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Peptidoglycan and β- glucan Detection SLP Reagent Set Silkworm Larvae Plasma Reagent

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 ℃) 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

Kit Contents

SLP Reagent Set

1.SLP reagent3 mL x 1 vial
Sensitivity: Endpoints (PG and β-glucan) determined by a visual test (after 60-minute incubation at 30 ℃) indicated for each lot
2. Substrate 3 mL x 1 vial
DOPA contained as a substrate
3. Substrate diluent 4 mL x 1 vial
Good's buffer contained;

SLP-HS Single Reagent Set

1. SLP-HS (Silkworm Larvae Plasma High Sensitive) 0.2 mL x 20 vials
Sensitivity: 10 pg/mL (PG), 1 pg/mL (β-glucan) (detected within 120 minutes with Toxinometer® at 30℃)
2. SLP diluent 1.0 mL x 20 vials
3. Standard (digested peptidoglycan from S. aureus ) 0.5 mL x 1 vial
Products
Code No. Product Name Application Quantity
297-51501 SLP Reagent Set For microorganism detection 3 mL
293-58301 SLP-HS Single Reagent Set For microorganism detection 20 tests

Topics

  • LAL Basic Knowledge
  • Lysate reagent selection flowchart

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