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Cytokine TNF-alpha bioactivity evaluation in-vitro test method

A cytokine and active body technology, applied in the field of biomedicine, can solve the problem of difficulty in meeting the needs of TNF-α clone screening, production release and stability research, difficulty in ensuring the stability and reliability of test results, and lack of detection system methods. To solve problems such as scientific verification research, etc., to achieve the effect of convenient high-throughput operation, easy promotion and high accuracy

Inactive Publication Date: 2015-06-24
MAB VENTURE BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, this cytological activity method has not been recognized or standardized, and the key experimental parameters include cell plating density, TNF-α and cell interaction time, color development time and color development methods, etc. The reports in different literatures are also inconsistent.
More importantly, in the reported TNF-α cytotoxic activity methods, there is a lack of methodological verification research on the detection system, including related evaluations on accuracy, precision, durability, etc., and it is difficult to guarantee the stability of the detection results. and reliability
The TNF-α cytotoxic detection method reported in the known literature is difficult to meet the needs of TNF-α clone screening, production release and stability research

Method used

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  • Cytokine TNF-alpha bioactivity evaluation in-vitro test method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 In vitro detection method for evaluating the biological activity of cytokine TNF-α

[0044] Using the reference product and the TNF-α test product with a titer of 75% and 125% as materials, the biological activity of the TNF-α test product at a titer of 75% and 125% was investigated using the steps of the above detection method.

[0045] The result is figure 1 As shown, the 4 parameters of the experimental curve of the TNF-α reference product and the test product are well fitted, and the curve fitting constant R 2 All met> 0.98, and the CV% of the OD value detected by 2 multiple holes at different concentrations of the added drug met 50 Comparison of the values, the cytotoxic biological activity of the test product at the 75% titer level was 76%, and the cytotoxic biological activity of the test product at the 125% titer level was 126%, both close to the theoretical value.

Embodiment 2

[0046] Example 2 Accuracy Evaluation Test

[0047] Using the reference product (same as Example 1) and the test product with different potency levels (50%, 75%, 100%, 125%, 150%) as materials, 2 experimenters (Analyst 1, 2) independently adopted The above-mentioned detection method detects the cytotoxic biological activity of the test products at these 5 potency levels.

[0048] The results are shown in Table 1. For 5 test products with potency levels of 50%, 75%, 100%, 125%, and 150% respectively, the test results of these test products by 2 experimenters using the method of the present invention are all satisfactory 4 The fitting constant R of the parameter fitting curve 2 >0.98, the CV% of 2 multiple holes meets <15%, and the average deviation (recovery rate) of the actual measured value of the test product with different potency levels by different personnel meets the theoretical value in the range of 95%-105% Inside. It shows that when detecting the cytotoxic biological acti...

Embodiment 3

[0051] Example 3 Precision Evaluation Test

[0052] Using reference products (same as Example 1) and test products with different potency levels as materials, the precision of the method of the present invention was evaluated. Two experimenters (Analyst 1, 2) used the above-mentioned detection method steps to test the cytotoxic biological activity of 5 test products with potency levels of 50%, 75%, 100%, 125%, and 150%, each in parallel Experiment 3 times to investigate the repeatability of the method. Two experimenters (Analyst 1, 2) used the above detection method steps to test the cytotoxic biological activity of the test product at the 75% titer level within 3 different days. The middle of the investigation method Precision.

[0053] As shown in Table 2, using the above detection method, different experimenters performed three parallel tests on each of the five potency levels of the test product, and the results all met the fitting constant R of the 4-parameter fitting curve o...

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Abstract

The invention belongs to the technical field of biological medicines and particularly relates to a cytokine TNF-alpha bioactivity evaluation in-vitro test method based on cytotoxic effect of cytokine TNF-alpha. The method is simple to operate, the expensive material and instrument are omitted, method verification can be completed, the requirements on method accuracy, precision and durability can be met completely, the activity test range can be 50% to 150%, the repeatability and intermediate precision test result are smaller than 10%, the test 4 parameter curve fitting constants R2 are larger than 0.99, and the double hole CV% is smaller than 15%. Thus, the cytokine TNF-alpha cytotoxic bioactivity can be tested rapidly, accurately and efficiently. In addition, the test method is stable and easy to implement, the promotion and high-throughput operation can be met completely, and the requirements of cytokine TNF-alpha bioactivity test on the basis of cytokine TNF-alpha cytotoxic effect and cytokine TNF-alpha bioactivity stability research are met.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an in vitro detection method for evaluating the biological activity of the cytokine TNF-α. Background technique [0002] Tumor necrosis factor (TNF-α) is secreted by monocytes, macrophages and T lymphocytes, and its main physiological effects include affecting cell apoptosis, proliferation, differentiation and cell function. After TNF-α binds to its receptor, it can initiate a series of intracellular events, such as acting on macrophages, promoting the release of inflammatory factors and chemical factors, and aggravating the inflammatory response; acting on endothelial cells, promoting the secretion of adhesion molecules and increasing Cell permeability; acting on fibroblasts and epithelial cells, affecting tissue remodeling and ion transport permeability. Studies have found that TNF-α has obvious cytotoxic effects on target cells, and the cell death rate is linearly r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 王少雄张宵吕品刘丹莉
Owner MAB VENTURE BIOPHARM CO LTD
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