François C, Neveu J, Sauvaire D, Bonnet P A, Tissier M H
French Health Products Safety Agency (Afssaps French OMCL) DLC, CBR department, 635 rue de la Garenne, 34740 Vendargues, France.
Pharmeur Sci Notes. 2006 Aug;2006(1):17-21.
The two conventional tests to detect pyrogen contaminants in injectable pharmaceutical drugs are the Rabbit Model and the Limulus amoebocyte lysate (LAL) test. To replace these models, a new system on human whole blood is developed, using the release of Interleukin 1 beta (IL1beta) after cell stimulation with gram-positive and gram negative pyrogens. The purpose of this study was to validate the ENDOSAFE-IPT kit using the quantitative ELISA enzyme immunoassay. The assay is divided into two parts: blood cell stimulation with Lipopolysaccharides (LPS) and Lipoteichoic acid (LTA) and quantitation of IL1beta using the ELISA method. In each assay, blood from a particular donor were stimulated with the Endotoxin Standard, and with a sample of a commercial antibiotic preparation (Clavulanic acid/Ticarcillin) spiked with the Endotoxin Standard. LTA from Bacillus subtilis and a sample of diphtheria toxoid were also used. At least, six assays were tested. A polynomial regression of the Endotoxin Standard series showed a correlation coefficient greater than 0.99. The spiked antibiotic sample recoveries were 50-121%. The LTA quantitation limit was 0.1 microg/ml and the range of detection of pyrogens from Gram positive diphtheria toxoid was 0.77 to 2.5 EEU/ml. The IL1beta production varied markedly between donors. However the coefficient of variation was less than 20 % intra-assay. In conclusion, the ENDOSAFE-IPT kit can be used for the quantitative and qualitative detection of pyrogens from Gram negative and Gram positive bacteria.
检测注射用药品中热原污染物的两种传统检测方法是兔模型和鲎试剂检测法。为了替代这些模型，开发了一种基于人全血的新系统，该系统利用革兰氏阳性和革兰氏阴性热原刺激细胞后白细胞介素1β（IL1β）的释放。本研究的目的是使用定量ELISA酶免疫测定法验证ENDOSAFE - IPT试剂盒。该测定分为两部分：用脂多糖（LPS）和脂磷壁酸（LTA）刺激血细胞，以及使用ELISA方法对IL1β进行定量。在每次测定中，用内毒素标准品以及添加了内毒素标准品的商业抗生素制剂（克拉维酸/替卡西林）样品刺激来自特定供体的血液。还使用了枯草芽孢杆菌的LTA和白喉类毒素样品。至少进行了六次测定。内毒素标准品系列的多项式回归显示相关系数大于0.99。加标抗生素样品的回收率为50 - 121%。LTA定量限为0.1微克/毫升，革兰氏阳性白喉类毒素热原的检测范围为0.77至2.5 EEU/毫升。不同供体之间IL1β的产生差异明显。然而，批内变异系数小于20%。总之，ENDOSAFE - IPT试剂盒可用于革兰氏阴性和革兰氏阳性细菌热原的定量和定性检测。
