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

鲎试剂被广泛用于检测医疗器械的细菌和真菌污染。但该方法存在以下缺陷：鲎试验无法检测到肽聚糖；由于脂多糖和- 1,3 -葡聚糖检测呈阳性，因此无法区分检测到的污染是细菌污染还是真菌污染；马蹄蟹的血细胞裂解液被用于鲎试验，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，专门检测微生物细胞壁成分。家蚕（Bombyx mori）血浆中含有由肽聚糖和- 1,3 -葡聚糖引发的酚氧化酶原级联。我们打算用级联来检测医疗器械的细菌和真菌污染。我们成功地开发了一种方法来定量肽聚糖或- 1,3 -葡聚糖在非常低的浓度，如几pg/ml。此外，我们还利用抗β - 1,3 -葡聚糖识别蛋白的抗体，制备了多肽特异性的蚕酚氧化酶原级联，该蛋白是酚氧化酶原级联的一个组成部分，与β - 1,3 -葡聚糖特异性相交。这些研究结果表明，酚氧化酶原级联是检测和定量医疗器械微生物污染，特别是革兰氏阳性菌检测的良好候选试剂。我们正在开发收集大量不触发级联的蚕血浆的方法，并稳定血浆中的级联，以便血浆从供应商运送到用户。

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.