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

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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.