Application of micellar electrokinetic capillary chromatography for identification, quantitative detection and unfolding analysis of interleukin-12.
10.12122/j.issn.1673-4254.2020.09.12
- Author:
Pengju BAO
1
;
Yao SUN
2
;
Haihua WANG
3
;
Xiaoju JIN
1
;
Genbao ZHANG
2
Author Information
1. School of Anesthesiology, Wannan Medical College, Wuhu 241002, China.
2. Department of Pathophysiology, Wannan Medical College, Wuhu 241002, China.
3. Department of Physiology, Wannan Medical College, Wuhu 241002, China.
- Publication Type:Journal Article
- Keywords:
disulfide bond;
interleukin-12;
micellar electrokinetic capillary chromatography
- From:
Journal of Southern Medical University
2020;40(9):1301-1306
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process.
METHODS:An uncoated fused-silica capillary (inner diameter 50 μm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states.
RESULTS:A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak.
CONCLUSIONS:This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.
