胶束毛细管电泳法检测IL-12的含量  

Application of micellar electrokinetic capillary chromatography for identification,quantitative detection and unfolding analysis of interleukin-12

作　　者：包鹏举 孙瑶[3,4] 王海华[2] 金孝岠[1] 张根葆 BAO Pengju;SUN Yao;WANG Haihua;JIN Xiaoju;ZHANG Genbao(School of Anesthesiology,Wannan Medical College,Wuhu 241002,China;Department of Physiology,Wannan Medical College,Wuhu 241002,China;Department of Pathophysiology,Wannan Medical College,Wuhu 241002,China;Institute of Snake Venom,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院麻醉学院,安徽芜湖241002 [2]皖南医学院生理学教研室,安徽芜湖241002 [3]皖南医学院病理生理学教研室,安徽芜湖241002 [4]皖南医学院蛇毒研究所,安徽芜湖241002

出　　处：《南方医科大学学报》2020年第9期1301-1306,共6页Journal of Southern Medical University

基　　金：安徽省高校科学研究基金重点项目(KJ2016A729);皖南医学院重点科研项目培育基金(WK2016Z02);皖南医学院重点项目科研基金(WK2019Z09)。

摘　　要：目的建立胶束毛细管电泳法鉴定白介素-12(IL-12)、检测其含量并分析IL-12去折叠的过程。方法以50μm i.d.×48.5 cm未涂层毛细管为色谱分离柱,分析影响IL-12分离效果的因素并建立IL-12标准曲线;将IL-12与抗IL-12抗体混匀液在38℃水浴槽中孵育,40 min后在相同条件下进行毛细管电泳,将其与IL-12、抗IL-12抗体的电泳结果比较以鉴定IL-12;将IL-12与DTT在60℃水浴槽中孵育不同时间,并研究IL-12在不同状态下的电泳结果分析IL-12去折叠的过程。结果建立了以含30 mmol/L十二烷基硫酸钠(SDS)的硼酸盐(pH=9.3,80 mmol/L)为缓冲液的胶束毛细管电泳在线扫集法;IL-12的峰面积与质量浓度具有良好的线性关系,线性相关系数为0.9991,线性范围为2~120 ng/L;随着IL-12与DTT孵育时间的延长,IL-12的二硫键逐渐被打开,图谱中可显示蛋白峰随之发生的变化。结论毛细管电泳法操作简单、灵敏度高,这为今后在肿瘤等疾病中快速检测IL-12的含量以及分析其含量变化的原因提供理论依据。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 50μm)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.Conclusion 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.

关 键 词：胶束毛细管电泳 白介素-12 二硫键 

分 类 号：R446.1[医药卫生—诊断学]