Development of a Reagent to Detect and Quantify Minute Amount of Cell Wall Components of Bacteria and Fungi.

检测细菌和真菌污染的医疗器械，目前广泛使用的检测方法是凝胶试验。然而，该检测方法存在以下缺点：河蟹试验不能检测到肽聚糖；由于内毒素和β-1，3-葡聚糖在试验中呈阳性，因此无法区分检测到的污染是细菌污染还是真菌污染；将马蹄蟹的血细胞裂解物用于河蟹试验，留下了未来马蹄蟹供应不足的可能性。本研究项目的目的是开发一种商用试剂来特异检测微生物细胞壁成分。家蚕(Bombyx Mori)血浆中含有由肽聚糖和β-1，3-葡聚糖触发的酚氧化酶原。我们打算利用级联检测医疗器械中的细菌和真菌污染。我们成功地建立了一种在很低的浓度下(如几个pg/ml)定量多肽多糖或β-1，3-葡聚糖的方法，并利用与β-1，3-葡聚糖特异相互作用的β-1，3-葡聚糖识别蛋白(β-1，3-葡聚糖识别蛋白)的抗体，使家蚕的多肽-1，3-葡聚糖原-1，3-葡聚糖链具有特异性。这些对酚氧化酶原级联的研究结果表明，该级联是一种很好的试剂，可用于医疗器械微生物污染的检测和定量，特别是革兰氏阳性菌的检测。我们正在开发收集大量不触发级联的家蚕血浆的方法，并稳定血浆中的级联，以便将血浆从供应商输送到用户。

The Limulus test is widely used to detect pollution of medical instruments with bacteria and fungi. However, the test method has drawbacks such as following : Peptidoglycan can not be detected by Limulus test ; As lipopolysaccharide and beta-1, 3-glucan are positive in the test, it cannot be differentiated whether the detected pollution is due to that of bacteria or fungi ; Hcmocyte lysate of horseshoe crab is employed to the Limulus test, leaving the possibility that we may not have enough supply with horseshoe crab in future.The purpose of this research project is to develop a commercially available reagent to specifically detect microbial cell wall components. The silkworm (Bombyx mori) plasma contains prophenoloxidase cascade which is triggered by peptidoglycan and beta-1, 3-glucan. We intended to use the cascade to detect pollution of medical instruments with bacteria and fungi. We succeeded in developing a method to quantify peptidoglycan or beta-1, 3-glucan at very low concentrations like several pg/ml. Furthermore, we could make silkworm prophenoloxidase cascade specific to peptidogly can by using antibody against beta-1, 3-glucan recognition protein, which is a component of prophenoloxidase cascade and specifically interects with beta-1, 3-glucan. These results on the studies of prophenoloxidase cascade indicated that the cascade is a good candidate as a reagent for detection and quantification of microbial pollution of medical instruments, especially for detection of Gram-positive bacteria. We are in the process to develop methods to collect large quantity of silkworm plasma of which cascade is not triggered and to stabilize the cascade in the plasma in order that the plasransported from supplier to users.