肝素对鲎试剂检测内毒素的干扰机制: 钙/镁离子螯合和内毒素结合作用

The interference principle of heparin on TAL testing endotoxin:chelation of calcium and magnesium ions and binding of endotoxin

目的 肝素作为抗凝药物,常用于临床存储血液样品中;而对肝素化血液作内毒素检测时,发现肝素对鲎试剂有干扰。方法 测定鲎试剂的蛋白组成和离子浓度、内毒素粒径及其与肝素的分子相互作用。结果 肝素浓度≥0. 500 mg/ml时,明显抑制鲎试剂对内毒素的检测,且鲎试剂酶促反应生成的凝固蛋白显著减少;加入肝素的鲎试剂中Ca^(2+)、Mg^(2+)浓度显著低于无肝素的鲎试剂,表明肝素与鲎试剂中的Ca^(2+)、Mg^(2+)发生螯合作用,从而干扰鲎试剂对内毒素的检测。另外,内毒素中加入0. 500 mg/ml肝素时,其粒径从(17. 11±0. 63) nm显著增加至(61. 99±0. 58) nm;肝素滴定内毒素时,ΔH>0且ΔH <|TΔS|,表明肝素与内毒素发生疏水结合作用。结论 肝素对鲎试剂检测内毒素的干扰机制包括Ca^(2+)、Mg^(2+)螯合和内毒素结合作用。

Objective Heparin,an anticoa^uiani,is often used to store biooci samples clinically.However,we found that heparin interfered with Tachypleus Amebocyte Lysate(TAL)in testing endotoxin of heparinized blood.Methods This paper determined the protein composition and ionic concentration of TAL,the particle size of endotoxin and its molecular interaction with heparin.Results It was found that when heparin concentration was ≥0.500 mg/ml,the detection of TAL on endotoxin was obviously inhibited,and the production of coagulin protein in the enzymatic reaction of TAL was significantly reduced;the concentration of calcium and magnesium ions in the heparin-added TAL was prominently lower than in the heparin-free TAL,indicating that the heparin chelates with the calcium and magnesium ions in the TAL,so as to interfere with the detection of TAL on endotoxin.On the other hand,when 0.500 mg/ml heparin was added into endotoxin,the particle size increased remarkably from 17.11 nm±0.63 nm to 61.99 nm±0.58 nm;when heparin was titrated into endotoxin,ΔH>0 and ΔH<|TΔ S|,showing that the heparin has hydrophobic binding with the endotoxin.Conclusion These results demonstrate that the interference principle of heparin on TAL includes chelation of calcium and magnesium ions and binding of endotoxin.