportion of CD45RA positive cells among the CD3 positive cells comprises the following steps: obtaining the proportion c of CD45RO positive cells in the CD3 positive cells, and obtaining the proportion of CD45RA positive cells in the CD3 positive cells through 1-c calculation.

3. The assay of claim 1, wherein the PBMC sample is a human PBMC sample and the method of obtaining the proportion of CD3 positive cells in the PBMC sample comprises the steps of: obtaining the number d of CD45 positive cells and the number e of CD3 positive cells in the PBMC sample, and calculating the proportion of the CD3 positive cells in the PBMC sample according to the e/d.

4. The assay of claim 1, wherein the proportion of CD45RA positive cells in the PBMC sample is assayed using flow cytometry.

5. The detection method according to claim 4, wherein the flow cytometry comprises the steps of: PBMC samples were taken into flow tubes, stained with fluorescent antibodies, and placed in flow cytometry for detection and analysis.

6. The detection method according to claim 5, wherein the method for staining with a fluorescent antibody comprises the steps of: and mixing the PBMC sample with the fluorescent antibody, incubating for 10-30 min at room temperature in a dark place, adding a buffer solution, centrifuging for 1-5 min by 100-400 g, taking a precipitate, and then adding the buffer solution for resuspension.

7. The method of detecting according to any one of claims 1 to 6, wherein the reconstitution capability is a reconstitution capability exhibited in a mouse model humanized to the immune system.

8. A screening method for a drug for treating immunodeficiency diseases, which is characterized by comprising the following steps:

detecting the immune system reconstitution ability of PBMCs according to the detection method of any one of claims 1 to 7;

selecting PBMC with reconstruction index of more than 0.2 to inoculate in an experimental animal body to construct an experimental animal model;

drug candidates are tested using the experimental animal model.

9. A computer-readable storage medium for storing a computer instruction, a program, a set of codes, or a set of instructions which, when run on a computer, causes the computer to perform the detection method of any one of claims 1 to 7.

10. An electronic device, comprising:

one or more processors; and

a computer-readable storage medium for storing a computer instruction, program, set of codes or set of instructions which, when run on a computer, causes the one or more processors to implement the detection method of any one of claims 1 to 7.

Technical Field

The invention relates to the technical field of medicines, in particular to a method for detecting immune system reconstruction capability of PBMC and application thereof.

Background

The experimental animal model plays an important role in the development of medical life science. Rodent models are widely used for drug and vaccine research, but many drugs or vaccines cannot be effectively used for human diseases and cannot be clinically used due to species differences or differences between applied pathogens and pathogens infecting human beings. Parasitic, bacterial and viral infections are the major causative factors that compromise human health, and the infection and pathogenic characteristics of many pathogens are species-specific, such as hiv only infects human cells and not mouse cells. Due to the lack of ideal animal disease models, the intensive research on the corresponding disease mechanism and prevention and treatment is limited. Establishment of a humanized mouse model provides a new choice for the research of human diseases. Immune system humanized mice are mice in which the human immune system is reconstituted by transplantation of human hematopoietic stem cells or immune tissues or cells.

In the development of the tumor immune drugs which are currently in the future, an experimental animal model containing a human immune system needs to be constructed urgently, and the experimental animal model takes the human immune system reconstructed by human PBMC in a mouse as a model representative and can be well used for evaluating the drug effect of the tumor immune drugs at an immune check point. However, the difference in the reconstituability of Donor (Donor) from different individual sources in the population is so great that each Donor must be screened to obtain acceptable PBMC (peripheral blood mononuclear cells) for further drug effect experiments. The brief steps are that severe immunodeficiency mice are used, large dose of PBMC (5 multiplied by 10E 6/mouse) is injected into tail vein, after 2-4 weeks, the reconstruction ability of PBMC in the mice is evaluated, and the PBMC is positive to human CD45 as a cell signal indicator of the human immune system.

However, the process of detecting the reconstitution ability is time-consuming and high in cost, so that the selection and experiment time planning of Donor are inconvenient, the rapid establishment of a humanized experimental animal model used for drug development is hindered, and the development of a new anti-tumor immunity-related drug is not facilitated.

Disclosure of Invention

Based on this, it is necessary to provide an accurate and rapid method for detecting the immune system reconstitution ability of PBMC.

A method for detecting immune system reconstitution ability of PBMC, comprising the following steps:

obtaining a PBMC sample;

obtaining the proportion a of CD3 positive cells in the PBMC sample and the proportion b of CD45RA positive cells in the CD3 positive cells, and obtaining the proportion of CD45RA positive cells in the PBMC sample as a reconstruction index through a x b calculation;

the larger the reconstitution index value, the higher the immune system reconstitution capacity of the PBMC sample, and the Cut-off value of the reconstitution index is 0.2.

In one embodiment, the method for obtaining the proportion of CD45RA positive cells in the CD3 positive cells comprises the following steps: obtaining the proportion c of CD45RO positive cells in the CD3 positive cells, and obtaining the proportion of CD45RA positive cells in the CD3 positive cells through 1-c calculation.

In one embodiment, the PBMC sample is a human PBMC sample, and the method of obtaining a proportion of CD3 positive cells in the PBMC sample comprises the steps of: obtaining the number d of CD45 positive cells and the number e of CD3 positive cells in the PBMC sample, and calculating the proportion of the CD3 positive cells in the PBMC sample according to the e/d.

In one embodiment, the proportion of CD45RA positive cells in the PBMC sample is detected using flow cytometry.

In one embodiment, the flow cytometry comprises the steps of: PBMC samples were taken into flow tubes, stained with fluorescent antibodies, and placed in flow cytometry for detection and analysis.

In one embodiment, the method for staining with a fluorescent antibody comprises the following steps: and mixing the PBMC sample with the fluorescent antibody, incubating for 10-30 min at room temperature in a dark place, adding a buffer solution, centrifuging for 1-5 min by 100-400 g, taking a precipitate, and then adding the buffer solution for resuspension.

In one embodiment, the reconstitution capabilities are those embodied in a humanized mouse model of the immune system; preferably, the mouse used in the model is a T/B/NK cell combined deletion type mouse.

The invention also provides a screening method of the medicine for treating the immunodeficiency diseases, which comprises the following steps:

testing the PBMCs for immune system reconstitution capability according to the test method described above;

selecting PBMC with reconstruction index of more than 0.2 to inoculate in an experimental animal body to construct an experimental animal model;

drug candidates are tested using the experimental animal model.

The present invention also provides a computer-readable storage medium for storing a computer instruction, a program, a set of codes or a set of instructions which, when run on a computer, causes the computer to perform the detection method as described above.

The present invention also provides an electronic device, comprising:

one or more processors; and

a computer-readable storage medium for storing a computer instruction, program, set of codes or set of instructions which, when run on a computer, causes the one or more processors to implement the detection method as described above.

The traditional PBMC immune system reconstruction ability detection method can only be carried out by injecting PBMC into a mouse, which inevitably causes the following problems: (1) in vivo screening is economically expensive. The existing screening mode can be carried out only by depending on high-grade animal facilities capable of feeding severe immunodeficiency mice, each PBMC screening generally needs 3-5 severe immunodeficiency mice, and the economic cost is very high. (2) The screening time is long. The reconstruction process of PBMC in an animal body needs 2-3 weeks, so that the time for judging the reconstruction capability of one Donor is long. Therefore, determining the reconstitution capacity of PBMCs in mice requires a significant amount of time and economic cost.

The invention summarizes experience of multiple screening, defines the Reconstruction Index (RI) of each Donor, and when the RI is more than 0.2, the Donor can be considered to be well reconstructed within 20 days, so that the Donor can be separated from a mouse, and the condition of the Donor in the mouse can be accurately and quickly judged in an in-vitro mode. Therefore, the method greatly facilitates the selection of Donor and the planning of experiment time, reduces the cost, can quickly and well promote the establishment of humanized experimental animal models for drug research and development, and helps develop new anti-tumor immunity-related drugs. The detection method quantifies the PBMC reconstruction ability through the reconstruction index, does not depend on animal rooms, only needs conventional equipment of a tumor immunity laboratory, such as a flow cytometer and the like, and can be completely operated in vitro. The detection method of the present invention requires very little time, and results can be obtained within two hours using, for example, flow cytometry. Furthermore, if the PBMC supplier is able to provide the corresponding index data, flow cytometry can be omitted altogether and only calculations are necessary for the determination.

Drawings

FIG. 1 is a schematic representation of the relationship between T cell proliferative capacity and effector function;

FIGS. 2 to 10 show the results of flow cytometry in examples 1 to 9 using staining with CD3 fluorescent antibody and CD45RO fluorescent antibody, respectively;

FIGS. 11 to 12 show the results of flow cytometry in examples 10 to 11 using staining with CD3 fluorescent antibody and CD45RA fluorescent antibody, respectively;

FIGS. 13 to 19 show the results of flow cytometry in examples 12 to 18, respectively, using staining with CD3, CD45RO and CD45RA fluorescent antibodies;

FIG. 20 is a reconstruction curve of PBMCs of examples 1-9 in mice;

FIG. 21 is a reconstruction curve of PBMCs of examples 10-18 in mice;

FIG. 22 is a graph showing the determination of cut-off values and linearly dependent regression of the reconstruction index and the reconstruction scale in example 20;

FIGS. 23 to 34 show the results of flow cytometry for 12 PBMCs in example 21;

FIG. 35 is a correlation between reconstitution index and reconstitution ratio for 12 PBMCs in example 21.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The method for detecting the immune system reconstitution ability of PBMC of one embodiment of the invention comprises the following steps:

obtaining a PBMC sample;

acquiring the proportion a of CD3 positive cells in the PBMC sample and the proportion b of CD45RA positive cells in the CD3 positive cells, and calculating the proportion of CD45RA positive cells in the PBMC sample through a x b to serve as a reconstruction index;

the larger the reconstitution index value, the higher the immune system reconstitution ability of the PBMC sample, and the Cut-off value of the reconstitution index is 0.2.

In the development of new drugs for tumor immunization, particularly in the development of immune checkpoint drugs represented by PD1 antibody biomacromolecule drugs, a humanized immune system is required to test the drug effect of the biomacromolecule drugs. The human peripheral blood derived mononuclear immune cells (PBMC, the donated Donor is called Donor) are injected into severe immunodeficiency mice (T/B/NK cell combined deletion) through tail vein, so that a humanized animal model can be simply and rapidly constructed, and the in vivo efficacy test of most immune checkpoint drugs can be provided. PBMCs derived from healthy humans, however, have very different in vivo reconstitution capabilities, i.e., some donors (donors) can be well reconstituted, while some donors are difficult to reconstitute, and PBMC reconstitution essentially requires in vivo batch screening of animals to determine that each Donor cannot be reconstituted. The process of PBMC reconstitution in mice is actually the process of T cell expansion. PBMCs are depleted by the non-specific elimination of the mouse immune system shortly after entering the mouse, leaving only a fraction of human T cells. The part of the residual T cells gradually accumulates cytokines, especially IL2, keeps the T cells alive and supports the expansion of the T cells, and after the accumulation reaches a certain amount, the T cells are cloned and expanded, and finally, the existence of a considerable proportion of humanized T cells is achieved, namely the establishment of an experimental model of the humanized animal of the immune system is successful.

CD45RA + T (CD45RA positive T) is a cell population with strong T cell expansion and proliferation ability, i.e. naive T cells, which have great proliferation potential, and the corresponding CD45RO + T (CD45RO positive T) effector cells have strong effector functions but weak proliferation ability (as shown in fig. 1). Furthermore, the research on the naive T of the T cell shows that the reconstruction in the mouse has definite positive correlation with the ratio of the naive T (CD45RO negative, namely CD45RO-, is equal to CD45RA positive, namely CD45RA +) subgroup, and has obvious negative correlation with the CD45RO + memory T cell group. The invention summarizes experience of multiple screening, defines the reconstruction index RI of each Donor, and when the reconstruction index RI is more than 0.2, the Donor can be considered to be well reconstructed within 20 days, so that the Donor can be separated from a mouse, and the condition of the Donor in the mouse can be accurately and quickly judged in an in-vitro mode. Therefore, the method greatly facilitates the selection of Donor and the planning of experiment time, reduces the cost, can quickly and well promote the establishment of humanized experimental animal models for drug research and development, and helps develop new anti-tumor immunity-related drugs.

It is understood that the term "cut-off value" in medicine, as a cut-off value, a cut-off value or a cutoff value, means a positive judgment value or "threshold value", that is, if the detected value is greater than the cut-off value, the value is positive, and if the detected value is less than the cut-off value, the value is negative, that is, the reconstruction index is greater than 0.2, and thus the Donor is considered to be well reconstructed within 20 days. When the reconstruction index is used to determine the reconstruction capability of the unknown Donor, it is very clearly shown that Donor can be well reconstructed when it is greater than 0.2, and the possibility of good reconstruction is greatly reduced when it is less than 0.2. Therefore, the RI is used as an index for evaluating the reconstruction capability of the unknown Donor, which is very simple, rapid and convenient.

In one specific example, the method of obtaining the proportion of CD45RA positive cells among CD3 positive cells comprises the steps of: the proportion c of CD45RO positive cells among CD3 positive cells was obtained, and the proportion of CD45RA positive cells among CD3 positive cells was calculated from 1-c. The T cells can be divided into two groups of relative populations according to CD45RO and CD45RA, so that the proportion of CD45RA positive cells in CD3 positive cells can be obtained by subtracting the proportion of CD45RO positive cells in CD3 positive cells.

In one specific example, the PBMC sample is a human PBMC sample, and the method for obtaining a proportion of CD3 positive cells in the PBMC sample comprises the steps of: and acquiring the number d of CD45 positive cells and the number e of CD3 positive cells in the PBMC sample, and calculating the proportion of the CD3 positive cells in the PBMC sample according to the e/d. Human PBMCs were essentially CD45 positive, so the number of CD45 positive cells was equivalent to the number of whole cells.

In one particular example, the proportion of CD45RA positive cells in the PBMC sample is detected using flow cytometry. It is understood that the method for detecting the cell type and ratio is not limited thereto, and may be selected as desired.

In one particular example, flow cytometry comprises the steps of: PBMC samples were taken into flow tubes, stained with fluorescent antibodies, and placed in flow cytometry for detection and analysis.

In one specific example, the method of staining with a fluorescent antibody comprises the steps of: and mixing the PBMC sample with the fluorescent antibody, incubating for 10-30 min at room temperature in a dark place, adding a buffer solution, centrifuging for 1-5 min by 100-400 g, taking a precipitate, and then adding the buffer solution for resuspension.

In one specific example, the above described reconstitution capabilities are those exhibited in a humanized mouse model of the immune system. Preferably, the mouse used in the above model is a T/B/NK cell-deficient mouse.

The screening method of the drug for treating the immunodeficiency diseases, which is provided by the embodiment of the invention, comprises the following steps of:

testing the PBMCs for immune system reconstitution capability according to the test method described above;

selecting PBMC with reconstruction index of more than 0.2 to inoculate in an experimental animal body to construct an experimental animal model;

drug candidates were tested using the experimental animal models described above.

In a specific example, the immunodeficiency disorder is a tumor.

A computer readable storage medium of an embodiment of the invention stores a computer instruction, a program, a set of codes, or a set of instructions, which when run on a computer, causes the computer to perform the detection method as described above.

An electronic device according to an embodiment of the present invention includes: one or more processors; and a computer readable storage medium for storing a computer instruction, program, code set or instruction set which, when run on a computer, causes the one or more processors described above to implement the detection method as described above.

The application of the leukocyte differentiation antigen detection reagent in detecting the immune system reconstitution capability of PBMC of the embodiment of the invention includes a CD45RA antibody. In a specific example, the leukocyte differentiation antigen detection reagent further comprises one or more of a CD3 antibody, a CD45RO antibody, and a CD45 antibody.

The following are specific examples.

Firstly, the whole experimental process:

1.1 detecting the proportion of CD3, CD45RA and CD45RO cells by using flow cytometry;

1.2 calculate the product of the CD3 of total cells ratio and CD45RA + (or CD45RO-) of CD3 ratio to obtain the reconstruction index RI.

Donor with an RI of 1.3 or more and 0.3 is judged to have good reconstruction ability.

Secondly, the specific instruments, reagents and operations are as follows:

TABLE 1 Instrument information

TABLE 2 reagent consumables information

Name of reagent	Brand	Goods number
			1640 cell culture medium	Thermo	C11875500BT
Fetal bovine serum	BI	04-001-1A
			Trypsin-EDTA(0.25％)	Gibco	25200056
PBS	Gibco	C20012500BT
			Anti-CD45RA	eBioscience	11-0458-42
Anti-CD45RO	eBioscience	12-0457-42
			Anti-CD3	eBioscience	56-0038-42
PBMC	Miaoshun (good luck and happiness)	/

2.1 resuscitating cells:

(1) before cell recovery, a superclean platform is wiped by alcohol, ultraviolet irradiation is carried out for 15 minutes, a centrifugal machine is opened, and a water bath kettle is opened to 37 ℃;

(2) preparing a complete culture medium: 1640 medium with a final volume of 10% FBS and 1% penicillin streptomycin double antibody;

(3) preparing 10mL of complete culture medium for later use;

(4) and (3) thawing the cells: taking out the required cells from the liquid nitrogen, checking whether the cell numbers are correct or not, placing the cryopreservation tube into a water bath kettle to shake rapidly, and transferring the cells into 10mL of complete culture medium after thawing;

(5) and (3) washing the cells: centrifuging at the rotating speed of 300g for 10 minutes, and then removing a supernatant;

(6) cells were resuspended in PBS and count adjusted to a concentration of 1 × 10E6/mL for reconstitution in injected mice and for the Reconstitution Index (RI) by flow cytometry.

2.2 flow cytometry detection Reconstruction Index (RI) protocol:

(1) sample acquisition: taking 100 mu L of the standby cells to a flow tube, and adding the standby cells into the flow tube in three parts, wherein the three parts are numbered 1#, 2#, and 3 #;

(2) cell staining:

preparing a sample detection tube: adding 5 μ L of CD45RA, 5 μ L of CD45RO and 5 μ L of CD3 antibody into a 1# tube, keeping out of the sun for 20min at room temperature, adding 1mL of PBS, centrifuging at 300g for 3 min, and resuspending 100 μ L of PBS for running;

CD45RA FMO control tubes were configured: adding 5 μ L of CD45RA iso-type, 5 μ L of CD45RO and 5 μ L of CD3 antibody into a No. 2 tube, keeping away from light for 20min at room temperature, adding 1mL of PBS, centrifuging at 300g for 3 min, and resuspending 100 μ L of PBS for running flow;

CD45RA FMO control tubes were configured: adding 5 μ L of CD45RA, 5 μ L of CD45RO iso-type and 5 μ L of CD3 antibody into a 3# tube, keeping away from light for 20min at room temperature, adding 1mL of PBS, centrifuging at 300g for 3 min, and resuspending 100 μ L of PBS for running flow;

(3) sampling on an upper flow cytometer, and collecting cells with the number of CD3 door being more than or equal to 10000 events;

(4) results CD3, CD45RA, CD45RO cell population proportion analysis was performed using professional flow software Flowjo or flow machine self-contained analysis software.

(5) The Reconstruction Index (RI) is calculated according to the ratio as follows:

firstly, determining the proportion (a) of CD3, namely CD3 of total cells; and the CD45RA ratio (b), i.e., CD45RA of CD 3; and the CD45RO ratio (c), CD45RO of CD 3. In actual calculation, the ratio of CD45RO of CD3 is equal to about 1- (CD45RA of CD3), whereas the ratio of CD45RA of CD3 is equal to about 1- (CD45RO of CD 3). The reconstruction index RI ═ a × b or RI ═ a × (1-c), RI >0.2 is considered that the denor reconstructed well within a prescribed time (2 weeks), the significance level is set at α ═ 0.05, and the confidence interval is 95%.

Example 1

Number 11 Donor: PBMC from Donor No. 11 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 2.

The reconstruction index RI value was calculated as follows: CD3 accounted for 56.777% of total cell number, while CD45RA accounted for 64.432% of CD3, RI 56.777% x 64.432% was 0.365; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 35.519%, and RI is 56.777% × (1-35.519%) -0.366; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 2

Number 12 Donor: PBMC from Donor No. 12 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 3.

The reconstruction index RI value was calculated as follows: CD3 accounted for 40.244% of total cell number, whereas CD45RA accounted for 70.216% of CD3, RI 40.244% x 70.216% was 0.283; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 29.710%, and RI is 40.244% × (1-29.710%) -0.283; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 3

Number 13 Donor: PBMC from Donor # 13 were stained with fluorescent antibody and analyzed by flow cytometry, the results are shown in fig. 4.

The reconstruction index RI value was calculated as follows: CD3 accounted for 57.539% of total cell number, while CD45RA accounted for 62.016% of CD3, RI 57.539% x 62.016% was 0.357; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 37.915%, and RI is 57.539% × (1-37.915%) -0.357; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 4

Number 14 Donor: PBMC from Donor No. 14 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 5.

The reconstruction index RI value was calculated as follows: CD3 accounted for 53.365% of total cell number, while CD45RA accounted for 69.857% of CD3, RI 53.365% x 69.857% was 0.373; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 30.064%, and RI is 53.365% × (1-30.064%) -0.373; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 5

Number 15 Donor: PBMC from Donor No. 15 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 6.

The reconstruction index RI value was calculated as follows: CD3 accounted for 45.562% of total cell number, while CD45RA accounted for 59.682% of CD3, RI 45.562% x 59.682% was 0.272; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 40.250%, and RI is 45.562% × (1-40.250%) -0.272; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 6

Number 16 Donor: PBMC from Donor No. 16 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 7.

The reconstruction index RI value was calculated as follows: CD3 accounted for 50.882% of total cell number, whereas CD45RA accounted for 55.860% of CD3, RI 50.882% x 55.860% was 0.284; alternatively, the CD45RO ratio was calculated, CD45RO accounts for 44.065% of CD3, RI 50.882% × (1-44.065%) is 0.285, RI calculated using CD45RO and CD45RA were the same in percentile decimal places with a relative error of less than 1%.

Example 7

Number 17 Donor: PBMC from Donor No. 17 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 8.

The reconstruction index RI value was calculated as follows: CD3 accounted for 68.044% of total cell number, whereas CD45RA accounted for 46.268% of CD3, RI 68.044% × 46.268% ═ 0.315, or calculating CD45RO, CD45RO accounted for 53.656% of CD3, RI 68.044% × (1-53.656%) -0.315; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 8

Number 18 Donor: PBMC from Donor No. 18 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 9.

The reconstruction index RI value was calculated as follows: CD3 accounted for 44.210% of total cell number, while CD45RA accounted for 54.493% of CD3, RI 44.210% x 54.493% was 0.241; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 45.453%, and RI is 44.210% × (1-45.453%) -0.241; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 9

Number 19 Donor: PBMC from Donor No. 19 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 10.

The reconstruction index RI value was calculated as follows: CD3 accounted for 47.431% of total cell number, while CD45RA accounted for 29.061% of CD3, RI 47.431% x 29.061% was 0.138, or calculating the CD45RO ratio, CD45RO accounted for 70.879% of CD3, RI 47.431% x (1-70.879%) -0.138; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 10

Number 2 Donor: the results of fluorescence antibody staining and flow cytometry analysis of Donor # 2 PBMC are shown in FIG. 11.

The reconstruction index RI value was calculated as follows: CD3 accounted for 70.611% of total cells, whereas CD45RA accounted for 65.76% of CD3, RI 70.611% x 65.753% 0.464; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 34.237%, and RI is 70.611% × (1-34.237%) -0.464; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 11

Number 3 Donor: the results of the fluorescent antibody staining and the flow cytometry analysis of the PBMC of Donor No. 3 are shown in fig. 12.

The reconstruction index RI value was calculated as follows: CD3 accounted for 58.874% of total cell number, while CD45RA accounted for 64.207% of CD3, RI 58.874% x 64.207% 0.378; alternatively, the CD45RO ratio was calculated, CD45RO accounted for CD3 ratio was 35.778%, RI 58.874% × (1-35.778%) was 0.378, RI calculated using CD45RO and CD45RA were the same in percentile decimal and the relative error was less than 1%.

Example 12

Number 34 Donor: PBMC from Donor # 34 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 13.

The reconstruction index RI value was calculated as follows: CD3 accounted for 13.702% of total cell number, while CD45RA accounted for 53.492% of CD3, RI 13.702% x 53.492% was 0.073; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 46.274%, and RI is 13.702% × (1-46.274%) -0.074; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 13

Number 101 Donor: PBMC from Donor # 101 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 14.

The reconstruction index RI value was calculated as follows: CD3 accounted for 42.708% of total cell number, whereas CD45RA accounted for 59.561% of CD3, RI 42.708% x 59.561% was 0.254; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 40.329%, and RI is 42.708% × (1-40.329%) -0.255; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 14

Number 102 Donor: PBMC from Donor No. 102 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 15.

The reconstruction index RI value was calculated as follows: CD3 accounted for 64.252% of total cell number, while CD45RA accounted for 48.694% of CD3, RI 64.252% x 48.694% was 0.313; alternatively, the CD45RO ratio was calculated, CD45RO accounted for CD3 at 51.137%, RI 64.252% × (1-51.137%) at 0.314, RI calculated using CD45RO and CD45RA were identical in percentile decimal places with relative error less than 1%.

Example 15

Number 103 Donor: PBMCs from Donor # 103 were stained with fluorescent antibodies and analyzed by flow cytometry, and the results are shown in FIG. 16.

The reconstruction index RI value was calculated as follows: CD3 accounted for 53.361% of total cell number, while CD45RA accounted for 71.512% of CD3, RI 53.361% x 71.512% was 0.382; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 28.471%, and RI is 53.361% × (1-28.471%) -0.382; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 16

Number 104 Donor: PBMCs from Donor 104 were stained with fluorescent antibodies and analyzed by flow cytometry, and the results are shown in FIG. 17.

The reconstruction index RI value was calculated as follows: CD3 accounted for 44.720% of total cell number, while CD45RA accounted for 53.238% of CD3, RI 44.720% x 53.238% 0.238; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 46.670%, and RI is 44.720% × (1-46.670%) -0.238; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 17

Number 105 Donor: PBMC from Donor # 105 were stained with fluorescent antibody and analyzed by flow cytometry, and the results are shown in fig. 18.

The reconstruction index RI value was calculated as follows: CD3 accounted for 68.377% of total cell number, while CD45RA accounted for 60.064% of CD3, RI 68.377% x 60.064% was 0.410; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 39.819%, and RI is 68.377% × (1-39.819%) -0.411; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

Example 18

4093 number Donor: PBMC of No. 4093 Donor were stained with fluorescent antibody and analyzed by flow cytometry, the results are shown in FIG. 19.

The reconstruction index RI value was calculated as follows: CD3 accounted for 26.029% of total cell number, while CD45RA accounted for 58.490% of CD3, RI 26.029% x 58.490% was 0.152; or calculating the proportion of CD45RO, wherein the proportion of CD45RO in CD3 is 40.968%, and RI is 26.029% × (1-40.968%) -0.152; RI calculated using CD45RO and CD45RA, both identical in percentile decimal places with relative error less than 1%.

EXAMPLE 19 reconstitution of the immune System in animals with Donor (PBMC)

Taking NSG of American JAXSON Lab for 6-8 weeks^TMMice or mice with the same grade of T/B/NK cell severe deficiency are adaptively raised for one week, weighed and inoculated with PBMCs of examples 1-18, respectively, and the inoculation information is shown in Table 3 below.

TABLE 3 resuscitated PBMC Vaccination information

Animal strain	PBMC	Number of animals	Inoculation mode	Amount of cells seeded/	Volume/volume of cell suspension seeded
						NSG	Donor	5	Tail vein i.v.	5×106	0.1mL

The proportion of human CD45+ (i.e., hCD45+) cells in the blood of mice was measured 7 days before and after PBMC cell inoculation, and peripheral blood immune system reconstitution values were measured weekly after the first test, and the test protocol is shown in Table 4 below.

Table 4 peripheral blood PBMC reconstitution flow cytometry assay

Detecting the index	Analytical method
		hCD45，mCD45	Peripheral blood human PBMC reconstitution ratio (%) ═ hCD45+/(hCD45+mCD45)×100％

The reconstructed values of donor (pbmc) were recorded for each example and the results are shown in fig. 20 and 21.

EXAMPLE 20 determination of Cut-off value

The RI of each Donor is calculated, linear regression statistics is performed by combining the reconstruction ratio (see Table 5) of about 19-21 days in vivo, the value of the reconstruction ratio commonly used in the industry is greater than 20% and is taken as the limit for judging the success of immune reconstruction, and the intersection point of the two is taken as the set point of the cut-off value, as shown in FIG. 22. The calculated RI value of the boundary point is about 0.2, when RI is greater than 0.2 and the reconstruction ratio is less than 20%, there are 2 cases, RI is greater than 0.2 and the reconstruction ratio is greater than 20%, there are 13 cases, the positive ratio is 13/15-86.6% and the samples with RI less than 0.2 are all negative, if the reconstruction index is not used for screening, the success rate is 13/18-72.2%. Therefore, the RI is set to 0.2, which is a suitable in vitro screening standard, can completely eliminate Donor with poor reconstruction, and can improve the success rate of Donor reconstruction screening.

TABLE 5 in vivo Donor reconstruction of mice human CD45 reconstruction ratio of about 19-21 days

Example 21

Human PBMC Donor is basically positive to CD45, therefore, the cells circled by the marker of CD45 are equivalent to total cells when cut-off is set, according to COA (product analysis report) provided by Donor supplier, we extract a batch of CD3 of CD45 and CD45RA of CD3 which need modeling Donor in mice, calculate the RI thereof, and judge the in vivo reconstruction ability according to cut-off set as 0.2, as shown in FIGS. 23-34 as specific examples.

The 12 Donor mice were injected 5X 10E6 tail vein, and the reconstruction ratio of human CD45 to the sum of human CD45+ murine CD45 was measured 20 days later (see Table 6), and the correlation between the reconstruction index RI and the reconstruction ratio was analyzed in combination with the reconstruction index RI calculated from the COA report of Donor manufacturer. As shown in fig. 35, the correlation of cut-off determination was almost perfectly reproduced, where Donor with RI 0.2 filtered out the reconstruction failure, and 7 of 8 Donor with RI >0.2 could be reconstructed well, with a success ratio of 7/8 87.5%, and if the reconstruction index was not used for screening, the success ratio was 7/12 58.3%. In contrast, the reconstruction success rate can be significantly increased.

TABLE 6 human CD45 reconstitution ratio for Donor reconstitution in New batch of mice for about 19-21 days

Donor numbering	Reconstruction index, RI	Reconstruction ratio, RP
			9044#	39.95％×(23.781％+22.118％)＝0.183	16.834
8006A#	54.727％×(23.436％+28.871％)＝0.286	33.219
			4071#	58.104％×(40.323％+12.718％)＝0.308	41.542
0903C#	44.746％×(37.28％+14.24％)＝0.231	24.591
			0503C#	62.162％×(33.61％+27.42％)＝0.379	39.152
0027#	38.729％×(39.437％+20.928％)＝0.234	12.221
			3070#	51.678％×(35.687％+15.505％)＝0.265	40.887
7023#	26.964％×(37.616％+5.746％)＝0.117	10.583
			2066#	52.295％×(40.674％+16.086％)＝0.297	29.116
4089#	35.302％×(27.836％+12.770％)＝0.143	9.546
			2101#	38.960％×(26.960％+8.397％)＝0.138	15.715
9113#	57.689％×(24.977％+22.388％)＝0.273	22.485

In summary, the immune system reconstruction ability of PBMC can be accurately quantified through the Reconstruction Index (RI), and the Cut-off setting of RI 0.2 can be used to well screen out the Donor with poor reconstruction, so that the Donor with good reconstruction can be quickly selected, the construction process of the immune system humanized mouse model in the drug effect screening process of the tumor immune drug is greatly accelerated, and the construction success rate of the immune system humanized mouse model is remarkably improved. The detection method provided by the invention is beneficial to the selection of Donor and the planning of experiment time, simultaneously reduces the cost, can quickly and well promote the establishment of a humanized experimental animal model used for drug research and development, contributes to the development of new anti-tumor immunity-related drugs, and has good application value.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.