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Method for detecting biological activity of recombinant human keratinocyte growth factors

A keratinocyte and growth factor technology, applied in biological testing, material inspection products, etc., can solve the problems affecting repeatability and reproducibility, and achieve the effect of strong reproducibility, high accuracy and good repeatability

Active Publication Date: 2016-12-14
信立泰(苏州)药业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although the EdU labeling method has been widely used in the detection of cell proliferation, factors such as cell type, cell density, cell viability, cell proliferation rate, and cell culture medium (such as serum concentration) often affect the repeatability and reproducibility of the EdU labeling method.
At present, there is no literature report on the application of EdU labeling method to the detection of rhKGF-1 biological activity, but how to use EdU labeling method to truly and accurately reflect the proliferative activity of rhKGF-1 is a technical difficulty that needs to be solved urgently in the present invention

Method used

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  • Method for detecting biological activity of recombinant human keratinocyte growth factors
  • Method for detecting biological activity of recombinant human keratinocyte growth factors
  • Method for detecting biological activity of recombinant human keratinocyte growth factors

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1. A detection method for recombinant human keratinocyte growth factor—— rhKGF-1 concentration screening

[0053] 1) Culture of epithelial cells (4MBr-5, derived from ATCC, cell number CCL-208) of rhesus monkey lung bronchus

[0054] Ham’s F-12 complete medium containing a final concentration of 50 ng / ml recombinant human epidermal growth factor (rhEGF, derived from Sigma Company) and 10% fetal bovine serum (FBS, derived from Gibco Company) was used at 37°C, 5% CO 2 , cultured 4MBr-5 cells under the condition of saturated humidity. Passage 2-3 times per week.

[0055] 2), 4MBr-5 cell inoculation

[0056] 2.1) Take the 4MBr-5 cells in the logarithmic growth phase in step 1, discard the medium, wash with trypsin, add 0.5ml trypsin to digest the cells, place the culture bottle under a microscope for observation, and wait until the cells are all rounded, Add Ham's F-12 medium containing 10% FBS to stop the digestion, and collect the cells; then wash the culture f...

Embodiment 2

[0068] Example 2. A detection method for recombinant human keratinocyte growth factor—— rhKGF-1 concentration screening

[0069] 1) Culture of epithelial cells (4MBr-5, derived from ATCC, cell number CCL-208) of rhesus monkey lung bronchus

[0070] Using Ham’s F-12 complete medium containing a final concentration of 50 ng / ml recombinant human epidermal growth factor (rhEGF, Sigma Company) and 10% fetal bovine serum (FBS, Gibco Company), 37 ° C, 5% CO 2 4MBr-5 cells were cultured under the condition of saturated humidity. Passage 2-3 times per week.

[0071] 2), 4MBr-5 cell inoculation

[0072] 2.1) Take the 4MBr-5 cells in the logarithmic growth phase in step 1, discard the medium, wash with trypsin, add trypsin to digest the cells, put the culture flask under a microscope and observe, after the cells are all rounded, add Ham's F-12 medium containing 10% FBS was used to stop the digestion, and the cells were collected; the culture flask was washed with Ham's F-12 medium con...

Embodiment 3

[0085] Example 3. A detection method for recombinant human keratinocyte growth factor - cell density screening

[0086] 1), adopt the method identical with embodiment 2, cultivate 4MBr-5 cell;

[0087] 2), using the same method as in Example 2, the 4MBr-5 cell suspension obtained in step 1 was mixed with 5 × 10 4 / ml and 1×10 5 Cells / ml were inoculated in 96-well cell culture plates, 100 μl per well, 37°C, 5% CO 2 Cultivate for 22-24 hours under saturated humidity conditions.

[0088] 3), using the same method as in Example 2, prepare the test product 1 whose final concentration of rhKGF-1 is 100ng / ml, and carry out 3-fold serial concentration dilution, totally 10 dilutions, and prepare the test product 1 and its Add the EdU solution with a final concentration of 10 μM to each dilution concentration and mix well to prepare the loading sample;

[0089] 4), using the same method as in Example 2, wash the cells in step 2, and remove the culture medium;

[0090] 5) Using the sa...

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Abstract

The invention provides a method for detecting recombinant human keratinocyte growth factors (rhKGF-1). The method includes steps of 1), cultivating keratinocytes; 2) inoculating the keratinocytes and controlling the density of the keratinocytes; 3), treating rhKGF-1 samples, carrying gradient dilution on the concentration of the rhKGF-1 samples and adding 5-acetenyl-2'-deoxyuridine (EdU) into the rhKGF-1 samples with various rhKGF-1 concentration dilution degrees to prepare loading samples with the series of rhKGF-1 concentration dilution degrees; 4), washing the keratinocytes in keratinocyte plates obtained at the step 2) and removing cultivation media; 5), loading the loading samples obtained at the step 3) into keratinocyte cultivation plates obtained at the step 4) and cultivating and incubating the keratinocytes; 6), solidifying and permeabilizing the keratinocytes, and then carrying out 'Click-iT' reaction on the EdU; 7), arranging the keratinocytes treated at the step 6) in a multifunctional microplate reader, reading the fluorescence intensity (RFU) and computing the biological activity of the rhKGF-1. The method has the advantages of low detection background, high sensitivity, specificity and accuracy, good repeatability and the like. Besides, the method is particularly applicable to detecting the biological activity of the rhKGF-1 and establishing quality standards.

Description

technical field [0001] The invention belongs to the field of biological product quality detection and analysis, and in particular relates to a biological activity detection method of recombinant human keratinocyte growth factor. Background technique [0002] Human keratinocyte growth factor-1 (human keratinocyte growth factor-1, hKGF-1), that is, fibroblast growth factor-7 (fibroblast growth factor-7, FGF-7), hKGF-1 is produced by mesenchymal cells, through paracrine Specifically acting on epithelial cells, it is a cell growth factor that can stimulate embryonic development, promote epithelial cell proliferation and growth, participate in immune reconstruction, and tumor formation and development and other important biological functions. [0003] Intravenous injection Palifermin (trade name kepivance, containing recombinant hKGF-1 (rhKGF-1)) developed by American Amgen Company was approved by the US FDA in December 2004 for the reduction of malignant blood diseases undergoin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 高婧罗双儒代亚东覃安友黎耘张宝华李晓鹏李月华
Owner 信立泰(苏州)药业有限公司
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