The development of a method for detection and quantification of cell wall components of bacteria and fungi.

本项目的目的是开发一种检测和定量微量肽聚糖和β-1，3-葡聚糖的方法。虽然利用河蟹血细胞裂解上清液建立了测定内毒素和β-1，3-葡聚糖的方法，但该方法不能检测到肽聚糖。以前我们报道了家蚕血淋巴的血浆部分含有由微量的肽聚糖或β-1，3-葡聚糖触发的原酚氧化物级联反应。研究了血浆组份是否可以用于检测和定量肽聚糖，以及是否可以在性质变化最小的情况下进行商业供应。为了提高该方法的可行性，还对肽聚糖或β-1，3-葡聚糖激活原酚氧化酶级联反应的机理进行了研究。本课题的主要研究成果如下：1)建立了一种家蚕血淋巴加工方法，用于检测和定量检测家蚕血淋巴中的肽聚糖和β-1，3-葡聚糖。血浆组分现在可以在商业上以SLP试剂的名称获得。2)SLP试剂的原酚氧化酶级联可由肽聚糖和β-1，3-葡聚糖共同触发。由肽聚糖或β-1，3-葡聚糖特异性触发的SLP试剂是可取的。我们开发了一种制备肽聚糖或β-1，3-葡聚糖特异性SLP试剂的方法。这种提高家蚕血浆特异性的方法已有报道，但其缺点是血浆中的原酚氧化酶级联在血浆处理过程中被激活。新开发的方法降低了自发激活的风险。目前，我们成功地获得了被肽聚糖激活的家蚕血浆，其原酚氧化酶级联的敏感性是后者的1万倍。

The end of the present project is to develop a method to detect and quantify minute amounts of peptidoglycan and beta-1,3-glucan. Although such method for lipopolysaccharide and beta-1,3-glucan has been developed by using hemocyte lysate supernatant of horseshoe crab (Limulus), peptidoglycan could not be detected by the method. Previously we reported that plasma fraction of silkworm hemolymph contains prophenol oxidase cascade that is triggered by minute amount of peptidoglycan or beta-1,3-glucan. The plasma fraction was studied if it could be used for detection and quantification of peptidoglycan and if it could be supplied commercially with minimal variation of its property. Toimprove the feasibility of the method, the mechanism of the activation of prophenol oxidase cascade with peptidoglycan or beta-1,3-glucan was also studied. The major results obtained during the term of the present project are as follows :1) A method for processing silkworm hemolymph was developed for supplying plasma fraction for the detection and quantification of peptidoglycan and beta-1,3-glucan. The plasma fraction is now available commercially under a name of SLP-reagent.2) Prophenol oxidase cascade of SLP-reagent could be triggered by bothof peptidoglycan and beta-1,3-glucan. SLP-reagent which is specifically triggered by peptidoglycan or by beta-1,3-glucan is desirable. We developed a method to make SLP-reagent specific to peptidoglycan or beta-1,3-glucan. This kind of method for raising the specificity of silkworm plasma has already been reported, but it had weak point in that prophenol oxidase cascade in plasma get start of activation during the manipulation of plasma. The newly developed method diminished the risk for the spontaneous activation. At present we succeeded in obtaining silkworm plasma of which prophenol oxidase cascade is 10,000 times more sensitive for being activated by peptidoglycan.