CN111024949A - Biological activity analysis method of recombinant anti-VEGFR 2 monoclonal antibody and application thereof - Google Patents
Biological activity analysis method of recombinant anti-VEGFR 2 monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a biological activity analysis method of a recombinant anti-VEGFR 2 monoclonal antibody and application thereof, wherein the biological activity analysis method comprises the following steps: co-culturing the recombinant VEGFR-resistant 2 monoclonal antibody, human umbilical vein endothelial cells and VEGF reagent, adding CCK8 color development solution for continuous culture, reading a light absorption value, performing 4-parameter fitting according to the dose-effect relationship between the concentration of the recombinant VEGFR-resistant 2 monoclonal antibody and the light absorption value, and fitting a curve according to the EC50Values were evaluated for biological activity. The method is a biological activity analysis method specially aiming at the recombinant VEGFR 2-resistant monoclonal antibody, has high sensitivity, high accuracy and wide application range, can detect the recombinant VEGFR2 monoclonal antibody with different titer levels, and can detect the biological activity of an intermediate, a stock solution or a finished product of the recombinant VEGFR2 monoclonal antibody.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a biological activity analysis method of a monoclonal antibody and application thereof, in particular to a biological activity analysis method of a recombinant anti-VEGFR 2 monoclonal antibody and application thereof.
Background
VEGFR2 is highly expressed on tumor-associated endothelial cells. When VEGF is combined with VEGFR2 on tumor cell related endothelial cell, signal cascade reaction is initiated to cause endothelial cell proliferation and metastasis, and the permeability of blood vessel wall is increased, so that angiogenesis is caused, and sufficient oxygen and nutrition are provided for tumor occurrence and development. The VEGFR 2-resistant recombinant fully humanized monoclonal antibody can specifically block the interaction between vascular endothelial growth factor receptor 2(VEGFR-2) and ligand VEGF thereof, inhibit the activation of VEGFR2 and downstream signal conduction pathways, block the generation of tumor blood vessels, block the blood supply to tumor cells, inhibit the proliferation of the tumor cells and further cause the apoptosis of the tumor cells. Cyramza is the first global humanized monoclonal antibody drug targeting VEGFR2 (vascular endothelial growth factor receptor 2) developed by American etiquette, achieves the purpose of inhibiting tumors by inhibiting tumor angiogenesis, is approved to be on the market in the United states, Japan and European Union in 2014, is the only second-line targeting drug for treating gastric cancer without screening patients at present, is recommended to be the second-line first-line drug for gastric cancer, and can also be used for treating colorectal cancer and non-small cell lung cancer.
The recombinant anti-VEGFR 2 monoclonal antibody has great application value, related research on the recombinant anti-VEGFR 2 monoclonal antibody necessarily involves evaluation on the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody, and no special biological activity analysis method aiming at the recombinant anti-VEGFR 2 monoclonal antibody exists at present, so that the development of the biological cell activity method of the recombinant anti-VEGFR 2 monoclonal antibody is very meaningful.
Disclosure of Invention
In view of the defects of the prior art, the present invention aims to provide a method for analyzing the biological activity of a monoclonal antibody and applications thereof, and particularly provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody and applications thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, comprising: co-culturing the recombinant VEGFR-resistant 2 monoclonal antibody, human umbilical vein endothelial cells and VEGF reagent, adding CCK8 color development solution for continuous culture, reading a light absorption value, performing 4-parameter fitting according to the dose-effect relationship between the concentration of the recombinant VEGFR-resistant 2 monoclonal antibody and the light absorption value, and fitting a curve according to the EC50Values were evaluated for biological activity.
The biological activity analysis method is a biological activity analysis method specially aiming at the recombinant VEGFR-resistant 2 monoclonal antibody, has high sensitivity, high accuracy and wide application range, can detect the recombinant VEGFR-resistant 2 monoclonal antibody with different titer levels, and can detect the biological activity of an intermediate, a stock solution or a finished product of the recombinant VEGFR-resistant 2 monoclonal antibody.
Preferably, the living cell density of the human umbilical vein endothelial cells in the co-culture is 0.5X 105-1×105cells/mL, e.g. 0.5X 105cell/mL, 0.55X 105cell/mL, 0.6X 105cell/mL, 0.65X 105cell/mL, 0.7X 105cell/mL, 0.75X 105cell/mL, 0.8X 105cell/mL, 0.85X 105cells/mL or 1X 105cell/mL, etc., other values within the range can be selected, and are not described herein. Preferably 0.5X 105cells/mL.
The living cell density of the human umbilical vein endothelial cells is specifically selected to be 0.5 multiplied by 10 during the co-culture5-1×105In the cell/mL range, because different cell plating densities influence the signal value and the fitting condition of the 4-parameter fitting curve, the signal value is possibly lower due to insufficient or too much cell density, and the drug sensitivity is reduced; if the concentration is exceeded, the interval between the upper and lower plateaus of the fitted curve becomes smaller, which may be caused by decreased sensitivity to the action of the drug due to too many cells not contributing to sufficient interaction with the drug.
Preferably, the concentration of the VEGF reagent during the co-culture is 0.2-2 μ g/mL, such as 0.2 μ g/mL, 0.5 μ g/mL, 0.8 μ g/mL, 1 μ g/mL, 1.2 μ g/mL, 1.5 μ g/mL, or 2 μ g/mL, and other specific values within the range can be selected, which is not repeated herein. Preferably 0.5. mu.g/mL.
During the co-culture, the concentration of the VEGF reagent is specifically selected to be within the range of 0.2-2 mug/mL, because different VEGF concentrations can influence the signal value and the fitting condition of a 4-parameter fitting curve, the signal value is possibly low due to insufficient or too much VEGF concentration, and the drug sensitivity is reduced; and increasing the concentration, wherein the signal value is basically unchanged, and the interval between the upper platform and the lower platform is basically stable.
Preferably, the co-culture time is 60-70h, such as 60h, 61h, 62h, 65h, 66h, 67h, 68h or 70h, and other specific values within the range can be selected, which is not repeated herein.
The specific selection of the co-culture time is in the range of 60-70h, because the proliferation inhibition signal interval needs a long enough time to be expressed, time is wasted when the time range is exceeded, the time cost of detection work is increased, and the proliferation inhibition signal interval is smaller when the time range is less than the time range, which is not favorable for the precision and accuracy of the method.
Preferably, the temperature of the co-culture is 35-39 ℃, such as 35 ℃, 36 ℃, 37 ℃, 38 ℃ or 39 ℃, and other specific values in the range can be selected, which are not repeated herein.
Preferably, the time for continuing the culture by adding the CCK8 color development solution is 3.5-4h, for example, 3.5h, 3.6h, 3.7h, 3.8h, 3.9h or 4h, and other specific values within the range can be selected, which is not described herein again.
The specific selection of the time for continuing the culture by adding the CCK8 developing solution is 3.5-4h, because the development time is short, enough proliferation inhibition signal intervals are not detected, if the development time exceeds the time range, the time is wasted, the time cost of the detection work is increased, and if the development time is less than the time range, the proliferation inhibition signal intervals are small, which is not favorable for the precision and the accuracy of the method.
Preferably, the temperature for continuing the culture by adding the CCK8 developing solution is 35-39 ℃, for example, 35 ℃, 36 ℃, 37 ℃, 38 ℃ or 39 ℃, and other specific points in the range can be selected, which is not repeated herein.
Preferably, the reading light absorption value is read by using a microplate reader, and the detection wavelength is 450nm, and the reference wavelength is 620 nm.
As a preferred technical scheme of the invention, the method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody specifically comprises the following steps:
(1) digesting human umbilical vein endothelial cells in logarithmic growth phase to prepare single cell suspension, adjusting the living cell density of the single cell suspension by using an ECM complete culture medium, culturing for 5-7h at 35-39 ℃, removing supernatant, and continuously culturing for 12-18h by using an ECM culture medium of 0.1% FBS;
(2) adding recombinant anti-VEGFR 2 monoclonal antibody solutions with different concentrations into the cells incubated in the step (1), and culturing at 35-39 ℃ for 1.5-2.5 h;
(3) adding a VEGF reagent into the sample treated in the step (2), and culturing at 35-39 ℃ for 60-70 h;
(4) adding a CCK8 color development solution into the sample treated in the step (3), continuously culturing for 3.5-4h, reading a light absorption value by using an enzyme-labeling instrument, detecting the wavelength of 450nm and the reference wavelength of 620nm, carrying out 4-parameter fitting according to the dose-effect relation between the concentration of the recombinant anti-VEGFR 2 monoclonal antibody and the light absorption value, and carrying out biological activity evaluation according to the EC50 value of curve fitting.
The step (1) may be specifically: taking human umbilical vein endothelial cells in logarithmic growth phase, carrying out cell digestion according to a cell subculture process, eluting the digested cells with an ECM complete culture medium, centrifuging, then re-suspending the cells with the ECM complete culture medium to prepare a human umbilical vein endothelial single cell suspension, mixing uniformly, counting the cells, and determining the number of viable cells and the cell viability. The viable cell density was then adjusted to 0.5X 10 using ECM complete medium5cells/mL, 100 μ L/well were added to 96 well cell culture plates. The 96-well cell culture plate was placed in an incubator for culture. The supernatant was discarded, and ECM assay medium containing 0.1% FBS was added at 100 μ L/well, and the periphery was sealed with PBS, and incubated overnight in an incubator.
The recombinant anti-VEGFR 2 monoclonal antibody solutions with different concentrations in the step (2) mean that the final concentrations of the recombinant anti-VEGFR 2 monoclonal antibody are 300 mug/mL, 20 mug/mL, 2.5 mug/mL, 0.75 mug/mL, 0.2 mug/mL, 0.04 mug/mL and 0.001 mug/mL respectively, 7 concentrations of the standard curve are adopted, the linear section of the curve is ensured to be completely fitted, and 1 96-well plate can be utilized to the maximum extent for sample detection.
In another aspect, the present invention provides a method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody, which is applied to the detection of the biological activity of the intermediate, the stock solution or the finished product of the recombinant anti-VEGFR 2 monoclonal antibody.
Compared with the prior art, the invention has the following beneficial effects:
the biological activity analysis method is a biological activity analysis method specially aiming at the recombinant VEGFR-resistant 2 monoclonal antibody, has high sensitivity, high accuracy and wide application range, can detect the recombinant VEGFR-resistant 2 monoclonal antibody with different titer levels, and can detect the biological activity of an intermediate, a stock solution or a finished product of the recombinant VEGFR-resistant 2 monoclonal antibody.
Drawings
FIG. 1 is a graph of the fit of examples 2, 6-8;
FIG. 2 is a graph of the fit of examples 1 and 3-5.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The key reagents and consumables involved in the following examples are shown in table 1:
TABLE 1
The apparatus to which the following examples refer is shown in table 2:
TABLE 2
| Name of instrument and equipment | Model number | Manufacturer of the product | Instrument numbering |
| Enzyme-linked immunosorbent assay (ELISA) instrument | SpectraMax | MD | MV-C-MPR-002 |
| Biological safety cabinet | 1374/1379 | Thermo | MV-C-BSC-001 |
| Carbon dioxide incubator | 311 | Thermo | MV-B-CDI-002 |
| Cell counter | Cedex HiRes | Roche | MV-C-CVA-009 |
| Biological clean bench | BCM-1300A | Airtech | MV-C-BCB-009 |
| Centrifugal machine | 5810R | Eppendorf | MV-B-CTF-011 |
The experimental reagents involved in the following examples are as follows:
(1) complete medium: ECM complete Medium
The ECM complete medium was prepared by adding 25mL LFBS, 5mL P/S and 5mL ECGS in ECM medium set to 500mL ECM-basal medium and mixing well. Labeling, storing at 2-8 deg.C, and keeping the shelf life of 1 month without exceeding the shelf life of any reagent.
(2) ECM assay media
500mL of ECM-basal medium was added with 5mL of P/S to prepare ECM analysis medium.
(3) ECM assay medium with 0.1% FBS
ECM analysis medium was prepared by adding 0.1% FBS to ECM analysis medium and prepared as ready for use, containing 0.1% FBS.
(4) Sample diluent
ECM assay medium was diluted with 2% FBS. It is prepared as before use.
The following examples used human umbilical vein endothelial cells were first subcultured, specifically: placing a cell culture bottle in a biological safety cabinet, slightly sucking out the culture medium, adding 10mL of PBS to wash off residual culture medium on the surface of the cell, removing the PBS, adding 1-2mL of trypsin to ensure that the trypsin can cover a cell layer at the bottom of the bottle, standing and digesting at 37 ℃ for 1-2min, adding 10mL of ECM complete culture medium to elute digested HUVEC cells after the cells are completely digested (observing under a microscope, wherein the cell morphology becomes round and is separated from the wall), centrifuging at 1000rpm at room temperature for 5min, removing supernatant, then adding 5-20mL of ECM complete culture medium to resuspend the cells, and preparing HUVEC single cell suspension. Counting cells in 0.3mL cell suspension, inoculating appropriate number of cells in T75 cell culture flask, placing the flask in carbon dioxide incubator at 37 deg.C and 5% CO2And (5) culturing.
Example 1
The present embodiment provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, comprising the following steps:
(1) taking HUVEC cells in logarithmic growth phase, carrying out cell digestion according to a cell subculture process, eluting the digested cells with an ECM complete culture medium, centrifuging, then re-suspending the cells with the ECM complete culture medium to prepare HUVEC single cell suspension, mixing uniformly, taking 0.3mL of the suspension, counting the cells, and carrying out parallel operation for 2 times. The number of viable cells and the cell viability were determined from the mean of 2 cell counts. The viable cell density was then adjusted to 0.5X 10 using ECM complete medium5cells/mL, 100 μ L/well were added to 96 well cell culture plates. The 96-well cell culture plate was placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator for 6 h. The supernatant was discarded, and 100. mu.L/well of ECM assay medium supplemented with 0.1% FBS was sealed at the periphery with PBS and 5% CO at 37 ℃2Culturing in a constant temperature incubator overnight, and removing supernatant;
(2) each diluted concentration of Cyramza assay was added to the corresponding well of a 96-well cell culture plate at 50. mu.L/well using a multi-channel pipette (to give final concentrations of 300. mu.g/mL, 20. mu.g/mL, 2.5. mu.g/mL, 0.75. mu.g/mL, 0.2. mu.g/mL, 0.04. mu.g/mL, 0.001. mu.g/mL). After the sample loading operation was completed, the 96-well cell culture plate was placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator for 2 h. VEGF165 was diluted to 1. mu.g/mL with sample diluent and added to the corresponding wells of the plate at 50. mu.l/well using a multi-channel pipette at 37 ℃ with 5% CO2Culturing for 65h in a constant-temperature incubator;
(3) CCK8 reagent was added at 20. mu.L/well to 96-well cell culture plates at 37 ℃ with 5% CO2Culturing in a constant temperature incubator for 3.8 h;
(4) and (3) fitting data: and reading the light absorption value by using a microplate reader, and detecting the wavelength of 450nm and the reference wavelength of 620 nm. Performing 4-parameter fitting according to the dose-effect relationship between the concentration and the light absorption value of the sample to be measured, and performing curve fitting according to the sample50Values biological cell activity evaluation of the samples was performed.
Example 2
The present embodiment provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, comprising the following steps:
(1) taking H in logarithmic growth phaseUVEC cells are subjected to cell digestion according to a cell subculture process, the digested cells are eluted by using an ECM complete culture medium and centrifuged, then the cells are resuspended into HUVEC single cell suspension by using the ECM complete culture medium, the mixture is uniformly mixed, 0.3mL of the mixture is taken for cell counting, and the parallel operation is carried out for 2 times. The number of viable cells and the cell viability were determined from the mean of 2 cell counts. The viable cell density was then adjusted to 1X 10 using ECM complete medium5cells/mL, 100 μ L/well were added to 96 well cell culture plates. The 96-well cell culture plate was placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator for 6 h. The supernatant was discarded, and 100. mu.L/well of ECM assay medium supplemented with 0.1% FBS was sealed at the periphery with PBS and 5% CO at 37 ℃2Culturing in a constant temperature incubator overnight;
(2) each diluted concentration of Cyramza assay was added to the corresponding well of a 96-well cell culture plate at 50. mu.L/well using a multi-channel pipette (to give final concentrations of 300. mu.g/mL, 20. mu.g/mL, 2.5. mu.g/mL, 0.75. mu.g/mL, 0.2. mu.g/mL, 0.04. mu.g/mL, 0.001. mu.g/mL). After the sample loading operation was completed, the 96-well cell culture plate was placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator for 1.5 h. VEGF165 was diluted to 1. mu.g/mL with sample diluent and added to the corresponding wells of the plate at 50. mu.l/well using a multi-channel pipette at 37 ℃ with 5% CO2Culturing for 60h in a constant-temperature incubator;
(3) CCK8 reagent was added at 20. mu.L/well to 96-well cell culture plates at 37 ℃ with 5% CO2Culturing for 4h in a constant-temperature incubator;
(4) and (3) fitting data: and reading the light absorption value by using a microplate reader, and detecting the wavelength of 450nm and the reference wavelength of 620 nm. Performing 4-parameter fitting according to the dose-effect relationship between the concentration and the light absorption value of the sample to be measured, and performing curve fitting according to the sample50Values biological cell activity evaluation of the samples was performed.
Example 3
This example provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, which differs from example 1 only in that VEGF165 was diluted to 0.4. mu.g/mL with a sample diluent in step (2), and the rest remained the same.
Example 4
This example provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, which differs from example 1 only in that VEGF165 was diluted to 2. mu.g/mL with a sample diluent in step (2), and the rest remained unchanged.
Example 5
This example provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, which differs from example 1 only in that VEGF165 was diluted to 4. mu.g/mL with a sample diluent in step (2), and the rest remained unchanged.
Example 6
This example provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, which differs from example 2 only in that VEGF165 was diluted to 0.4. mu.g/mL with a sample diluent in step (2), and the rest remained the same.
Example 7
This example provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, which differs from example 2 only in that VEGF165 was diluted to 2. mu.g/mL with a sample diluent in step (2), and the rest remained unchanged.
Example 8
This example provides a method for analyzing the biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, which differs from example 2 only in that VEGF165 was diluted to 4. mu.g/mL with a sample diluent in step (2), and the rest remained unchanged.
Evaluation experiment 1:
the results of the fit for examples 1-8 are shown in Table 3 (in the table, A/D represents the interval of the proliferation inhibition curve, B represents the slope of the test standard 4-parameter fit curve, EC50Representing the half-effective concentration of the drug effect, R2Fitting degree representing curve fitting):
TABLE 3
The fitted curves for examples 1-8 are shown in FIGS. 1 and 2.
As can be seen from Table 3 and FIGS. 1-2: the curve obtained by the method for analyzing the bioactivity of the recombinant VEGFR 2-resistant monoclonal antibody has good fitting effect, and R is2The values are all higher than 0.950, and the signal value and the drug sensitivity are high. Comparing example 1 with example 2, when the cell density is higher, the interval between the upper and lower platforms becomes smaller, which may be caused by decreased sensitivity to the action of the drug due to too many cells not facilitating sufficient interaction with the drug; in comparative examples 3 to 5 or examples 6 to 8, the concentration of VEGF was increased to 0.5. mu.g/mL, and then the concentration was increased, the signal value was substantially unchanged, and the interval between the upper and lower plateaus was also substantially stable.
Evaluation experiment 2:
to investigate whether the assay method of the present invention is suitable for active samples with different titer levels, Cyramza assay samples were diluted with sample diluent (2% FBS assay medium) to prepare active samples with different titer levels (50%, 75%, 100%, 125% and 150% active titer level samples, respectively), and the assay method as described in example 1 was used to perform experiments to examine the recovery of assay results. The results are shown in Table 4.
TABLE 4
From the data in table 4, it can be seen that: the method for analyzing the biological activity of the recombinant VEGFR-resistant 2 monoclonal antibody has the advantages of high recovery rate, high accuracy and wide application range, and can be used for detecting the recombinant VEGFR-resistant 2 monoclonal antibodies with different titer levels.
The applicant states that the present invention is illustrated by the above examples to show the method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody and the application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for analyzing biological activity of a recombinant anti-VEGFR 2 monoclonal antibody, comprising: co-culturing the recombinant VEGFR-resistant 2 monoclonal antibody, human umbilical vein endothelial cells and VEGF reagent, adding CCK8 color development solution for continuous culture, reading a light absorption value, performing 4-parameter fitting according to the dose-effect relationship between the concentration of the recombinant VEGFR-resistant 2 monoclonal antibody and the light absorption value, and fitting a curve according to the EC50Values were evaluated for biological activity.
2. The method of claim 1, wherein the co-culture is performed at a viable cell density of 0.5 x 10 for human umbilical vein endothelial cells5-1×105cell/mL; preferably 0.5X 105cells/mL.
3. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to claim 1 or 2, wherein the concentration of the VEGF reagent is 0.2 to 2 μ g/mL when the co-culture is performed; preferably 0.5. mu.g/mL.
4. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to any one of claims 1 to 3, wherein the co-culturing time is 60 to 70 hours.
5. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to any one of claims 1 to 4, wherein the co-culturing temperature is 35 to 39 ℃.
6. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to any one of claims 1 to 5, wherein the incubation is continued for 3.5 to 4 hours with the addition of a CCK8 chromogenic solution.
7. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to any one of claims 1 to 6, wherein the temperature for continuous culture by adding a CCK8 developing solution is 35 to 39 ℃.
8. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to any one of claims 1 to 7, wherein the reading of the absorbance is performed by using a microplate reader at a detection wavelength of 450nm and a reference wavelength of 620 nm.
9. The method for analyzing the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody according to any one of claims 1-8, wherein the method specifically comprises the steps of:
(1) digesting human umbilical vein endothelial cells in logarithmic growth phase to prepare single cell suspension, adjusting the living cell density of the single cell suspension by using an ECM complete culture medium, culturing for 5-7h at 35-39 ℃, removing supernatant, and continuously culturing for 12-18h by using an ECM culture medium of 0.1% FBS;
(2) adding a recombinant anti-VEGFR 2 monoclonal antibody solution into the cells incubated in the step (1), and culturing at 35-39 ℃ for 1.5-2.5 h;
(3) adding a VEGF reagent into the sample treated in the step (2), and culturing at 35-39 ℃ for 60-70 h;
(4) adding a CCK8 color development solution into the sample treated in the step (3), continuously culturing for 3.5-4h, reading a light absorption value by using an enzyme-labeling instrument, detecting the wavelength of 450nm and the reference wavelength of 620nm, carrying out 4-parameter fitting according to the dose-effect relation between the concentration of the recombinant anti-VEGFR 2 monoclonal antibody and the light absorption value, and carrying out biological activity evaluation according to the EC50 value of curve fitting.
10. Use of the method of any of claims 1-9 for the analysis of the biological activity of the recombinant anti-VEGFR 2 monoclonal antibody for the detection of the biological activity of an intermediate, stock solution or finished product of the recombinant anti-VEGFR 2 monoclonal antibody.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094351A (en) * | 2020-09-11 | 2020-12-18 | 北京东方百泰生物科技股份有限公司 | Detection antibody of anti-VEGFR-2 monoclonal antibody, application and detection method |
| CN112903646A (en) * | 2021-01-25 | 2021-06-04 | 上海睿钰生物科技有限公司 | Method and system for detecting antibody affinity and application thereof |
| CN113480653A (en) * | 2021-08-06 | 2021-10-08 | 北京戴域生物技术有限公司 | Application of pharmaceutical composition containing gamma delta T cells in treating cancer |
| CN116718781A (en) * | 2023-08-09 | 2023-09-08 | 苏州依科赛生物科技股份有限公司 | Method for detecting cell adhesion promoting activity of recombinant human vitronectin |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003075840A2 (en) * | 2002-03-04 | 2003-09-18 | Imclone Systems Incorporated | Human antibodies specific to kdr and uses thereof |
| CN101245106A (en) * | 2007-12-04 | 2008-08-20 | 澳赛尔斯生物技术（上海）有限公司 | Anti-VRGF acceptor monoclone antibody, preparation method and application thereof |
| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
| CN103012589A (en) * | 2012-12-11 | 2013-04-03 | 上海赛伦生物技术有限公司 | Human anti-vascular endothelial cell growth factor antibody and application thereof |
| CN103113469A (en) * | 2012-10-17 | 2013-05-22 | 中国药科大学 | Single-chain antibody for resisting vascular endothelial growth factor receptor 2 |
| CN105628910A (en) * | 2014-11-07 | 2016-06-01 | 上海中信国健药业股份有限公司 | Biological activity detection method for VEGF targeted therapy drugs |
| CN106188296A (en) * | 2016-07-19 | 2016-12-07 | 中山康方生物医药有限公司 | The monoclonal antibody of one class vascular endothelial growth factor receptor VEGFR2 and encoding gene thereof and application |
| CN106674349A (en) * | 2017-03-07 | 2017-05-17 | 北京东方百泰生物科技有限公司 | Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody |
| CN106892980A (en) * | 2017-01-25 | 2017-06-27 | 长春金赛药业有限责任公司 | Anti-vegf R2 monoclonal antibodies and its application |
| CN107328943A (en) * | 2017-07-28 | 2017-11-07 | 东曜药业有限公司 | A kind of detection method of VEGF biological activity and application |
-
2019
- 2019-12-31 CN CN201911417826.7A patent/CN111024949A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003075840A2 (en) * | 2002-03-04 | 2003-09-18 | Imclone Systems Incorporated | Human antibodies specific to kdr and uses thereof |
| CN101245106A (en) * | 2007-12-04 | 2008-08-20 | 澳赛尔斯生物技术（上海）有限公司 | Anti-VRGF acceptor monoclone antibody, preparation method and application thereof |
| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
| CN103113469A (en) * | 2012-10-17 | 2013-05-22 | 中国药科大学 | Single-chain antibody for resisting vascular endothelial growth factor receptor 2 |
| CN103012589A (en) * | 2012-12-11 | 2013-04-03 | 上海赛伦生物技术有限公司 | Human anti-vascular endothelial cell growth factor antibody and application thereof |
| CN105628910A (en) * | 2014-11-07 | 2016-06-01 | 上海中信国健药业股份有限公司 | Biological activity detection method for VEGF targeted therapy drugs |
| CN106188296A (en) * | 2016-07-19 | 2016-12-07 | 中山康方生物医药有限公司 | The monoclonal antibody of one class vascular endothelial growth factor receptor VEGFR2 and encoding gene thereof and application |
| CN106892980A (en) * | 2017-01-25 | 2017-06-27 | 长春金赛药业有限责任公司 | Anti-vegf R2 monoclonal antibodies and its application |
| CN106674349A (en) * | 2017-03-07 | 2017-05-17 | 北京东方百泰生物科技有限公司 | Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody |
| CN107328943A (en) * | 2017-07-28 | 2017-11-07 | 东曜药业有限公司 | A kind of detection method of VEGF biological activity and application |
Non-Patent Citations (10)
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094351A (en) * | 2020-09-11 | 2020-12-18 | 北京东方百泰生物科技股份有限公司 | Detection antibody of anti-VEGFR-2 monoclonal antibody, application and detection method |
| CN112903646A (en) * | 2021-01-25 | 2021-06-04 | 上海睿钰生物科技有限公司 | Method and system for detecting antibody affinity and application thereof |
| CN113480653A (en) * | 2021-08-06 | 2021-10-08 | 北京戴域生物技术有限公司 | Application of pharmaceutical composition containing gamma delta T cells in treating cancer |
| CN113480653B (en) * | 2021-08-06 | 2022-05-06 | 朱有建 | Application of pharmaceutical composition containing gamma delta T cells in treating cancer |
| CN116718781A (en) * | 2023-08-09 | 2023-09-08 | 苏州依科赛生物科技股份有限公司 | Method for detecting cell adhesion promoting activity of recombinant human vitronectin |
| CN116718781B (en) * | 2023-08-09 | 2023-10-03 | 苏州依科赛生物科技股份有限公司 | Method for detecting cell adhesion promoting activity of recombinant human vitronectin |
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