Method for testing vascellum esoderma inhibin bioactivity

A technology of vascular endothelium and detection method, which is applied in the field of biological product activity detection, can solve problems such as difficult operation, unclear method description, large experimental error, etc., achieve specificity and repeatability, less human influence factors, and overcome repetition poor sex effect

Inactive Publication Date: 2008-09-03
SHANDONG SIMCERE BIO PHARMA CO LTD
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Problems solved by technology

[0004] 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 the proliferation and migration of different cells such as HUVEC, HDVEC, HMEC, and ECV304 to detect its cell proliferation inhibitory activity, but the selected experiment Materials and test results vary greatly, and many test results have poor repeatability
[0005] In addition, we learned from the China Institute for the Control of Biological Products that some manufacturers in China 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 and has large errors. , poor repeatability and difficult operation
[0006] Taking the HMEC cell migration inhibition method as an example, the detection of recombinant human endostatin (rhEndostatin) cell proliferation inhibition activity has the following shortcomings: (1) In the experiment, no active standard was used to correct the error, and there were many influencing factors in the experiment , resulting in large experimental errors and poor repeatability; (2) Since the method of manually counting the stained migrating cells under a microscope was used in the experiment, the field of view selected for counting and the determination of cells were greatly influenced by human factors; (3 ) The calculation method of the experimental results is unscientific; (4) The method provided in the appendix of the procedure is not clearly described, and there are many shortcomings that are inconsistent with the actual operation
Due to the above defects, the HMEC cell migration inhibition method is not objective enough to evaluate the biological activity of endostatin, and has the disadvantages of large errors and poor repeatability
[0007] In view of the above shortcomings of the above method, the cell proliferation inhibitory activity of endostatin is difficult to be uniformly evaluated objectively, and cannot meet the needs of large-scale production and quality control of multi-batch products. Therefore, it is objectively necessary to provide a method with strong specificity and repeatability. A good detection method for endostatin activity

Method used

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  • Method for testing vascellum esoderma inhibin bioactivity
  • Method for testing vascellum esoderma inhibin bioactivity
  • Method for testing vascellum esoderma inhibin bioactivity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Use 30mM acetic acid-sodium acetate buffer system with pH5.5±0.5 to perform ultrafiltration dialysis on the purified recombinant human endostatin protein solution, and prepare 90.9ml recombinant human endostatin solution with a concentration of 9.9mg / ml , add 60ml of 20% mannitol, add 4.20ml of acetic acid-sodium acetate buffer solution of about 1.5M pH5.5, and add water for injection to 300ml. Sterilize and filter through a 0.22μm microporous membrane, pack into vials, the liquid level is 1-1.5cm higher than the bottom of the bottle, add butyl rubber stopper, place the liquid in a freeze-drying box, and the temperature of the product drops to - 40°C, keep it for 3-4 hours, vacuumize, heat the partition, raise the temperature of the product to -20°C, keep it for 8 hours, continue heating to raise the temperature to 25°C, keep it for 6 hours, until the vacuum degree does not change much , take it out after vacuum pressing, and roll the cap. Preparation specifications: re...

Embodiment 2

[0037] Example 2 sample detection

[0038] 2.1 Detection steps

[0039] 2.1.1 Cell culture: HUVEC cells were purchased from Sciencell, and the corresponding medium was also purchased from the company. According to the instructions, add FBS (fetal bovine serum in Chinese), ECGS (a mixture containing various factors that promote the growth of endothelial cells extracted from the bovine pituitary gland), P / OSolution (a mixture of penicillin and streptomycin) to the ECM medium , at 37°C, 5% CO 2 HUVEC cells were cultured in an incubator and subcultured to the fifth generation. After the cells were in good condition and entered the logarithmic growth phase, they were ready to be inoculated.

[0040] 2.1.2 Inoculation: Digest the cells with 0.25% trypsin, centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend with medium, and count live cells with a hemocytometer under a microscope. Adjust the cell density to 5000 cells / ml, and add 160 μl of cell suspension to ea...

Embodiment 3

[0066] Repeatability evaluation of embodiment 3 detection method

[0067] The repeatability evaluation of the detection method was evaluated by intra-assay repeatability and inter-assay repeatability. Intra-batch repeatability: refers to the results of a sample being tested 5 times in parallel on the same 96-well plate on the same day; inter-batch repeatability: refers to the results of a sample being tested 5 times on different days.

[0068] The results showed that the half-effective concentration of recombinant human endostatin samples (EC 50 ) within the batch relative standard deviation (RSD) was 37.8% (see Table 3), recombinant human vascular endostatin half-effective concentration (EC 50 ) between batches relative standard deviation (RSD) was 45.0% (see Table 4). The intra-batch and inter-batch recombinant human endostatin samples had good repeatability, and the error was controlled below 45.0%.

[0069] Table 3 Intra-assay repeatability detection of biological activ...

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Abstract

The invention discloses a method for testing vascular endothelial inhibitor biological activity, the method utilizes vascular endothelial inhibitor standard and sample with the same concentration gradient after dilution in multiple proportions to inhibit HUVEC cell proliferation, and detects the proliferate inhibition activity of vascular endothelial inhibitor to HUVEC cells with trace enzyme reaction colorimetry, and then analyzes biological activity of angiogenesis inhibition of vascular endothelial inhibitor. The method uses the vascular endothelial inhibitor standard to correct testing errors of vascular endothelial inhibitor samples in different batches, which causes the cell proliferation inhibition activity testing results of vascular endothelial inhibitor have specificity and repeatability, and overcomes the defects that existed in prior vascular endothelial inhibitor activity testing method that the repeatability is bad, the method can not be unified, and the method can not meet the needs of large scale production and multiple-batch quality control.

Description

technical field [0001] The invention relates to the technical field of biological product activity detection, in particular to a detection method for the biological activity of vascular endostatin. Background technique [0002] There are many kinds of angiogenesis inhibitors, among which the angiogenesis inhibitors targeting the proliferation of vascular endothelial cells are an important class, and they are represented by angiostatin and endostatin. Endostatin is an enzyme cleavage product with a molecular weight of 20kDa at the carboxy-terminal of collagen XVIII. It has the activity of inhibiting the proliferation, migration and angiogenesis of vascular endothelial cells. It is the strongest endogenous angiogenesis inhibitor currently known. [0003] At present, vascular endostatin (endostatin) mainly uses DNA recombination engineering technology to prepare recombinant vascular endostatin, and uses prokaryotic or eukaryotic expression system to clone, express and purify th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/25C12Q1/18
Inventor 王鸿梅陈倩洁姜静刘武
Owner SHANDONG SIMCERE BIO PHARMA CO LTD
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