CN113025571B - Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application - Google Patents
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Abstract
The invention relates to a human peripheral blood T lymphocyte processing method, preparation and application of immunogen preparation and antiserum, specifically utilize discarded leukocyte filter disc to separate and purify T lymphocyte, and carry on the enrichment culture, carry on surface mark identification comparison and activity comparison of antigen to T lymphocyte after separating and culturing; then, the high-activity T lymphocytes after the amplification culture are used as immunogen, the healthy pig is used for preparing antiserum, the titer after the immunity is evaluated by the results of an E rosette inhibition experiment and a cell toxicity experiment, and an immunization program is formulated, so that the antiserum can be prepared in a large quantity; and collecting the anti-plasma for separating plasma, and preparing the ALG product. The method can solve the limitation problem that fresh whole blood needs to be collected every time in the titer detection of the raw materials and the finished product titer detection of the anti-human T lymphocyte immunoglobulin, and can also solve the bottleneck problem of the raw materials of the anti-human T lymphocyte immunoglobulin.
Description
Technical Field
The invention relates to the technical field of blood products, in particular to a treatment method of human peripheral blood T lymphocytes, an immunogen preparation and application of antiserum.
Background
The indications of human lymphocyte immunoglobulin (ALG) are mainly used for the prevention and treatment of immunological rejection in clinical organ transplantation, the prevention of graft-versus-host reaction in bone marrow transplantation, and the treatment of aplastic anemia and other diseases.
The immune globulin product for resisting human lymphocyte conventionally adopts human thymus lymphocyte as immunogen to immunize pig to obtain raw material serum of immune protein product for resisting human lymphocyte, and then the raw material serum is purified and refined to obtain pig immune globulin preparation for resisting human lymphocyte.
However, human thymic lymphocytes are difficult to obtain as raw materials, and the normalization problem of titer detection is poor. In addition, when thymocytes are conventionally adopted as antigens, the antigen components are complex, and the immune process can generate hybrid antibodies such as erythrocyte antibodies, thymic tissue antibodies, platelet antibodies and the like, so that the subsequent production and purification process of the ALG product is complex.
Disclosure of Invention
Based on the above, there is a need for a method for treating human peripheral blood T lymphocytes, and an immunogen preparation, and an antiserum preparation and application thereof.
The technical scheme of the invention is as follows:
the technical idea of the invention is to separate, purify and multiply culture T lymphocytes by using a waste leukocyte filter disc, and carry out antigen surface marker identification comparison and activity comparison on the separated T lymphocytes and the cultured T lymphocytes; then taking the high-activity T lymphocytes after the amplification culture as immunogen to immunize healthy pigs, preparing antiserum, evaluating the titer after immunization by using the results of an E rose rosette inhibition experiment and a cell toxicity experiment, and formulating an immunization program to prepare the antiserum in large quantity; and collecting the anti-plasma for separating plasma, and preparing the ALG product.
Specifically, the invention provides a human peripheral blood T lymphocyte processing method, which comprises the following steps: collecting the white blood cells in the white blood cell filter disc, and treating the white blood cells by adopting a lymphocyte separation solution to obtain separated and purified mononuclear cells; and performing activity identification on the separated and purified mononuclear cells, and performing identification and cell counting on main surface antigen molecules. And (3) carrying out proliferation culture on the separated and purified mononuclear cells to obtain high-activity T lymphocytes (the activity is more than 90%).
The human peripheral blood T lymphocyte processing method also comprises the step of cell cryopreservation: the mononuclear cells subjected to surface antigen molecule identification and cell counting are collected and subjected to batch cryopreservation to obtain the cells for titer detection, so that the limitation problem that fresh whole blood needs to be collected for each detection is solved, and the normalization problem of the cells for titer detection can be solved.
The invention also provides a T lymphocyte immunogen preparation which is an oil-in-water antigen preparation prepared by mixing a mineral oil adjuvant, a molecular adjuvant and high-activity T lymphocytes. Preferably, the mineral oil adjuvant is preferably selected from RM502 component a or RM503 component a of tetracaine, and the molecular adjuvant is preferably selected from RM502 component B or RM503 component B.
The invention also provides a preparation method of the T lymphocyte immunogen preparation, which comprises the following steps: and taking the high-activity T lymphocyte as an antigen, uniformly mixing the antigen and a mineral oil adjuvant according to the mass ratio of 1:1 under the aseptic condition, adding a molecular adjuvant, and oscillating to form the oil-in-water antigen preparation.
The invention also provides a production method of antiserum containing anti-human lymphocyte immunity protein, which comprises the following steps: and immunizing a healthy pig by injecting the immunogen preparation into the neck side muscle, wherein the immunization program comprises 1 time of basic immunization and 2 times of boosting immunization, the immunization time interval is 21-28 days, and the anti-plasma containing the anti-human T cell immune protein is collected on 9-14 days after the 2 nd boosting immunization, so that the vaccine is obtained. Preferably, the method further comprises the step of performing antibody titer analysis on the anti-plasma.
The invention also provides a production method of the anti-human lymphocyte immunity protein, which takes the white blood cells collected by the white blood cell filter disc as raw materials, utilizes the proliferated and cultured high-activity T lymphocyte immunity animal to produce the obtained antiserum, separates, purifies and refines to obtain the anti-human lymphocyte immunity protein.
The invention has the beneficial effects that:
the method for treating the human peripheral blood T lymphocytes can obtain high-activity in-vitro proliferated T lymphocytes by utilizing a waste leukocyte filter disc, prepare an immunogen preparation by matching with a specific mineral oil adjuvant and a molecular adjuvant, and obtain antiserum by injecting immune pigs by using cervical side muscles, can solve the limitation problem that fresh whole blood needs to be collected each time in titer verification of an anti-human T lymphocyte immunoglobulin preparation raw material and finished product titer verification, can solve the problem of cell normalization for titer verification, and can solve the problem of bottleneck of the anti-human T lymphocyte immunoglobulin raw material. Meanwhile, the T lymphocytes cultured by proliferation are not doped with erythrocytes, platelets and the like, so that the subsequent immune healthy pigs as antigens hardly produce erythrocyte antibodies and platelet antibodies, tissue antibodies and plasma antibodies, and the subsequent production and purification process of ALG products can be simplified.
Drawings
FIG. 1 is a photograph of a leukocyte filter disk in example 1.
FIG. 2 is a photograph of a time-division layer for lymphocyte separation in example 1.
FIG. 3 is a photograph showing the culture conditions of the cryopreserved cells of test groups Nos. 2 to 6.
Fig. 4 and 5 are flow cytometric views of example 2.
Figure 6 is a flow cytometric map of negative and positive controls.
Fig. 7 and 8 are graphs for evaluating lymphocytes in example 3.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. 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.
Example 1 isolation, cryopreservation and quality evaluation of lymphocytes
As shown in FIG. 1, a leukocyte filter disc (obtained from Central blood station in Wuhan City) was placed in a biosafety cabinet, the leukocyte filter disc was back-flushed with 0.9% physiological saline, and the back-flushed physiological saline containing leukocytes was collected in a sterile bottle.
15mL of lymphocyte separation solution (manufacturer model: Fujian san Yi) is pipetted into a separation tube (specification: 50 mL), and 15-30mL of diluted blood sample is added. Centrifuging for 10min under the condition of 1200g, and after centrifugation, layering the sample in the separation tube from top to bottom: the plasma-leucoderma layer-separating medium-interlayer-separating medium-erythrocyte sediment, the sample layering is shown in figure 2. The tunica albuginea layer was then pipetted into a new sterile centrifuge tube (50 mL), PBS buffer (pH 7.2-7.4) was added to 45mL, the washed cell sample was resuspended with a pipette, and the platelet was removed by centrifugation at 300g for 5min at room temperature. Washing is repeated once to obtain mononuclear cells with higher purity according to 1 x 107The cells were frozen at a density of/mL or cultured. The frozen stock solution is fetal bovine serum containing 10% DMSO.
The frozen lymphocytes are used as cells for detection, and particularly used as an E rosette inhibition test and a control test to judge whether the cells are feasible or not. Lymphocytes were taken as assays at different times (2 weeks per interval) in parallel with 2 controls each, and the assays were repeated 5 times with the results shown in table 1 below:
TABLE 1 cryopreservation of lymphocytes for E rosette experiments
As can be seen from the results in Table 1, it was possible to use cryopreserved lymphocytes as cells for assay and to ensure the consistency between batches established for each assay control.
EXAMPLE 2 proliferation culture conditions for lymphocytes and identification of surface antigens
This example investigated the effect of culture conditions on lymphocytes cultured for in vitro proliferation and investigated the lymphocyte proliferation medium (culture environment: 37 ℃ C., 5% CO)2Incubator) for cell proliferation cycle, status, density, activity, and surface antigen molecular identification. The investigation of some of the experimental examples is shown in Table 2 below:
TABLE 2 proliferation culture exploration test condition statistics table
Among them, the culture conditions of the cryopreserved cells of test groups Nos. 2 to 6 are shown in FIG. 3, which shows: obvious lymphocyte aggregation can occur in 3-6 days, the cell growth reaches a peak after 8-10 days of culture, the cell growth is in a plateau period after 14 days, and the cell activity is detected to be more than 90% by trypan blue staining after 21 days.
As can be seen from the above table: in the culture process, the recovery period of 3-4 days is needed for the recovery of the cryopreserved lymphocytes, the proliferation peak period is changed, the proliferation times are slightly lower than those of fresh cells, and the cells can only proliferate by about 60-80 times.
In addition, it is worth to be noted that the present invention mainly relates to a method for comprehensively and systematically identifying and studying the change of T lymphocyte cell surface antibodies before and after proliferation culture and the surface antibodies against thymocytes, etc. by obtaining human peripheral blood T lymphocytes using a discarded leukocyte filter disc and performing proliferation culture, optimizing the culture conditions of the T lymphocytes. The inventor team finds out in a large number of research and test processes that: the main condition for rapid proliferation and culture of T lymphocytes is that RPMI1640 added with CD3 monoclonal antibody, IL-2 and IGF-1 contains unnecessary amino acid, and HSA (preferably 0.5 g/L-1.5 g/L) is used for maintaining high activity in the proliferation and culture process. The RPMI1640 culture medium containing non-essential amino acid is better than the common RPMI1640 basic culture medium, and has the advantages of higher multiplication factor and longer growth period.
Meanwhile, the T lymphocytes proliferated and cultured by adopting the preferable culture solution are not doped with red blood cells, platelets and the like, and the subsequent immune healthy pigs as antigens hardly produce red blood cell antibodies and platelet antibodies, and further do not produce tissue antibodies and plasma antibodies.
In this example, the cryopreserved lymphocytes in example 1 and the proliferated and cultured lymphocytes in examples 2 to 6 were separately washed by resuspension in 1% BSA PBS buffer (pH 7.2 to 7.4), centrifuged at 1000rpm for 5min, the supernatant was discarded, the washing was repeated 2 to 3 times, and the cell density was adjusted to 7 10 in flow cytometry buffer6Cell suspension/mL. And (3) adding 5-10 mul of antibodies (types: CD2, CD3, CD4, CD8, CD25, CD56 and the like) into 100 mul of cell suspension, reacting for 20min at normal temperature in a dark place, and centrifuging at 1000rpm for 5min to remove supernatant. After washing once with 2mL of 1% BSA in PBS buffer (pH. apprxeq.7.2-7.4), the supernatant was removed by centrifugation at 1000rpm for 5 min. Then 0.5mL of 1% BSA in PBS buffer was added for resuspension and flow cytometry was used for detection.
The results of the assays are shown in table 3 below, where the mean of the initial lymphocyte CD3+ isolated was about 59.81%, CD2+ CD3+ about 61.8%, CD3+ CD4+ about 32.73%, CD3+ CD56+ about 18.45%, CD3+ CD8+22.15%, and CD3+ CD25 was about 8.23% of the lymphocytes analyzed.
Lymphocytes cultured for 14 days were again tested and analyzed for CD3+ averaging about 98.8% of the lymphocytes analyzed, CD2+ CD3+ about 97.5%, CD3+ CD4+ about 18.32%, CD3+ CD56+ about 2.91%, CD3+ CD8+75.31%, and CD3+ CD25 about 10.6%. The flow analysis is shown in FIGS. 4 and 5.
The cultured lymphocytes were collected, and a small number of the cells were collected and analyzed by flow cytometry for 17 kinds of lymphocyte molecules such as CD2, CD3, CD4, CD8, CD44, CD45, CD98, CD38, CD99, CD107a, etc., as compared with Jurkat cells (from Procel corporation) and peripheral lymphocytes, and the results are shown in Table 4 below:
TABLE 3 flow cytometry analysis of the major surface antigen molecules of lymphocytes
TABLE 4 statistical table of surface antigen alignments
Remarking: (-) not detected.
As can be seen from the above table, there was not much difference between the peripheral blood and leukocyte filter disc antigen-expressing molecules. However, compared with Jurkat cells, the expression levels of CD4, CD8, CD25 and CD44 are all higher than those of Jurkat cells; compared with thymocytes, CD4, CD50 and CD107a are slightly lower, and CD3, CD44, CD82 and CD49d are slightly higher; the expression level of CD107a and CD50 in Jurkat cells is higher than that of peripheral blood and filter discs.
Example 3 antigen preparation, pig immunization method and potency detection
The lymphocytes in the 2-6 test groups after proliferation culture for 14 days are centrifuged at 1500rpm for 5min, collected, washed with 0.9% NaCl physiological saline for 2-3 times, and counted for death and activity by using a cell counter, wherein the cell activity must be more than 90%.
Preparing antigen cell amount according to the weight of healthy pigs: 1 hundred million proliferated lymphocytes are added per 5-8 kg. Taking out the mineral oil adjuvant and the antigen under aseptic conditions, rapidly mixing the mineral oil adjuvant and the antigen uniformly according to the mass ratio of 1:1, adding 10 mu L of molecular adjuvant according to the immune dose of each pig, mixing uniformly, and shaking for 10-20min to form the oil-in-water antigen. Wherein, the formulation for preparing the oil-containing antigen preparation is as follows 5:
TABLE 5 dosage scale for formulations of different antigen preparations
Multiple intramuscular injections were made on both sides of the neck of pigs (breed: Changbai). The first immunization was performed 21 days later for the 1 st booster, and the last booster was performed 21 days after the 2 nd immunization. The titer is detected by blood sampling before and after each immunization, and the fluctuation level of the antibody titer is continuously monitored after the 2 nd boosting immunization. Namely, the plasma collected from the different antigen preparations of this example was subjected to antibody titer analysis using the E rosette inhibition assay and the lymphocyte toxicity assay. For a detailed method of the rose inhibition test and the lymphocyte toxicity test, please refer to the three parts of the China pharmacopoeia (Tonghe 3515 and Tonghe 3516) 2020 edition.
The result of an E rose rosette inhibition test shows that after 3-1 test groups are adopted to immunize healthy pigs with expanded peripheral blood lymphocytes for the first time, a strong immune reaction is generated, 2 pig plasma titer detections are qualified, and the plasma antibody levels at 6 th to 15 th days after 2 times of boosting immunity are in a peak period and are all qualified by 100%; the inhibition rate of E rosette began to decrease after 18 days, and the results are shown in Table 6 and FIG. 7. The titer results of the lymphotoxin after 2 boosts showed that the antibody level was highest at day 9 and 87.5% was acceptable; the late phase began to decline and the results are shown in table 7 and figure 8.
Meanwhile, the flow cytometry is used for analyzing the lymphotoxin experimental result of the immunized plasma, so that the positive and negative controls are established, the lymphocyte death rate of all immunized porcine plasma diluted by 500 times is high and reaches over 78 percent, and the highest lymphocyte death rate reaches 92.8 percent, which is shown in the table 8.
TABLE 6 results of inhibition assay using 3-1 Immunity porcine plasma E Rose Ring
TABLE 7 analysis of the results of the cytotoxic assay using 3-1 of 3-immunized porcine plasma after immunization
Results of the flow-type cytotoxicity assay on 3-immune porcine plasma samples of Table 83-1 diluted 500 times
Example 4 formulation of pig immunization program
According to the results of the titer test in example 3, the immunization program was defined as: 3-1 antigen immune preparation is adopted, and the immune program comprises 1 basic immune and 2 boosting immune with the interval of 21-28 days. And collecting the anti-plasma 9-14 days after the 2 nd boosting immunization.
The above immunization program is applied to immunize 10 pigs again, the result of the test of the plasma resistance titer of the 10 pigs meets the standard of the preparation of the anti-human T cell pig immunoglobulin raw material in the 2020 pharmacopoeia, and the result statistics are shown in the following table 9:
TABLE 93 anti-plasma (diluted 1000-fold) E rosette inhibition assay test results collected after immunization
Example 5 application
One application is to prepare assay cell banks batch-wise using the cells studied in example 1 above to solve the problem of cell normalization for potency assays.
The second application is that the preparation of the antigen preparation studied in the examples 3-1 and 3-2 and the establishment of the immunization program in the example 4 are used for producing a large amount of anti-human T cell immune plasma, and then the production is carried out according to the process of anti-human T cell pig immunoglobulin, and the large-scale application can be carried out. The T lymphocyte cultured by proliferation under specific conditions is used as an antigen, so that erythrocyte antibodies and platelet antibodies are hardly produced, tissue antibodies and plasma antibodies are not produced, the production process can be simplified, the product cost is reduced, and the benefit is brought to vast patients.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. A method for processing human peripheral blood T lymphocytes is characterized by comprising the following steps:
collecting white blood cells in a white blood cell filter disc, treating the white blood cells by adopting lymphocyte separation liquid, sucking a white blood cell layer after centrifugation, carrying out heavy suspension by adopting PBS buffer solution, centrifuging and washing repeatedly to obtain separated and purified mononuclear cells;
collecting mononuclear cells qualified by activity identification, and performing batch cryopreservation;
and performing proliferation culture on the mononuclear cells qualified by activity identification, wherein the proliferation culture adopts an RPMI1640 non-genetic amino acids medium containing CD3 monoclonal antibody, IL-2, IGF-1 and Human Serum Album (HSA) to obtain the T lymphocytes with the activity of more than 90% and without doping red blood cells and platelets.
2. A T lymphocyte immunogen preparation which is an oil-in-water antigen preparation prepared by mixing a mineral oil adjuvant, a molecular adjuvant and T lymphocytes prepared by the treatment method according to claim 1.
3. A method of preparing a T lymphocyte immunogen preparation according to claim 2, comprising the steps of:
taking the T lymphocyte prepared according to the method 1 as an antigen, uniformly mixing the antigen and a mineral oil adjuvant according to the mass ratio of 1:1 under the aseptic condition, adding a molecular adjuvant, and oscillating to form an oil-in-water antigen preparation.
4. Use of the highly active T lymphocytes obtained by the method for treating human peripheral blood T lymphocytes according to claim 1 or the T lymphocyte immunogen preparation according to claim 2 for the production of an anti-human T cell immunity protein.
5. A method for producing antiserum containing anti-human T cell immune protein, comprising the steps of:
a healthy pig is immunized by injecting the T lymphocyte immunogen preparation of claim 2 into the neck and the immunization program is 1 basal immunization and 2 booster immunizations, the immunization time interval is 21-28 days, and the anti-plasma containing the anti-human T cell immune protein is collected on 9-14 days after the 2 booster immunizations.
6. The method for producing antisera containing an anti-human T-cell immunity protein according to claim 5, further comprising a step of analyzing the antibody titer of said anti-plasma.
7. A process for producing an anti-human lymphocyte immunity protein, which comprises using the antiserum obtained by the production process according to claim 5, which is separated and purified from leukocytes collected on a leukocyte filter as a raw material, to obtain the anti-human lymphocyte immunity protein.
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