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Quantitative determination method for activity of nerve growth factor

A nerve growth factor, quantitative determination technology, applied in the direction of material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of poor repeatability, cumbersome operation, unsuitable quality control requirements, etc., and achieve the effect of good accuracy and precision

Active Publication Date: 2013-10-30
STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional chick embryo dorsal root ganglion method is established based on the characteristics of NGF inducing ganglion growth and stimulating axon production, which has the advantages of strong specificity, etc. Not every ganglion after separation can be stimulated to grow axons, and the degree of growing axons is not the same. As long as there is one terminal stimulation effect in multiple ganglia, the potency can be judged. The operation is cumbersome, the test period is long, As a result, the subjective judgment cannot be quantified and the repeatability is poor, which is not suitable for the quality control requirements of this product. At the same time, as a semi-quantitative method, it also has certain limitations.

Method used

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  • Quantitative determination method for activity of nerve growth factor
  • Quantitative determination method for activity of nerve growth factor
  • Quantitative determination method for activity of nerve growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. NGF activity detection method

[0042] 1. Cultivation of TF-1 cells for detection

[0043] Culture TF-1 cells with complete medium, place at 37°C, 5% CO 2 Cultivate in an incubator until the logarithmic growth phase, the cell shape is plump, and the number meets the needs. Wash the cells with RPMI 1640 medium, centrifuge at 750rpm for 5min, change the medium and then centrifuge, repeat washing 3 times to completely remove the serum in the culture medium and rhGMCSF. The cells were resuspended in RPMI 1640 medium, and the concentration of the cell suspension was adjusted to 2×10 5 cells / ml for later use.

[0044] 2. In vitro activity detection of NGF

[0045] 2.1 Dilution of standards and samples to be tested

[0046] 2.1.1 Preparation of standard products for each dilution:

[0047] Dissolve the mouse nerve growth factor standard (1000AU / ml) in 1ml RPMI 1640 medium, and pre-dilute to 200AU / ml, which is the standard D1, and then compare and dilute, a total of 8 ...

Embodiment 2

[0085] 1. NGF activity detection method

[0086] 1. Cultivation of TF-1 cells for detection

[0087] Culture TF-1 cells with complete medium, place at 37°C, 5% CO 2 Cultivate in an incubator until the logarithmic growth phase, the cell shape is plump, and the number meets the needs. Wash the cells with RPMI 1640 medium, centrifuge at 750rpm for 5min, change the medium and then centrifuge, repeat washing 3 times to completely remove the serum in the culture medium and rhGMCSF. Cells were incubated with RPMI 1640 medium at 37°C, 5% CO 2 After culturing in the incubator for 18-20 hours, adjust the concentration of the cell suspension to 2×10 5 cells / ml for later use.

[0088] 2. In vitro activity detection of NGF

[0089] Same as Example 1.

[0090] 3. Test results

[0091] 3.1 Use the built-in software of the M5 microplate reader for data analysis and result processing, and select the 4-parameter method (4-P) as the standard curve.

[0092] 3.2 For the test values ​​of s...

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Abstract

The invention discloses a quantitative determination method for activity of a nerve growth factor. The method comprises the following steps of: (1) washing TF-1 cells in a logarithmic phase by a basal culture medium that contains no serum and recombined human granulocyte-macrophage colony stimulating factors, and then resuspending the TF-1 cells to obtain TF-1cell suspension liquid; (2) respectively adding the TF-1cell suspension liquid into a gradient-diluted nerve growth factor standard sample and a sample to be tested, and incubating at 37 DEG C in presence of 5% of CO2; then respectively adding an indicating agent to detect the absorbance / fluorescence value and drawing a standard curve line; and (3) calculating the activity concentration of the nerve growth factor of the sample to be tested according to the standard curve line and the absorbance / fluorescence value of the sample. The quantitative determination method for activity of the nerve growth factor disclosed by the invention has good accuracy and precision.

Description

technical field [0001] The invention relates to a quantitative assay method for nerve growth factor activity. Background technique [0002] As for the determination of the activity of nerve growth factor (hereinafter referred to as NGF), the method currently adopted in the industry is the chicken embryo dorsal root ganglion method. The traditional chick embryo dorsal root ganglion method is established based on the characteristics of NGF inducing ganglion growth and stimulating axon production, which has the advantages of strong specificity, etc. Not every ganglion after separation can be stimulated to grow axons, and the degree of growing axons is not the same. As long as there is one terminal stimulation effect in multiple ganglia, the potency can be judged. The operation is cumbersome, the test period is long, As a result, the subjective judgment cannot be quantified and the repeatability is poor, which is not suitable for the quality control requirements of this product...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 谭淑萍蒋立新周志文
Owner STAIDSON (BEIJING) BIOPHARMACEUTICALS CO LTD
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