摘要
目的建立重组人白细胞介素12(rhIL-12)的生物活性检测方法,观察rhIL-12诱导健康人外周血单个核细胞(PBMC)产生γ干扰素(IFN-γ)的水平与剂量和时间之间的关系,揭示其变化规律。方法rhIL-12供试品刺激健康人PBMC分泌IFN-γ,经国际标准品校正,建立rhIL-12生物活性测定方法。以1 ng/mL浓度rhIL-12诱导健康人PBMC,分别于24、48、72、96和120 h 5个时间点用酶联免疫吸附试验(ELISA)测定培养上清的IFN-γ水平。测定0.1、0.5和2.5 ng/mL 3个浓度rhIL-12诱导人PBMC 72 h后培养上清的IFN-γ水平,用Prism 5.0统计软件的重复测量方差分析法对所得数据进行统计学处理。结果rhIL-12供试品的生物活性曲线呈典型S型,IFN-γ含量为最大浓度一半时的供试品浓度(ED50)为(1.008±0.238)ng/mL,供试品rhIL-12效价为7 938 IU/mL。测定1ng/mL浓度rhIL-12刺激健康人PBMC后5个时间点培养上清中IFN-γ显示,不同个体高峰出现时间有所不同,在高峰出现前均呈现递增趋势。高、中、低3个浓度rhIL-12分别诱导健康人PBMC产生的IFN-γ水平与浓度之间呈显著量效关系,且在0.1 ng/mL的极低浓度即可诱生高水平IFN-γ。结论rhIL-12供试品生物活性良好,与国际标准品比对基本一致,不同人群对rhIL-12的应答高峰存在个体差异,以诱生IFN-γ作为评价rhIL-12生物活性的指标具有明显的量效关系。
Objective To establish a method for detecting hioactivity of recombinant human interleukin-12 （rhiL- 12） in order to observe the relationship between induction dose/time and the level of interferon-γ （IFN-γ） in the trials of peripheral blood mononuclear cells （ PBMC ） from heahhy subjects which were induced by rhIL-12, and reveal the change rules. Methods The tested rhIL-12 was used for stimulating PBMC from healthy subjects to produce IFN-γ. The method for detecting rhiL-12 bioactivity was established after calibrating by international standard rhIL-12. The IFN- γ levels in culture supernatant of PBMC from healthy subjects which were stimulated by 1 ng/mL rhIL-12 at 5 time points of 24, 48, 72, 96 and 120 h were analyzed by enzyme-linked immunosorbent assay （ELISA）. The IFN-γ levels in culture supernatant of PBMC in response to 0. 1 , 0.5 and 2.5 ng/mL rhiL-12 72 h stimulation were determined, respectively. The statistical analysis of data were performed by one-way analysis of variance with Prism 5.0. Results An S-curve of tested rhiL-12 bioactivity could achieve. 50% effective dose （ED50） was （1. 008 ±0.238） ng/mL, and the potency of tested rhlL-12 was 7 938 IU/mL. The IFN-3, levels detected in culture supernatant of PBMC from healthy subjects in response to 1 ng/mL rhIL-12 stimulation at 5 time points indicated that IFN-γ peak values in different subjects showed up at varied times with increasing tendency before the summit forming. There was a significant dose- effect correlation between IFN-γ production of PBMC from healthy subjects and the high dose, middle dose and low dose of rhIL-12. Furthermore, a very low dose of 0. 1 ng/mL also could induce high IFN-γ level. Conclusions The bioactivity of tested rhIL-12 is good, which is almost identical with that of international standard rhIL-12. There are individual variations among different people in response to rhlL-12 stimulation and there is a significant dose-effect correlation, evaluating the bioactivity index of rhIL-12 with induced IFN-γ.
出处
《检验医学》
CAS
北大核心
2009年第8期586-590,共5页
Laboratory Medicine
关键词
重组人白细胞介素12
Γ干扰素
细胞免疫
Interleukin-12, human,recombinant
Interferon-γ
Immunity, cellular
