Wako Pure Chemical
 
작성일 : 12-08-20
[Wako Pure Chemical] SLP(Silkworm Larvae Plasma) Reagent Set
Peptidoglycan and β-glucan Detection kit
SLP(Silkworm Larvae Plasma) Reagent Set
Peptidoglycan and β-glucan Detection kit
 

Principle
 
The hemolymph of the silkworm (Bombyx mori ) contains a self-defense mechanism termed "the prophenoloxidase
cascade system (ProPO)," which is triggered by peptidoglycan (PG) and (1 → 3)-β-D-glucan (β-glucan), consequently
activating prophenoloxidase (PO) in the system. It is postulated that serial serine proteases are involved in the PO
activation; however, this has not yet been elucidated. This cascade system is believed to participate in the melanin
formation observed in the insect's body fluids as a self-defense mechanism.
SLP reagent is a lyophilized product prepared under sterile conditions from the silkworm hemolymph, which contains all
the ProPO factors involved in the cascade system. The reagent, activated by PG and β-glucan, oxidizes the DOPA (L-3,4-
dihydroxyphenylalanine) in the substrate and forms a melanin pigment. Since PG is a component found in most bacterial
cell walls and β-glucan, in many fungal cell walls, SLP enables the detection of various microorganisms by measuring the
melanin pigment formation.
The activation mechanism of SLP is shown in Figure 1. PG or β-glucan binds to the respective recognition protein (PGRP
or GRP), which initiates the ProPO cascade reactions, and consequently activates prophenoloxidase. The activated
prophenoloxidase then oxidizes the DOPA in the substrate, thus forming black melanin pigment. Since endotoxin, which
can be detected by horseshoe crab (Limulus polyphemus ) amebocytes (LAL reagent), does not activate the SLP
cascade system, it cannot be detected with SLP. However, it can detect PG, which cannot be detected by the LAL
reagent (Figure 1).
The SLP reagent strongly reacts with PG and β-glucan with β-1,3-glucoside bond, derived from various bacteria.
However, it is barely activated by lipopolysaccharide (LPS) (endotoxin), a cell wall component of gram-negative bacteria.
 
 
 
 
Since PG is found not only in gram-positive but also gram-negative bacteria, the SLP reagent responds to a wide range of
bacteria, regardless of their gram-staining classification. It also reacts with fungus-derived β-glucan, which suggests that it
can respond widely to general microorganisms. Therefore, the concurrent use of LAL and SLP reagents, which are
activated by endotoxin and β-glucan respectively, enable the type of microorganism in a sample to be predicted.
 
 
Features
 
  • Sensitive quantitation of peptidoglycan and β-glucan
  • Visual detection without the use of any special apparatus is possible.
  • Sensitive and accurate quantification of peptidoglycan and β-glucan is possible by using a microplate reader or Toxinometer®, a parallel turbidimetric time analyzer.
  • Stable for a minimum 18 months, in lyophilized (2 to 10 °C) form
 
Applications
 
  • Elucidation of the peptidoglycan structure-activity relationship, biosynthesis, metabolism and pathogenic significance
  • Water contamination measurement
  • Microbial contamination testing of the dialysis solution
  • Detection for fungal compounds in pharmaceuticals and medical devices, biologics and genetically-engineered products
  • Elucidation of the biological defense mechanism of insects
 
Ordering informations ;
 
1. SLP Reagent Set
  • SLP reagent       : 3 mL × 1 vial   * Sensitivity: Endpoints (PG and β-glucan) determined by a visual test (after 60-minute incubation at 30 °C) indicated for each lot
  • Substrate         : 3 mL × 1 vial   * DOPA contained as a substrate
  • Substrate diluent : 3 mL × 1 vial   * Good's buffer contained
 
2. SLP-HS Single Reagent Set
  • SLP-HS (Silkworm Larvae Plasma High Sensitive) : 0.2 mL × 20 vials   * Sensitivity: 10 pg/mL (PG), 1 pg/mL (β-glucan) (detected within 100 minutes with Toxinometer® at 30 °C)
  • SLP diluent 1.0 mL × 20 vials
  • Standard (digested peptidoglycan from S. aureus ) 0.5 mL × 1 vial
 
 
 

Measurement Method
 
Several methods for measuring PG and β-glucan using SLP are available: Toxinometer® measurement, microplate reader
measurement and visual determination.
The method using a microplate reader or Toxinometer® uses the pigment quantity (absorbance change) generated by
the activation, and measures the activation time (Ta or onset time) of the reactions from the start to the point at which the
absorbance reaches its predetermined threshold, quantifying the SLP activation substance (PG or β-glucan). The
principles used in these methods are the same as those for measuring endotoxin using a Toxinometer® (see Figure 2).
Figure 4 shows a measurement example using a Toxinometer®, while Figure 3 shows the microplate reader
measurement principle and Figure 5, an example of the same.
The visual determination refers to the procedure of checking the color change observed in the reaction mixture after a
certain reaction period has elapsed. This visual determination method is similar to that used in the LAL gelation test;
however, this is easier and more reliable, since the color change is more distinct and there is no vibration influence.
 
 
 
 
 
▣ 관련 페이지 ; Wako Pure Chemical