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

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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.