体外新鲜人血法检测热原相关因子IL-1β的条件优化及初步方法学研究
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篇名: 体外新鲜人血法检测热原相关因子IL-1β的条件优化及初步方法学研究
TITLE:
摘要: 目的:建立热原相关因子白细胞介素1β(IL-1β)的检测方法并优化其条件,同时进行初步方法学研究。方法:采用体外新鲜人血法。将5、2、0.8、0.32 EU/ml的细菌内毒素标准溶液加入稀释血液中,同时用稀释后的RPMI 1640作为空白对照,培养后采用酶联免疫吸附(ELISA)试验测定血液中释放的IL-1β含量。考察不同的稀释剂(RPMI 1640培养基、灭菌生理盐水)、胎牛血清添加与否和血液最终稀释体积分数(40%、20%、10%、8.3%)以及血液保存时间(2、5、6、8、26 h)与细菌内毒素释放的IL-1β含量的相关性,计算相关系数及检出限;将不同稀释倍数的清开灵注射液和金纳多注射液供试品及其干扰溶液分别加入稀释血液中,检测其回收率。结果:使用RPMI 1640培养基、40%的终体积分数稀释血液的结果更灵敏(检出限为0.128 EU/ml,r=0.993),是否加入胎牛血清对结果无明显影响;血液在4 ℃保存26 h内,检出限均为0.128 EU/ml,r值均大于0.990;清开灵注射液和金纳多注射液稀释10、32倍浓度及更多倍数时对该方法无干扰作用,回收率在68%~118%之间。结论:本研究建立的体外新鲜人血检测方法可用于热原物质的检测,并为中药注射液的热原检测提供了试验依据。
ABSTRACT: OBJECTIVE: To establish the detection method for pyrogen-related factor interleukin 1β (IL-1β) through optimizing detection condition, and to conduct preliminary methodology study. METHODS: The in vitro fresh human whole blood detection method was used. The bacterial endotoxin standard solution (5, 2, 0.8, 0.32 EU/ml) were added into diluted blood; using diluted RPMI 1640 as blank control, the content of IL-1β in blood sample was determined by ELISA after incubation. The relationship of the addition of different attenuants (RPMI 1640 culture, sterilized normal saline) and fetal bovine serum, final dilution volume fraction (40%, 20%, 10% and 8.3%) and storage duration (2, 5, 6, 8, 26 h) with the contents of endotoxin IL-1β were investigated, and related coefficient and detection limits were calculated. Different dilution times of Qingkailing injection and Ginaton injection samples and interference solutions were added into diluted blood to detect their recovery. RESULTS: The results indicated that RPMI 1640 media and 40% diluted blood was more sensitive (detection limit was 0.128 EU/ml, r=0.993); while the addition of fetal bovine serum didn’t influence the results. The detection limits of blood sample storied at 4 ℃ for 26 h were 0.128 EU/ml (r>0.990). When Qingkailing injection and Ginaton injection were diluted 10, 32 and more times, the detection method was not interfered and the recovery ranged 68%-118%. CONCLUSIONS: Established in vitro fresh human whole blood detection method can be used for the detection of pyrogen, and provides trial evidence for the pyrogen detection of TCM injection.
期刊: 2016年第27卷第19期
作者: 王文佳,陈志明,庞智慧,方海顺,何华红,李薇
AUTHORS: WANG Wenjia,CHEN Zhiming,PANG Zhihui,FANG Haishun,HE Huahong,LI Wei
关键字: 新鲜人血;热原;白细胞介素1β;体外试验;清开灵注射液;金纳多注射液
KEYWORDS: Human fresh blood; Pyrogen; Interleukin 1β; in vitro test; Qingkailing injection; Ginaton injection
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