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鲎的变形细胞溶解物能与内毒素发生反应,但其中含有一种G 因子能与(1→3)β—D—葡聚糖发生反应,所以传统的鲎试验对内毒素都不是特异的。作者用从鲎变形细胞溶解物中除去G 因子的方法,制备出一种特异的内毒素鲎色原试剂—Endospecy,并创立了一种用微量滴定板测定全血内毒素的新方法。方法:所有玻璃器皿都必须加热250℃两小时,试剂121℃高压消毒20～90min,去除内毒素。加标本用的吸样器吸器和试剂分配器空针都必须无内毒素和无β-葡聚糖。一次性96孔微量滴定板亦应无内毒素和β-葡聚糖。取无热原塑料空针(含肝素5单位/ml 血)静脉抽血,注入玻璃管内,放混合器上猛烈振荡,使其溶血。检测前放-80℃保存。取全血50μl,加0.25%Tritonx-100(0.66mol/LHNo_3)溶液250μl,充分振荡后,置37℃孵育5min。然后3500rpm 离心5min。取25μl 上清液加入微量滴定板孔内,加入0.5mol/L NaoH25μl 中和。每孔加入Endospecy(溶于2.2ml0.2mol/LPH8.0Tris-HCl
Dendritic cells can react with endotoxins but contain a factor G that reacts with (1 → 3) β-D-glucan, so traditional limulus test is not specific for endotoxin. The authors used a method to remove G-factor from limulus amebocyte lysates to produce Endospecy, a specific endotoxin reagent, and created a new method for the determination of whole blood endotoxin using microtiter plates. Methods: All glassware must be heated at 250 ℃ for two hours, reagents 121 ℃ autoclave 20 ~ 90min, removal of endotoxin. Sample extractors for sample addition and reagent dispenser empty needles must be endotoxin-free and free of beta-glucans. Disposable 96-well microtiter plates should also be free of endotoxin and β-glucan. Take pyrogen free plastic needle (containing heparin 5 units / ml blood) venous blood, into the glass tube, put the mixer violent oscillation, so hemolysis. Pre-test release -80 ℃ preservation. Take whole blood 50μl, add 0.25% Tritonx-100 (0.66mol / LHNo_3) solution 250μl, shake well and incubate at 37 ℃ for 5min. Then centrifuged at 3500 rpm for 5 min. Take 25μl supernatant was added to the microtiter plate wells, adding 0.5mol / L NaoH25μl neutralization. Endospecy was added to each well (dissolved in 2.2 ml 0.2 mol / LPH8.0 Tris-HCl