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Analytical method for evaluating in-vitro biological activity of human follicle stimulating hormone

A human follicle-stimulating hormone, biologically active technology, applied in the field of protein medicine and bioengineering, can solve problems such as health hazards, complicated experimental operations, and high requirements for experimental personnel, and achieve reliable data results, improve stability, and avoid time-consuming. Effect

Inactive Publication Date: 2015-04-29
BEIJING TIDE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the defects of the rat ovary weight gain method mentioned above, some researchers tried to use the method of in vitro cell viability assay to detect the activity of follicle hormone. The cells need to be modified by gene transfection, and then the activity is measured by radioimmunoassay. The experimental operation of these methods is also complicated, and they need to be exposed to isotopes, which have high requirements for experimenters and are harmful to health. It has not been well and widely used, and cannot replace the rat ovary weight gain method

Method used

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  • Analytical method for evaluating in-vitro biological activity of human follicle stimulating hormone
  • Analytical method for evaluating in-vitro biological activity of human follicle stimulating hormone
  • Analytical method for evaluating in-vitro biological activity of human follicle stimulating hormone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Evaluation of analytical methods for in vitro biological activity of human follicle-stimulating hormone international standard

[0050] (1) Cell recovery and passage:

[0051] Take the cell line out of the liquid nitrogen tank, quickly put it in a 37°C water bath, and shake it gently to melt it quickly; transfer it into a centrifuge tube in an ultra-clean bench, add 10% FBS+DF12 medium to re- Suspend cells; centrifuge at 800~1000rpm for 5 minutes, discard the supernatant, add cell culture medium, mix by pipetting, and transfer to a cell culture bottle; the cell name, operation date, and cell generation are marked on the wall of the bottle.

[0052] (2) Cell plating

[0053] Digest the human ovarian granulosa cell tumor cells in the logarithmic growth phase with trypsin, discard the trypsin, add 10% FBS+DF12 medium to stop the digestion, and resuspend the cells; count with a cell counter and adjust The cell concentration was 2×105 cells / ml; 100 μl / well cells ...

Embodiment 2

[0063] Example 2 In vitro biological activity evaluation of recombinant human follicle-stimulating hormone

[0064] (1) Cell recovery and passage

[0065] Take the cell line out of the liquid nitrogen tank, quickly put it in a 37°C water bath, shake gently to melt it quickly; transfer it to a centrifuge tube in an ultra-clean bench, add 10% FBS+DF12 medium to resuspend Cells; centrifuge at 800rpm for 5 minutes, discard the supernatant, add cell culture medium, mix by pipetting, and transfer to a cell culture bottle; the cell name, operation date, and cell generation are marked on the wall of the bottle.

[0066] (2) Cell plating

[0067] Digest the human ovarian granulosa cell tumor cells in the logarithmic growth phase with trypsin, discard the trypsin, add 10% FBS+DF12 medium to stop the digestion, and resuspend the cells; count with a cell counter and adjust Cell concentration to 2 x 10 5 cells / ml; 100 μl / well of cells was added to a 96-well plate at 37°C, 5% CO 2 Culti...

Embodiment 3

[0078] Example 3 Evaluation of the in vitro biological activity of commercially available Narfen

[0079] (1) Cell recovery and passage:

[0080] Take the cell line out of the liquid nitrogen tank, quickly put it in a 37°C water bath, and shake it gently to melt it quickly; transfer it into a centrifuge tube in an ultra-clean bench, add 10% FBS+DF12 medium to re- Suspend cells; centrifuge at 800~1000rpm for 5 minutes, discard the supernatant, add cell culture medium, mix by pipetting, and transfer to a cell culture bottle; the cell name, operation date, and cell generation are marked on the wall of the bottle.

[0081] (2) Cell plating

[0082] Digest the human ovarian granulosa cell tumor cells in the logarithmic growth phase with trypsin, discard the trypsin, add 10% FBS+DF12 medium to stop the digestion, and resuspend the cells; count with a cell counter and adjust Cell concentration to 2×105 cells / ml; add 100 μl / well cells into 96-well plate, 37°C, 5% CO 2 Cultivate ove...

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Abstract

The invention discloses an analytical method of utilizing adherent cell-human ovarian granular cell tumor cell strain (KGN) to evaluate in-vitro biological activity of human follicle stimulating hormone. The method comprises the following steps: using the human follicle stimulating hormone to stimulate the human ovarian granular cell tumor cell, measuring the content of generated progesterone, so as to reflect the activity of the human follicle stimulating hormone. The analytical method of using the human ovarian granular cell tumor adherent cell to evaluate the in-vitro biological activity of the human follicle stimulating hormone, disclosed by the invention, is effective, stable and quick, and avoids the limitations of long time consumption, complicated operation and poor result repeatability in the animal in-vivo ovarian weight increment method; meanwhile, the progesterone content is determined by a commercial enzyme linked immunosorbent assay (ELISA) kit, so that the biological activity of the human follicle stimulating hormone can be reflected truly and more accurately.

Description

technical field [0001] The invention belongs to the field of protein medicine bioengineering and technology, and specifically relates to a novel analytical method for reflecting the biological activity of human follicle-stimulating hormone by stimulating the content of progesterone produced by human ovarian granulosa cell tumor cells (KGN) by human follicle-stimulating hormone. Background technique [0002] According to Appendix XII of the Pharmacopoeia of the People's Republic of China (2010 Edition), the human follicle-stimulating hormone bioassay method is to compare the effect of urinary gonadotropin standard substance and the test product on the ovarian weight gain of young rats, so as to determine the test product. Potency of human follicle-stimulating hormone. The experiment selected young female rats aged 19-23 days and weighing 36-50 g, and randomly divided them into 6 groups according to body weight, with 8 rats in each group, and subcutaneously injected high, medi...

Claims

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Application Information

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IPC IPC(8): G01N33/74
CPCG01N33/74
Inventor 贾慧齐连权梁艳常翠云王鑫周丽莹肖萱
Owner BEIJING TIDE PHARMA
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