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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 the bioassay of recombinant human interleukin-12 (rhIL-12) and observe the effect of rhIL-12 on the level and dose and time of IFN-γ production in peripheral blood mononuclear cells (PBMCs) Between the relationship, reveal the law of change. Methods rhIL-12 was used to stimulate the secretion of IFN-γ in PBMC from healthy people. The rhIL-12 bioassay method was established by international standards. PBMCs were induced by rhIL-12 at a concentration of 1 ng / mL. IFN-γ levels in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) at 24, 48, 72, 96 and 120 h, respectively. The levels of IFN-γ in culture supernatants of human PBMCs induced by rhIL-12 at concentrations of 0.1, 0.5 and 2.5 ng / mL for 3 h were measured and the data were statistically analyzed by repeated measures ANOVA with Prism 5.0 statistical software. Results The biological activity curve of rhIL-12 was typical S type. The concentration of ED50 was (1.008 ± 0.238) ng / mL when the concentration of IFN-γ was half of the maximum concentration. The effect of rhIL-12 The price is 7 938 IU / mL. The level of IFN-γ in supernatant of 5-day stimulation of 1ng / mL rhIL-12 stimulated PBMCs of healthy individuals showed that the peak time of different individuals appeared to be different, and showed an increasing trend before the peak appeared. The high, middle and low concentrations of rhIL-12 induced a significant dose-response relationship between the level of IFN-γ and the concentration of PBMC induced by rhIL-12, and the high level of IFN- γ. Conclusion The biological activity of rhIL-12 is good, which is consistent with that of international standards. There are individual differences in the response peak of rhIL-12 in different populations, and the induction of IFN-γ as an indicator to evaluate the biological activity of rhIL-12 is obvious The dose-effect relationship.