CN110964695B - Cell strain and method for detecting rhIL-12 in-vitro activity by proliferation of cell strain - Google Patents
Cell strain and method for detecting rhIL-12 in-vitro activity by proliferation of cell strain Download PDFInfo
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Abstract
The invention provides a cell strain and a method for detecting the in vitro activity of rhIL-12 by proliferation of the cell strain. The preservation number of the cell strain is CCTCC NO: c2019242; the T3E3 cell strain is stimulated to proliferate by using rhIL-12 standard products and rhIL-12 test products with different gradient concentrations respectively, and the in vitro activity of rhIL-12 can be effectively detected by combining with MTS detection method. The detection method has good accuracy and high precision, and can be suitable for activity detection of rhIL-12 stock solution and preparation.
Description
Technical Field
The invention belongs to the technical field of cell activity detection, and relates to a cell strain and a method for detecting the in vitro activity of rhIL-12 by proliferation thereof.
Background
Human interleukin-12 (IL-12) is an important cytokine in humans that stimulates lymphocyte proliferation, induces cytotoxic activity of CTL and NK cells, and promotes secretion of cytokines such as IFN-gamma, TNF-alpha, GM-CSF, etc. The biological product 1 new medicine recombinant human interleukin-12 (rhIL-12) independently developed by the company has the function of comprehensively recovering blood images, and can be used for leucocyte, erythrocyte, thrombocytopenia and acute radiation diseases caused by tumor radiotherapy and chemotherapy. rhIL-12 is a high molecular glycoprotein, and has the complexity and difficulty shared by biological products in quality research and quality control as a medicine, in particular to the establishment of a biological activity detection method of rhIL-12, which is a difficulty which must be overcome in medicine development.
At present, the reported method for detecting the activity of rhIL-12 at home and abroad mainly comprises an IFN-gamma induction method and a PBMC proliferation method, and a standard method for detecting the biological activity of rhIL-12 is not recorded in Chinese pharmacopoeia. IFN-gamma induction methods are reported in the literature and patents to induce IFN-gamma production by stimulation of PBMC or PHA-activated PBMC with rhIL-12, indirectly reflecting rhIL-12 activity by comparison with a standard. Another literature reports that a reliable bioassay for determining its biological activity was established by stimulating NK-derived NKG cell lines to produce IFN-gamma using rhIL-12. The PBMC proliferation method mentioned in the literature is to stimulate PHA activated PBMC with rhIL-12, and reflect the biological activity of rhIL-12 by detecting the proliferation of cells and comparing with a standard. The IFN-gamma induction method is a cell method and an ELISA method, the systematic error of the method is larger, the error range of the detection value is larger, and the difference between ELISA kit batches, the difference between different strips of the same plate and the operation requirement on experimenters are high, so that the detection result of the activity of the sample can sometimes have larger fluctuation, and the result is inaccurate and reliable. The PBMC proliferation method involves only cell culture, and the method has less ELISA detection than the IFN-gamma induction method. The cells used in the proliferation method reported at present are PBMC, and as PBMC cells are derived from human bodies, the time for obtaining available cells is long (whether pathogens such as viruses exist or not) and the PBMC are mixed cells, the PBMC cells have large batch-to-batch differences and the like, the PBMC cells cannot be popularized and used as a batch detection release method for industrial production, and the application of the method is limited.
Disclosure of Invention
Based on the drawbacks of the prior art, an object of the present invention is to provide a cell line obtained by screening, which has a preservation number of: cctccc NO: C2019242. the cell strain of the invention has been preserved, the preservation date: 2019.11.09; preservation unit: china Center for Type Culture Collection (CCTCC); deposit unit address: the university of martial arts, chinese, postal code 430072; preservation number: cctccc NO: c2019242; classification naming: human T lymphocytes T3E3 (Human T lymphocyte T E3).
Another object of the present invention is to provide a method for detecting the in vitro activity of rhIL-12 by proliferation of the cell line. The method is different from an IFN-gamma induction method, and meets the conditions of relatively more accurate and reliable test, simpler and safer test, system error reduction as much as possible, error reduction caused by human factors and the like.
It is a further object of the invention to provide the use of the cell line for detecting rhIL-12 in vitro activity.
The aim of the invention is achieved by the following technical means:
in one aspect, the invention provides a cell strain, named as a T3E3 cell strain, with a preservation number of CCTCC NO: C2019242.
the T3E3 cell strain is obtained by screening by the inventor, and is specifically:
human Peripheral Blood Mononuclear Cells (PBMC) of normal people are taken and cultured by a complete culture medium, and clone screening is carried out to obtain a clone which has better proliferation and proliferation response to rhIL-12 stimulation, and the clone is named as T3E 3. T3E3 cells can be stably passaged within the test passages. The phenotype of T3E3 cells analyzed by flow cytometry was additionally: CD3 + CD4 + CD8 － CD56 - CD16 - 。
The T3E3 cell strain has better proliferation response to rhIL-12 stimulation, and the proliferation method of T3E3 cells can replace the conventional IFN-gamma induction method and PBMC proliferation method, so that the complexity of combination of a cell method and an ELISA method in the gamma-interferon induction method is avoided, the difference between batches of ELISA kits, the difference between different strips of the same plate and the high operation requirement on experimenters are avoided, and the possibility that activity detection sometimes has larger fluctuation is avoided; avoiding the risks of cell acquisition (whether pathogens such as viruses exist or not) in the PBMC proliferation method, and the defects that the time for obtaining the available activated PBMC is long, the PBMC is mixed cells, the batch-to-batch differences are large, the cell sources are difficult, the stable cell libraries for detection are difficult to establish and the like.
In another aspect, a method for detecting rhIL-12 in vitro activity, the method comprising the steps of:
respectively stimulating T3E3 cell strains to proliferate by using rhIL-12 standard products and rhIL-12 test products with different gradient concentrations;
obtaining the OD value of proliferation of the T3E3 cell strain by adopting an MTS detection method;
and (3) taking an OD value as an ordinate and a dilution multiple as an abscissa, and fitting by four parameters to obtain an activity curve regression equation, so as to obtain the in vitro activity of the rhIL-12 test sample.
The method directly stimulates cells through rhIL-12, and then MTS is used for detecting proliferation of the cells to obtain the biological activity of rhIL-12.
In the above method, preferably, before the stimulation of the T3E3 cell line, the method further comprises subculturing the T3E3 cell line, specifically:
subculturing the T3E3 cell strain in a complete culture medium to obtain a cell suspension;
centrifuging the cell suspension to remove the supernatant, cleaning the cell suspension with a semi-complete culture medium, and then supplementing the semi-complete culture medium for culture;
then adding PHA to activate the cells, and removing PHA after activation.
In the invention, rhIL-2 is removed from the culture medium in the culture of the T3E3 cell strain before the stimulation, so that the proliferation response of the rhIL-12 stimulated cells is more obvious. The inventors found in the study that rhIL-12 has a much lower effect on promoting proliferation of T3E3 cells in culture containing rhIL-2 than on promoting proliferation of T3E3 cells in culture without rhIL-2. Activation by PHA addition promotes T cell mitosis, and the T3E3 cells of the invention are CD4 + Is activated and expresses high affinity IL-12 receptor (IL-12R) on the cell surface, namely IL-12 specifically recognizes and binds to IL-12R expressed on the surface of activated T cell membrane and promotes cell proliferation through a series of signal paths, so PHA activation is the premise of the cell strain responding to IL-12 proliferation and can make the cell more sensitive to rhIL-12 stimulation.
In the above method, preferably, the culture is performed by taking a semi-complete medium from which rhIL-2 is removed one day before PHA activation. The inventors have found that removal of rhIL-2 one day prior to PHA activation allows the cells to adapt, and that subsequent stimulation of cell proliferation by rhIL-12 is better.
In the above method, preferably, the complete medium is a medium comprising Alpha MEM medium, 7.5% -17.5% horse serum, 7.5% -17.5% fetal bovine serum, 0.1mM beta-mercaptoethanol, 0.02mM folic acid and rhIL-2 with a final concentration of 100-250U/ml;
more preferably, the complete medium is a medium composed of Alpha MEM medium supplemented with 12.5% horse serum, 12.5% fetal bovine serum, 0.1mM beta-mercaptoethanol, 0.02mM folic acid and rhIL-2 at a final concentration of 200U/ml.
In the above method, preferably, the semi-complete medium is a medium composed of Alpha MEM medium added with 7.5% -17.5% horse serum, 7.5% -17.5% fetal bovine serum, 0.1mM beta-mercaptoethanol and 0.02mM folic acid;
more preferably, the semi-complete medium is a medium composed of Alpha MEM medium added with 12.5% horse serum, 12.5% fetal bovine serum, 0.1mM beta-mercaptoethanol and 0.02mM folic acid.
In the method, preferably, rhIL-12 standard and rhIL-12 test with different gradient concentrations are diluted in an exponential gradient according to the initial stimulation concentration; preferably 3 n Diluting the gradient multiple, wherein n is an integer greater than or equal to 0; it is further preferred to dilute 9 gradients.
In the method, preferably, the initial stimulation concentration of the rhIL-12 standard substance or the rhIL-12 test substance is 1-15 ng/ml; more preferably 5ng/ml.
In the above method, preferably, the T3E3 cells have a density of 0.5 to 1.5.times.10 when stimulated 6 cells/ml; more preferably 1X 10 6 cells/ml。
The above method, preferably, the conditions under which the stimulation is performed are: 37 ℃,5% CO 2 Culturing for 24-72 h; preferably, the culture is performed for 48 hours.
Preferably, the method for obtaining the OD value of proliferation of the T3E3 cell strain by adopting the MTS detection method comprises the following steps:
after the cell proliferation stimulation is finished, MTS is added for incubation, and an enzyme-labeled instrument is used for detecting OD value after the incubation is finished.
In the above method, preferably, the incubation condition is 35-39 ℃ and the incubation time is 2-5 h;
more preferably, the incubation conditions are 36-38deg.C and the incubation time is 3-4 h.
In the above method, preferably, the OD value is taken as an ordinate, the dilution multiple is taken as an abscissa, and four parameters are used for fitting to obtain an activity curve regression equation, so that the specific method for obtaining the in vitro activity of the rhIL-12 test sample comprises the following steps:
taking OD value as ordinate and dilution multiple as abscissa, fitting with four parameters to obtain regression equation of activity curve, top value and Bottom value and R 2 A value;
the in vitro activity of the rhIL-12 test sample is obtained by the following formula:
pr is the biological activity of a standard substance, and the unit is U/ml; ds is the pre-dilution of the test sample; dr is the standard pre-dilution; es is the dilution multiple of the half-effective amount of the sample corresponding to the standard sample; er is the dilution multiple of the half-effective amount of the standard substance.
In still another aspect, the invention also provides the use of the T3E3 cell strain described above or the method for detecting rhIL-12 in vitro activity described above for detecting rhIL-12 in vitro activity.
The invention has the beneficial effects that:
(1) The invention directly stimulates T3E3 cells obtained by screening through rhIL-12, and then uses MTS to detect the proliferation of the cells to obtain the biological activity of rhIL-12, thereby avoiding the complexity of combining a cell method and an ELISA method in a gamma-interferon induction method, avoiding the possibility of larger fluctuation of activity detection caused by batch-to-batch difference of ELISA kits, different battens of the same plate and high operation requirement on experimenters.
(2) The cell strain T3E3 cells obtained by screening are used for detecting the biological activity of rhIL-12, so that the defects that the risk of obtaining cells in a PBMC proliferation method (whether pathogens exist such as viruses and the like) and the time for obtaining available activated PBMC are long, the PBMC are mixed cells, the difference among batches is large, the cell sources are difficult, the stable cell bank for detection is difficult to establish and the like are avoided.
(3) The method for detecting the biological activity of rhIL-12 by using the T3E3 cell proliferation method has the advantages of good accuracy and high precision, and can be suitable for activity detection of rhIL-12 stock solution and preparations.
Drawings
FIG. 1A is a graph of T3E3 cell doubling time in example 1.
FIG. 1B is a graph showing the T3E3 cell viability in example 1.
FIG. 2A is a micrograph (100X) of T3E3 cells of example 1 prior to passaging.
FIG. 2B is a cell micrograph (100X) of T3E3 cells passaged for 48h in example 1.
FIG. 3A is a graph showing the activity of rhIL-12 stock solution cell proliferation assay in example 1.
FIG. 3B is a graph showing the activity of rhIL-12 preparation in example 1 by cell proliferation assay.
FIG. 4 is a graph showing the activity of rhIL-12 in stimulating T3E3 cells in comparative example 1.
FIG. 5 is a graph showing the proliferative activity of rhIL-12 in comparative example 2 to stimulate PHA-unactivated Jurkat cells.
FIG. 6 is a graph showing the proliferation activity of Jurkat cells activated for 24h with rhIL-12 stimulated PHA (10. Mu.g/ml) in comparative example 2.
Culture preservation for patent procedures:
the T3E3 cell strain of the invention;
preservation date: 2019.11.09;
preservation unit: china Center for Type Culture Collection (CCTCC);
deposit unit address: the university of martial arts, chinese, postal code 430072;
preservation number: cctccc NO: c2019242;
classification naming: human T lymphocytes T3E3 (Human T lymphocyte T E3).
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention. The reagent materials and the like used in the examples of the present invention are commercially available unless otherwise specified.
Term noun interpretation:
biological Activity: the ability of a product to produce a specific biological effect refers to the ability of a cytokine to promote proliferation and differentiation of target cells, enhance anti-infective and cell killing effects, promote or inhibit expression of other cytokines and membrane surface molecules, promote inflammatory processes, affect cellular metabolism, and the like.
Specific activity: specific activity refers to the unit of biological activity per milligram of protein, an important indicator of recombinant protein drugs as opposed to chemical drugs.
Interleukin: is a class of cytokines produced by and acting on a variety of cells. At least 38 interleukins, named IL-1-IL-38, are found to play an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and are involved in various physiological and pathological reactions of the body.
IFN-. Gamma.: gamma interferon, also known as type II interferon or immune interferon, is produced by mitogen-stimulated T lymphocytes. Interferon is a highly effective antiviral bioactive substance, and is a lymphokine with broad immunomodulating effect.
TNF- α: tumor necrosis factor is produced mainly by activated mononuclear/macrophages, can kill and inhibit tumor cells, promote neutrophil phagocytosis, resist infection, cause fever, induce the synthesis of acute phase proteins of hepatocytes, promote the differentiation of myeloid leukemia cells into macrophages, promote cell proliferation and differentiation, are important inflammatory factors, and participate in pathological damage of certain autoimmune diseases.
GM-CSF: granulocyte-macrophage colony stimulating factor has stimulating effect on the proliferation and differentiation process from marrow stem cell to mature granulocyte. Can promote differentiation of marrow stem cells into granulocytes (including neutrophils and eosinophils), erythroid, megakaryocyte, granulocyte and monocyte, promote proliferation and maturation of hematopoietic stem cells, has no obvious effect on basophils, and has less effect on erythroid late hematopoietic cells.
PBMC: peripheral Blood Mononuclear Cell peripheral blood mononuclear cells refer to cells having a single nucleus in peripheral blood, and include lymphocytes, monocytes, and the like.
PHA: phytohemagglutinin (Phytohaemagglutinin) is a lectin found in plants, particularly leguminous plants, belonging to the class of high molecular glycoproteins, and is a complex of oligosaccharides (composed of galactose, N-acetylglucosamine and mannose) and proteins, with the activity of promoting mitosis and inducing secretion of interferons.
NK cells: natural killer cells (natural killer cell) are important immune cells of the body, and are involved not only in anti-tumor, anti-viral infection and immunomodulation, but also in the development of hypersensitivity reactions and autoimmune diseases in some cases.
ELISA detection: enzyme Linked Immunosorbent Assay ELISA refers to a qualitative or quantitative method of adsorbing a soluble antigen or antibody to a solid carrier such as polystyrene to perform an immune reaction.
MTS cell proliferation assay: MTS is a novel MTT (thiazole blue) analogue, and in the presence of a coupling agent, dehydrogenases in living cells are converted into a liquid soluble formazan compound, and the absorption value of the formazan compound at 490nm can be directly measured on a 96-well plate, and the color shade and the cell number are in a linear relationship, so that the proliferation condition of cells is reflected.
Example 1:
the present embodiment provides a method for detecting rhIL-12 in vitro activity, comprising the steps of:
(1) Subculture of T3E3 cells
The T3E3 cells preserved by the screening method are adopted for about 48 hours to pass through the generation. T3E3 cells were stably passaged within the test passages, as shown in FIGS. 1A and 1B, with an average doubling time of 17.25h, an average cell viability of 95.72% and CV values of < 10%.
And preheating the prepared and packaged semi-complete culture medium (Alpha MEM culture medium+12.5% horse serum+12.5% fetal bovine serum+0.1 mM beta-mercaptoethanol+0.02 mM folic acid), and adding rhIL-2 to obtain the final concentration of 200U/ml, namely the complete culture medium. The brands and the numbers of the components of the culture medium are shown in the following table 1.
Table 1:
mu.l of cell suspension trypan blue was counted and the cell passage density was calculated to be 3X 10 based on the viable cell density 5 cell/ml and final volume after passage desired cell suspension volume, in biosafety cabinet, sucking desired cell suspension into T75 flask, adding desired volume of complete medium, then placing into incubator, 37℃and 5% CO 2 Culturing. Cell microscopy images before and after 48h of subculture are shown in FIGS. 2A and 2B.
(2) rhIL-2-free culture of T3E3 cells
Appropriate amount of cell suspension was taken according to the test requirement and centrifuged at 800rpm for 5min. The supernatant was removed and washed once with 10ml of semi-complete medium and centrifuged at 800rpm for 5min. Removing supernatant, re-suspending with 5ml semi-complete medium, sampling, counting, transferring cell suspension to culture dish, and culturing according to cell density of 3×10 5 cells/ml, half-complete medium was supplemented. Placing into incubator at 37deg.C with 5% CO 2 Culturing for 24h.
(3) PHA activation
The T3E3 cell suspension cultured in the semi-complete medium was removed, PHA (Sigma, catalog Number L8902) was directly added to a final concentration of 10. Mu.g/ml, and the mixture was pipetted 3 times and homogenized. 37 ℃,5% CO 2 Culturing for 24h.
(4) Preparation of cell suspensions
Taking PHA activated cell suspension, removing supernatant, centrifuging at 800rpm for 5min, washing with semi-complete medium for 2 times to remove PHA, centrifuging at 800rpm for 5min, suspending with appropriate amount of semi-complete medium, trypan blue counting, and regulating cell concentration to 2×10 with semi-complete medium 6 cells/ml. The above cell suspension was added to a 96-well plate with a multi-channel pipette, 50. Mu.l per well.
(5) rhIL-12 stimulation
(1) rhIL-12 working liquid preparation
The rhIL-12 standard and the rhIL-12 test are respectively diluted to 10ng/ml by a semi-complete culture medium, and then diluted by 3 times of gradient sequentially, wherein the total dilution gradient is 9. The test sample is in the form of stock solution and preparation.
(2) Preliminary dilution of rhIL-12 standard and test
Dilution of rhIL-12 standard: 30 mu l of rhIL-12 standard substance with the concentration of 100ng/ml after 1 mu g/ml is taken, added into a 1.5ml centrifuge tube added with 270 mu l of semi-complete culture medium, evenly mixed, diluted by 10 times, and the concentration after dilution is 100ng/ml, namely mother liquor of the rhIL-12 standard substance.
Dilution of rhIL-12 stock solution test sample: according to the concentration of the rhIL-12 stock solution, adjusting the concentration of the rhIL-12 stock solution to 0.1mg/ml by using a semi-complete culture medium; then the semi-complete culture medium is diluted by 10 times in turn to a final concentration of 100ng/ml, namely rhIL-12 stock solution to be tested mother solution.
Dilution of rhIL-12 preparation test article: taking 50 mu l of 15 mu g/ml rhIL-12 preparation, adding the preparation into a 1.5ml centrifuge tube added with 700 mu l of semi-complete culture medium, uniformly mixing, diluting 15 times, and obtaining the diluted concentration of 1 mu g/ml; then, 100 mu l of 1 mu g/ml rhIL-12 preparation diluent is taken and added into a 1.5ml centrifuge tube added with 900 mu l of semi-complete culture medium, and the mixture is uniformly mixed and diluted by 10 times, and the concentration after dilution is 100ng/ml, namely rhIL-12 preparation test sample mother liquor.
(3) Gradient dilution in rhIL-12 standard and test 96-well plate
The 96-well plate was labeled, 3 multiplex wells per sample, and the first dilution well was specifically performed as follows:
rhIL-12 standard: 18 μl of rhIL-12 standard stock solution (100 ng/ml) +162 μl of semi-complete medium;
rhIL-12 test: 18 μl of rhIL-12 stock solution or preparation test stock solution (100 ng/ml) +162 μl of semi-complete medium;
in a 96-well plate, 60 μl of rhIL-12 standard substance and test substance mixed in the first row of holes is added into 120 μl of semi-complete culture medium in the second row of holes, and mixed; 60 μl of rhIL-12 standard substance and test substance in the second row of holes is added into 120 μl of semi-complete culture medium in the third row of holes, and mixed uniformly; 3-fold gradient dilutions were performed sequentially, totaling 9 dilutions.
(6) Cell stimulation
Taking 50 μl of diluted rhIL-12 standard substance and test substance from 96-well plate, adding into 96-well plate with 50 μl of cell suspension, and final culturing with final cell density of 1×10 at 100 μl 6 cells/ml. The initial stimulation concentrations of the standard and the test sample are 5ng/ml, and the initial stimulation concentrations are 3-time gradient dilution, and 9 dilution gradients are total (the dilution gradient tables and the arrangement of the rhIL-12 standard and the test sample are shown in the table 2). 3 multiplex wells were made at each concentration, and the perimeter of the test wells was blocked by 100 μl PBS (phosphate buffer) to prevent edge well effects and water evaporation, 37deg.C, 5% CO 2 Culturing for 48h.
Table 2:
(7) MTS detection
After the stimulation was completed, 10. Mu.l MTS (Bei Bo organism, cat. No. BB-4204-3) was added to each well with 10. Mu.l of 8-lane pipettor; incubation is carried out at 37℃for 3.5-4h. Taking out the 96-well plate every 1h during incubation, and shaking and uniformly mixing for 30s by using an enzyme-labeled instrument. OD at 490nm was measured after incubation using SpectraMax M2 microplate reader.
(8) Data processing
Data were processed using GraphPad Prism 6 software, OD values as ordinate, dilution as abscissa, fitting with four parameters to obtain activity curves, top and Bottom values and R 2 Value, test standard requires R 2 The value ∈ 0.90. And (3) injection: the individual abnormal data can be removed during data processing, the abnormal data is marked, and the basis for judging the abnormal data is described.
The dilution multiple Er of the standard semi-effective amount is obtained by the following steps: the (Top+bottom)/2 value of the standard corresponds to the dilution factor under the standard fitting activity curve regression equation.
The dilution factor Es of the test sample corresponding to the half-effective amount of the standard sample is obtained by the following method: the (Top+bottom)/2 value of the standard corresponds to the dilution factor under the regression equation of the test sample fitted activity curve.
Wherein Pr is the biological activity of the standard, i.e. rhIL-12 standard biological activity=1×10 4 U/ml；
Ds is the pre-dilution of the test sample;
dr is the standard pre-dilution;
es is the dilution multiple of the half-effective amount of the sample corresponding to the standard sample;
er is the dilution multiple of the half-effective amount of the standard substance.
The experimental setup was as follows:
a batch of stock solution and a batch of preparation produced by my department were selected and tested three times at three different times, wherein the stock solution and preparation were repeated three times in the second and third tests, respectively.
The experimental results are as follows:
the biological activities of the stock solutions and formulations were tested by three runs and the results are summarized in tables 3 and 4.
Table 3:
table 4:
from the results of Table 3 and Table 4, it can be seen that the average value of the specific activities of the stock solutions is 1.24X10 7 U/mg, CV value 8.5%; the biological activity of the preparation has an average value of 2.09×10 5 U/ml, CV value was 15.9%.
And (3) accuracy detection:
the rhIL-12 standard and the test (stock and preparation) were compared using an in vitro activity assay for induction of T3E3 cell proliferation by rhIL-12, and the measured data were subjected to a four-parameter curve fit, plotted as the logarithm of IL-12 concentration versus absorbance of the sample (as shown in FIGS. 3A and 3B).
As can be seen from FIGS. 3A and 3B, a typical S-shaped curve has a correlation coefficient value (R 2 ) The dose response curves of the stock solution and the preparation are respectively 0.94/0.95 and 0.93/0.96, and have parallelism with the dose response curve of the rhIL-12 standard substance, so that the method is applicable to the detection of the biological activity of rhIL-12.
And (3) precision detection:
the biological activities of rhIL-12 stock and formulations were as shown in tables 3 and 4 above, with three experiments at three different times, respectively, using rhIL-12 to induce T3E3 cell proliferation.
Repeatability aspect:
the preparation and stock solution were repeatedly assayed 3 times in parallel in the second test, and the calculated biological activities are shown in Table 5.
Table 5:
as can be seen from table 5: the biological activity results of the stock solution and the preparation, which are repeatedly detected at the same time, have better repeatability, and CV values are less than 10%.
Intermediate precision aspect:
the results of the biological activity calculations for the stock solutions and formulations tested at three different times (three trials) are shown in table 6.
Table 6:
as can be seen from table 6: the biological activity results of the stock solution and the preparation detected at different times have better intermediate precision, and CV values are less than 20%.
From the above, it can be seen that: the method for detecting the biological activity of rhIL-12 by adopting the T3E3 cell proliferation method has good accuracy and high precision, and is indicated to be suitable for activity detection of rhIL-12 stock solution and preparations.
Comparative example 1:
and (3) comparing whether rhIL-2 exists in the culture medium in the subculture process before T3E3 cell stimulation. The experimental results are shown in table 7 and fig. 4. Table 7 shows the OD values of T3E3 cell proliferation MTS assays stimulated by rhIL-12 in the presence or absence of rhIL-2. FIG. 4 is a graph showing the activity profile of rhIL-12 in stimulating T3E3 cells (rhIL-2 was removed 1 day prior to activation, and the concentration of rhIL-12 ranged from 16.7 to 0.002 ng/ml).
Table 7:
note that: the OD values in the tables are the values after subtraction of the background OD value of the control group (rhIL-12 concentration of 0 ng/ml).
From the experimental results in table 7, it can be seen that: the promoting effect of rhIL-12 on T3E3 cell proliferation in culture containing rhIL-2 is far lower than that of T3E3 cell proliferation in culture without rhIL-2. In addition, the effect of removing rhIL-2 one day before PHA activation was more remarkable (as shown in FIG. 4). The reason is probably that the growth of the T3E3 cell strain depends on rhIL-2, and in the presence of rhIL-2, the effect of rhIL-2 on promoting T3E3 cell proliferation covers the effect of rhIL-12 on promoting T3E3 cell proliferation, and the effect is more obvious when the cells are subjected to adaptive culture by removing the rhIL-2 on the day before activation.
Comparative example 2:
the T3E3 cells obtained by screening of the invention are compared with conventional T lymphocytes, and the specific steps are as follows:
jurkat, clone E6-1 (hereinafter referred to as Jurkat) cell purchase from ATCC [ ]
TIB152 TM ) The cells are derived from acute T cell leukemia, and the cell type is T lymphocyte, and the cells grow in suspension.
Jurkat cells are existing human T lymphocytes. The following related experiments were performed with respect to whether existing human T lymphocytes can also be used for proliferation assay of rhIL-12 in vitro activity according to the methods of the embodiments of the present invention:
rhIL-12 (concentration range 5-0.0008 ng/ml) stimulated PHA unactivated and PHA (10 μg/ml) activated Jurkat cells for 24 hours, after 48h of culture, MTS method detected proliferation of cells, and ELISA reader detected OD at 490 nm. Data were processed with GraphPad Prism 6 software, OD values on the ordinate and dilution factors of rhIL-12 on the abscissa, and four parameters were used for fitting to obtain activity curves, and experimental results are shown in FIGS. 5 and 6. FIG. 5 is a graph of the proliferative activity of rhIL-12 to stimulate PHA-unactivated Jurkat cells. FIG. 6 is a graph showing the proliferation activity of Jurkat cells stimulated with rhIL-12 to activate PHA (10. Mu.g/ml) for 24h.
As can be seen from FIGS. 5 and 6, there is no obvious dose-response relationship between the in vitro activity of rhIL-12 detected after the Jurkat cells are not activated by PHA and are activated by PHA (10. Mu.g/ml) for 24 hours, i.e. the in vitro activity of rhIL-12 can not be detected by the proliferation method using the existing Jurkat cells. A good cell proliferation and rhIL-12 dose response curve (as shown in FIG. 3) was obtained using the method of the present invention.
From the above, it can be seen that: the invention directly stimulates T3E3 cells obtained by screening through rhIL-12, and then uses MTS to detect the proliferation of the cells to obtain the biological activity of rhIL-12, thereby avoiding the complexity of combining a cell method and an ELISA method in a gamma-interferon induction method, avoiding the possibility of larger fluctuation of activity detection caused by batch-to-batch difference of ELISA kits, different battens of the same plate and high operation requirement on experimenters. The cell strain T3E3 cells obtained by screening are used for detecting the biological activity of rhIL-12, so that the defects that the risk of obtaining cells in a PBMC proliferation method (whether pathogens exist such as viruses and the like) and the time for obtaining available activated PBMC are long, the PBMC are mixed cells, the difference among batches is large, the cell sources are difficult, the stable cell bank for detection is difficult to establish and the like are avoided. The method for detecting the biological activity of rhIL-12 by using the T3E3 cell proliferation method has the advantages of good accuracy and high precision, and can be suitable for activity detection of rhIL-12 stock solution and preparations.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the invention, but any modifications, improvements and equivalents within the spirit and scope of the method are intended to be included in the scope of the present invention.
Claims (18)
1. A cell line with a preservation number of CCTCC NO: C2019242.
2. a method of detecting rhIL-12 activity in vitro comprising the steps of:
stimulating the cell strain of claim 1 to proliferate with rhIL-12 standard and rhIL-12 test with different gradient concentrations;
obtaining an OD value of cell strain proliferation by adopting an MTS detection method;
taking an OD value as an ordinate and a dilution multiple as an abscissa, and fitting by four parameters to obtain an activity curve regression equation, so as to obtain the in vitro activity of the rhIL-12 test sample;
wherein, before stimulating the cell strain, the method further comprises subculturing the cell strain, specifically:
subculturing the cell strain in a complete culture medium containing rhIL-2 to obtain a cell suspension;
centrifuging the cell suspension to remove supernatant, cleaning with semi-complete culture medium without rhIL-2, and supplementing semi-complete culture medium without rhIL-2 for culturing;
then adding PHA to activate the cells, and removing PHA after activation.
3. The method according to claim 2, wherein the complete medium is a medium consisting of Alpha MEM medium supplemented with 7.5% -17.5% horse serum, 7.5% -17.5% fetal bovine serum, 0.1mM beta-mercaptoethanol, 0.02mM folic acid and rhIL-2 at a final concentration of 100-250U/ml;
the semi-complete culture medium is a culture medium formed by adding 7.5% -17.5% of horse serum, 7.5% -17.5% of fetal bovine serum, 0.1mM of beta-mercaptoethanol and 0.02mM of folic acid into Alpha MEM culture medium.
4. The method according to claim 3, wherein the complete medium is a medium consisting of Alpha MEM medium supplemented with 12.5% horse serum, 12.5% fetal bovine serum, 0.1mM beta-mercaptoethanol, 0.02mM folic acid and rhIL-2 at a final concentration of 200U/ml;
the semi-complete culture medium is a culture medium formed by adding 12.5% of horse serum, 12.5% of fetal bovine serum, 0.1mM of beta-mercaptoethanol and 0.02mM of folic acid into Alpha MEM culture medium.
5. The method of claim 2, wherein the rhIL-12 standard and rhIL-12 test at different gradient concentrations are diluted exponentially with the initial stimulus concentration.
6. The method of claim 5, wherein the rhIL-12 standard and rhIL-12 test at different gradient concentrations are at 3 according to the initial stimulation concentration n And diluting the gradient multiple, wherein n is an integer greater than or equal to 0.
7. The method of claim 6, wherein 9 gradients are diluted.
8. The method according to claim 5, wherein the initial stimulation concentration of the rhIL-12 standard or rhIL-12 test is 1-15 ng/ml.
9. The method of claim 8, wherein the initial stimulation concentration of the rhIL-12 standard or rhIL-12 test is 5ng/ml.
10. The method according to claim 8, wherein the cells have a density of (0.5 to 1.5). Times.10 when stimulated 6 cells/ml。
11. The method of claim 10, wherein the cells have a density of 1 x 10 when stimulated 6 cells/ml。
12. The method of claim 2 or 8, wherein the conditions under which stimulation is performed are: 37 ℃,5% CO 2 Culturing for 24-72 h.
13. The method of claim 12, wherein culturing is for 48 hours.
14. The method of claim 2, wherein the OD value of cell line proliferation is obtained by MTS assay:
after the cell proliferation stimulation is finished, MTS is added for incubation, and an enzyme-labeled instrument is used for detecting OD value after the incubation is finished.
15. The method according to claim 14, wherein the incubation is carried out at 35-39 ℃ for a period of 2-5 hours.
16. The method according to claim 14, wherein the incubation is carried out at a temperature of 36-38 ℃ for a time of 3-4 hours.
17. The method of claim 2, wherein the OD value is taken as an ordinate, the dilution factor is taken as an abscissa, and four parameters are used for fitting to obtain an activity curve regression equation, and the specific method for obtaining the in vitro activity of the rhIL-12 test sample is as follows:
taking OD value as ordinate and dilution multiple as abscissa, fitting with four parameters to obtain regression equation of activity curve, top value and Bottom value and R 2 A value;
the in vitro activity of the rhIL-12 test sample is obtained by the following formula:
pr is the biological activity of a standard substance, and the unit is U/ml; ds is the pre-dilution of the test sample; dr is the standard pre-dilution; es is the dilution multiple of the half-effective amount of the sample corresponding to the standard sample; er is the dilution multiple of the half-effective amount of the standard substance.
18. Use of the cell line of claim 1 or the method of detecting the in vitro activity of rhIL-12 of claim 2 for detecting the in vitro activity of rhIL-12.
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