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A method for detecting biological activity of recombinant human endostatin

A technology of vascular endothelium and detection methods, applied in the direction of color/spectral characteristic measurement, measuring devices, instruments, etc., can solve problems such as large errors, many influencing factors, and difficult operations, and achieve rigorous results and overcome large errors.

Active Publication Date: 2020-04-03
SHANDONG SIMCERE BIO PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The method for detecting the cell proliferation inhibitory activity of Endostatin in the prior art is mainly based on the possible mechanism of action of Endostatin, using Endostatin to inhibit HUVEC (human umbilical vein endothelial cells), HMEC (human microvascular endothelial cells), ECV304 (human umbilical vein endothelial cells) and other different cell proliferation and migration to detect its cell proliferation inhibitory activity, but the error of the selected experimental materials and test results is relatively large, and many of the test results have poor repeatability
[0006] Domestic manufacturers also use Endostatin to inhibit the growth of mouse tumors to evaluate its biological activity. However, due to the large difference in the size of mouse tumors, the in vivo assay method lacks regularity, has the disadvantages of large errors, poor repeatability, and difficult operation.
[0007] The existing detection of recombinant human endostatin (rhEndostatin) cell proliferation inhibitory activity has the following disadvantages: (1) The protein content of the sample is required to be at least greater than 5mg / ml, and low-concentration samples cannot be assayed by this method; (2) The activity assay results need to be Carry out four-parameter fitting, there are many influencing factors in the experiment, and there may be situations where the EC50 cannot be obtained by fitting; (3) The error range of the activity detection result is relatively large, ranging from 50% to 200%

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  • A method for detecting biological activity of recombinant human endostatin
  • A method for detecting biological activity of recombinant human endostatin
  • A method for detecting biological activity of recombinant human endostatin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 sample detection

[0065] 1.1 Detection steps

[0066] 1.1.1 Cell culture: HUVEC cells, HUVEC cells were completely cultured with ECM based on 37°C, 5% CO 2 The cells were cultured and subcultured to 5 passages in an incubator, and the cells were ready to be inoculated when the cells were in good condition.

[0067] 1.1.2 Vaccination:

[0068] Digest the cells with 0.25% trypsin-0.02% EDTA for about 1 min, observe under the microscope, immediately add culture medium to stop the digestion when the cells become round, pipette, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend with the medium , live cells counted with a hemocytometer under a microscope. Adjust the cell density to about 5000 cells / ml, add it into a 96-well culture plate, and add 160 μl of cell suspension to each well.

[0069] 1.1.3 Sample preparation and dosing

[0070] According to the activity of the standard recombinant human endostatin, reconstitute the standar...

Embodiment 2

[0120] Embodiment 2 precision evaluation

[0121] Precision evaluation includes intra-plate repeatability evaluation, inter-plate repeatability evaluation and intermediate precision.

[0122] Intra-plate repeatability inspection method: measure 2 parts of the same test solution in the same 96-well plate, and calculate the RSD% (relative standard deviation) of the specific activity of the sample in the plate to be 7.9%.

[0123] Inter-plate repeatability investigation method: the same person measured the same test solution 5 times in five 96-well plates, and the RSD% of the specific activity of the samples between the plates was calculated to be 16.0%.

[0124] Intermediate precision inspection method: 2 people conducted this test separately at different times, respectively tested the specific activity of the same test product solution 3 times, and calculated the RSD of the specific activity of the test product measured by 2 people for a total of 6 times was 7.3%.

[0125] The...

Embodiment 3

[0128] Embodiment 3 durability evaluation

[0129] Evaluate the factors that may cause the deviation of the activity determination experiment: the influence of different cell numbers in each well, the influence of different cell culture time, and the results all meet the requirements.

[0130] 3.1 Different cell numbers: adjust the cell numbers to 4000 and 6000 / ml respectively, detect the sample activity, and calculate its specific activity. The curves of the standard product and the test product are parallel, and the specific activity RSD of the test product under the three conditions is 1.4%, which meets the requirements.

[0131] 3.2 Different culture time: cultured for 72h, 89h and 96h respectively after adding the drug, tested the activity of the sample, and calculated its specific activity. The curves of the standard product and the test product are parallel, and the specific activity RSD of the test product under the three conditions is 10.6%, which meets the requireme...

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Abstract

The invention relates to a method for detecting the biological activity of recombinant human endostatin. The method comprises the following steps: inhibiting the proliferation of HUVEC by using concentration gradient points in a linear titer range in the activity detection results of a sample and a standard substance respectively, detecting the proliferation inhibition activity of the endostatin on the HUVEC by MTS colorimetry, and analyzing the biological activity of the recombinant human endostatin against angiogenesis by a quantitative response parallel line assay technology. The activity detection method is suitable for detecting low-concentration samples, significantly improves the method repeatability, and reduces the method error range.

Description

technical field [0001] The invention relates to the technical field of biological product bioassay statistical methods, in particular to a detection method for the biological activity of vascular endostatin. Background technique [0002] Vascular endostatin (Endostatin) is a kind of substance that O'Relly isolated from the culture medium of mouse endothelial cell line EOMAD in 1997 and has the effect of inhibiting vascular endothelial cells (O'Relly, M.S., etal.Cell.88: 299-285, 1997). Amino acid sequence analysis The substance is a degradation fragment of the carboxyl terminal of collagen XVIII, with a molecular weight of about 20kD. [0003] Recombinant human endostatin (rhEndosratin) and recombinant mouse endostatin have 85% homology in amino acid sequence. In 1996, the American company Entremed used yeast as the expression system to produce recombinant human endostatin. Produced by recombinant DNA technology and using Escherichia coli as the expression system, compare...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31G01N33/535
CPCG01N21/314G01N33/535G01N2021/3148
Inventor 姜桂香刘珊珊刘晓艳陈倩洁陈虹
Owner SHANDONG SIMCERE BIO PHARMA CO LTD
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