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Method for detecting in-vitro activity of recombined human interleukin-12 by using cell with interleukin-12 receptor and application

A technology for interleukin and receptors, which is applied in the field of detection of recombinant human interleukin-12 in vitro activity, can solve the problems of poor accuracy, accuracy and repeatability, and achieve improved accuracy and repeatability, high sensitivity, and passage Unlimited number of effects

Inactive Publication Date: 2014-06-04
QINGDAO KANGLITAI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The first technical problem to be solved by the present invention is to provide a detection accuracy method for the problem of poor accuracy, precision and repeatability of the in vitro activity detection results of rhIL-12 due to individual differences in the prior art. Method for detecting recombinant human interleukin-12 activity in vitro using stable cells with interleukin-12 receptors with high accuracy and good repeatability

Method used

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  • Method for detecting in-vitro activity of recombined human interleukin-12 by using cell with interleukin-12 receptor and application
  • Method for detecting in-vitro activity of recombined human interleukin-12 by using cell with interleukin-12 receptor and application
  • Method for detecting in-vitro activity of recombined human interleukin-12 by using cell with interleukin-12 receptor and application

Examples

Experimental program
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Effect test

Embodiment 1

[0048] (1) Take out a cryopreserved cell with interleukin-12 receptor from the liquid nitrogen tank, thaw the cell quickly and recover it, culture it statically in the culture medium and pass it to the logarithmic growth phase, and put the activated leukocyte IL-12 receptor cells were transferred to 24-well plates.

[0049] (2) Use different concentrations of recombinant human interleukin-12 standard products to co-culture with the above-mentioned activated cells with interleukin-12 receptors in the medium, and recombinant human interleukin-12 standard products in the co-culture system The concentration in the medium is: 0.032~51.2ng / ml, to induce cells with interleukin-12 receptors to produce gamma interferon, take the amount of gamma interferon produced as the ordinate, and take recombinant human interleukin-12 standard The different concentrations of different concentrations are the abscissa, and the S-type standard curve is obtained. From the S-type standard curve, the con...

Embodiment 2

[0053] (1) Take out a cryopreserved NK92 cell from the liquid nitrogen tank, thaw the cells quickly, replace the cryopreservation solution, and inoculate in the culture medium, at 37°C, 5% CO 2 , static culture to the logarithmic growth phase, subculture, according to the density of NK92 cells in the medium is 2 × 10 5 After the resuspension, the NK92 cells in the logarithmic growth phase were resuspended in the medium, and the activated NK92 cells were transferred to a 24-well plate, and then 0.5ml of the IL-12 sample was mixed with the medium. solution or culture medium (control), the final density of NK92 cells was 1×10 5 cells / ml / well. The frozen NK92 cells were purchased from the American Type Culture Collection (ATCC), numbered CRL-2407.

[0054] The recombinant human interleukin-12 standard used in this example was purchased from NIBSC (The National Institute for Biological Standards and Control), and its activity was 10000 IU / μg.

[0055] Dilute the recombinant huma...

Embodiment 3

[0065] (1) Take out a cryopreserved NK92 cell from the liquid nitrogen tank, thaw the cells quickly, replace the cryopreservation solution, and inoculate in the culture medium, at 37°C, 5% CO 2 , static culture to the logarithmic growth phase, subculture, according to the density of NK92 cells in the medium is 3 × 10 5 After resuspension, statically activate the culture, resuspend the NK92 cells in the logarithmic growth phase with medium, transfer the activated NK92 cells to a 24-well plate, and then add 0.5ml of IL-12 sample and medium Mixed solution or culture medium (control), the final density of NK92 cells is 1.5×10 5 cells / ml / well. The frozen NK92 cells were purchased from the American Type Culture Collection (ATCC), numbered CRL-2407. The medium is MEM medium 5 with 0.1mM β-mercaptoethanol, 0.02mM folic acid, 100U / ml rhIL-2, 12.5wt% horse serum and 12.5wt% fetal bovine serum.

[0066] The recombinant human interleukin-12 standard used in this example was purchased f...

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Abstract

The invention discloses a method for detecting the in-vitro activity of recombined human interleukin-12 by using a cell with an interleukin-12 receptor and application. The method comprises the following steps of (1) taking out the frozen cell with the interleukin-12 receptor from a liquid nitrogen tank, and performing recovery activation cultivation till the logarithm growth period is reached; (2) inducing the cell with the interleukin-12 receptor through a recombined human interleukin-12 standard substance under different concentrations to generate gamma interferon to obtain the concentration value ED50s of the recombined human interleukin-12 standard substances; (3) inducing the cell with the interleukin-12 receptor through a recombined human interleukin-12 protein to be detected under different concentrations to generate the gamma interferon to obtain a concentration value ED50r of the recombined human interleukin-12 protein to be detected; and (4) obtaining the in-vitro activity Pr of the recombined human interleukin-12 protein to be detected according to a formula Pr=Ps*ED50s / ED50r.

Description

technical field [0001] The invention relates to a technology for detecting the activity of recombinant human interleukin-12, in particular to a method for detecting the activity of recombinant human interleukin-12 in vitro by using cells with interleukin-12 receptors. Background technique [0002] Cells with interleukin-12 receptors can be induced by interleukin-12 to induce intracellular signal transduction. NK-92 is an interleukin-2-dependent natural killer cell line. Experiments have proved that NK92 cells, under the induction of interleukin-12, regulate the expression of cell surface receptors, enhance the binding with tumor cell surface ligands, inhibit and kill tumor cells. Or NK92 cells directly inhibit tumor cell growth by secreting immune factors (eg: interferon) and other cytokines. Interleukin-12 (IL-12), a heterodimeric molecule, is a cytokine produced by monocytes / macrophages and B lymphocytes. [0003] Recombinant human interleukin-12 (rhIL-12) is the coded ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6869G01N2333/5434G01N2333/57
Inventor 赵毅李友海李健章方良蒋忻坡黄冰卢世香裴秀芝王媛媛丁会芹
Owner QINGDAO KANGLITAI PHARMA
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