用聚阳离子固定化纤维素珠吸附法进行鲎试剂检测内毒素

Limulus amebocyte lysate assay for endotoxins by an adsorption method with polycation-immobilized cellulose beads.

作者信息

Sakata Masayo, Inoue Tomofumi, Todokoro Masami, Kunitake Masashi

机构信息

Department of Applied Chemistry & Biochemistry, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan.

出版信息

Anal Sci. 2010;26(3):291-6. doi: 10.2116/analsci.26.291.

DOI:10.2116/analsci.26.291

PMID:20215676

Abstract

To assay lipopolysaccharides (LPSs) in solutions containing Limulus amebocyte lysate (LAL)-inhibiting or LAL-enhancing compounds, we developed a selective endotoxin (LPS) assay using poly(epsilon-lysine)-immobilized cellulose beads (PL-Cellufine) and LAL. The PL-Cellufine can adsorb LPSs in a solution containing certain compounds (NaCl, proteins and amino acids) at an ionic strength of mu = 0.05-0.4 at neutral pH. The LPSs adsorbed on the PL-Cellufine were separated from the compounds by centrifugation and then the PL-Cellufine was suspended in LPS-free water. The LPS activities of the suspension are directly assayed by a turbidimetric time assay with the LAL reagent. The accuracy of the adsorption method was high compared with those of common solution methods. As for the common method, the apparent recovery of LPS from the compounds was 40-95%. This suggests that these compounds inhibit the LAL procedure. By contrast, the adsorption method showed good LPS recovery (88-120%) in all cases, without being inhibited or enhanced by the compounds.

摘要

为了测定含有鲎试剂（LAL）抑制或增强化合物的溶液中的脂多糖（LPS），我们开发了一种使用聚（ε-赖氨酸）固定化纤维素珠（PL-纤维素）和LAL的选择性内毒素（LPS）测定法。PL-纤维素可以在中性pH值下，离子强度为μ = 0.05 - 0.4的含有某些化合物（氯化钠、蛋白质和氨基酸）的溶液中吸附LPS。通过离心将吸附在PL-纤维素上的LPS与化合物分离，然后将PL-纤维素悬浮于无LPS的水中。通过用LAL试剂进行比浊时间测定法直接测定悬浮液的LPS活性。与常用的溶液方法相比，吸附法的准确性较高。对于常用方法，从化合物中LPS的表观回收率为40 - 95%。这表明这些化合物抑制了LAL程序。相比之下，吸附法在所有情况下均显示出良好的LPS回收率（88 - 120%），不受这些化合物的抑制或增强。