Method for detecting activity of recombinant human interleukin 12 (rhIL-12) protein

A technology of interleukin and cells, which is applied in the field of detection of recombinant human interleukin-12 protein activity, can solve the problems of experimental stability, repeatability, and sensitivity, and achieve broad application prospects, accurate and reliable results, and easy operation Effect

Inactive Publication Date: 2010-08-11
UNIV OF SCI & TECH OF CHINA
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  • Application Information

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Problems solved by technology

At present, NK cells and T cells used for activity determination are mainly derived from normal human peripheral blood or neonatal umbi...

Method used

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  • Method for detecting activity of recombinant human interleukin 12 (rhIL-12) protein
  • Method for detecting activity of recombinant human interleukin 12 (rhIL-12) protein
  • Method for detecting activity of recombinant human interleukin 12 (rhIL-12) protein

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preparation example Construction

[0032] Preparation of NKG cells:

[0033] 1. Peripheral blood sample: Take 10ml of peripheral blood from a patient with non-Hodgkin's lymphoma, anticoagulate with heparin, and obtain the consent of the patient.

[0034] 2. Separation of peripheral blood mononuclear cells: After diluting the anticoagulated peripheral blood specimen with 10ml sterile PBS solution, carry out density gradient centrifugation with lymphocyte separation medium, 2200 rpm, 30 minutes, 20 degrees; For mononuclear cells, wash 3 times with PBS solution, 1500 rpm, 10 minutes, 20 degrees; use 250 μl sterile PBS solution to resuspend the cells, count, and the cell concentration is 1.1×10 7 / 250μl, store at 4 degrees, and set aside.

[0035]3. Separation and purification of NK cells: Add 20 μl of non-specific mouse IgG to the above cell solution at 4 degrees for 30 minutes to block non-specific binding. Add 100 μl CD56 MicroBeads to the cell solution, shake and mix, 4 degrees, 30 minutes, and mix once every...

Embodiment 1

[0041] Embodiment 1, detect the activity of recombinant human interleukin-12 protein

[0042] 1. Cell activation and preparation

[0043] Inoculate NKG cells (human natural killer cells) CGMCC No.2901 in cell culture medium I at 37°C, 5% CO 2 Cultured under certain conditions until the logarithmic growth phase; during this period, the cells can be subcultured depending on the growth of the cells.

[0044] The cell culture medium I is composed of α-MEM medium, fetal bovine serum, horse serum and recombinant human interleukin-2, and the ratio of α-MEM medium, fetal bovine serum, horse serum and recombinant human interleukin-2 For: 8ml α-MEM medium: 1ml fetal bovine serum: 1ml horse serum: 1000IU recombinant human interleukin-2.

[0045] Get the NKG cells in the logarithmic growth phase in a 50ml sterile centrifuge tube, 800rpm, 10min, centrifuge, discard the supernatant, wash twice with PBS solution, suspend in 1640 complete medium (1640 culture medium containing 10% fetal bov...

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Abstract

The invention discloses a method for detecting the activity of the recombinant human interleukin 12 (rhIL-12) protein, comprising the following steps: (1) stimulating the natural killer cell (NKC) which have the collection number of 2901 in the China General Microbiological Culture Collection Center (CGMCC) to genera Gamma interferon by using the standard rhIL-12 protein of different concentration and detecting the amount of the Gamma interferon to obtain a standard curve; (2) doing the experiment with the rhIL-12 protein to be detected in the activity by using the same method adopted in the step (1) so as to obtain the concentration of the rhIL-12 protein to be detected in the activity, which corresponds to half of the maximum amount of the Gamma interferon; and (3) calculating the activity of the rhIL-12 protein. The method for detecting the activity of the rhIL-12 protein can be operated easily, rapidly and stably and has low cost and high stability, repeatability, sensitivity and specificity, thereby having broad application prospect in the field of the detection of the activity of the rhIL-12 protein and laying a good foundation for the clinical use and the medicinal detection of the rhIL-12 protein.

Description

technical field [0001] The invention relates to a method for detecting activity of recombinant human interleukin-12 protein. Background technique [0002] Human Interleukin-12 (Human Interleukin 12, hIL-12) is a heterodimeric cytokine composed of two subunits p35 and p40 linked by multiple pairs of disulfide bonds. hIL-12 is also known as NK cell stimulating factor (NKSF), hIL-12 has strong anti-tumor and anti-viral effects, this effect is mediated by the body's natural killer cells (Natural killer cells, NK cells), hIL-12 -12 also plays an important role in the development, differentiation, maturation and activation of cytotoxic T cells, helper T cells, and B cells. . This series of biological functions makes people realize that hIL-12 may play an important role in clinical application. At present, recombinant human interleukin-12 (Recombinant Human Interleukin 12, rhIL-12) has entered the stage of clinical trials for anti-tumor, anti-viral diseases and allergic asthma a...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577C12Q1/02
Inventor 田志刚程民魏海明郑晓东孙汭
Owner UNIV OF SCI & TECH OF CHINA
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