Reagents and Related Products for Detection of
Microbial Cell Components
Endotoxin

Peptidoglycan(1 → 3)-β-D-glucan

Revised in October 2019

Contents
Basics of Bacterial Endotoxins Test ……………………………… 2 ‒5
PYROSTAR™ ES-F Single Test VialPYROSTAR™ES-F Multi Test Vial ……………………………………… 6
PYROSTAR™ ES-F (2 ML) with CSE
PYROSTAR™ ES-F (2 ML) without CSE ……………………………… 7
PYROSTAR™ ES-F (5.2 ML) with CSE
PYROSTAR™ ES-F (5.2 ML) without CSE …………………………… 8
PYROSTAR™ ES-F/Plate ……………………………………………… 9
The Limulus Color KY Series ………………………………………… 10
SLP-HS Single Reagent SetⅡ ………………………………… 11 ‒ 12
Toxinometer® ET-7000 ……………………………………………

13

Toximaster® Software ………………………………………………… 14
MPR Endotoxin Measurement System for BT ……………………… 15
Endotoxin Test Related Accessories …………………………… 16 ‒18
Endotoxin Extracting Solution ……………………………………… 19
Endotoxin Indicator …………………………………………………… 20

Contribution to Measurement Technology- Acquisition of FDA approval …… 21

Microbial Cell Wall Components
Bacteria

Fungi

Gram Positive

Gram Negative

Yeasts, Molds, etc.

Peptidoglycan

Endotoxin

β-glucan• Peptidoglycan(30 ‒ 70 %)• Polysaccharide• Teichoic acid

• LPS (endotoxin)• Lipoprotein• Peptidoglycan(10 %)• β-glucan• Chitin• Mannan• Galactomannan

Limulus Amebocyte Lysate (LAL)
Silkworm Larvae Plasma (SLP)LAL Gel-clot Reagents
Bland NameTypeSINGLE TESTPYROSTAR™

Product Name

Gel-clot Sensitivity

QuantityPYROSTAR™ ES-F SINGLE TEST

0.015, 0.03 EU/mL

・25 tests + 1 vial CSE (500ng/vial)

PYROSTAR™ ES-F MULTI KIT

0.015, 0.03, 0.06,
0.125, 0.25 EU/ｍL

・80 tests + 1 vial CSE (500ng/vial)・200 tests + 1 vial CSE (500ng/vial)

PYROSTAR™ ES-F MULTI KIT, BULK

0.015, 0.03, 0.06,
0.125, 0.25 EU/mL

・2,000 tests
・5,000 tests

MULTI TEST

Page

6～8p

LAL Turbidimetric Reagents
Bland NameTypeSINGLE TESTFor Toxinometer

PYROSTAR™

MULTI TESTFor Toxinometer

MULTI TESTFor Microplate Reader

Product Name

Quantitative Range

QuantityPYROSTAR™ ES-F SINGLE TEST

0.001-10 EU/mL

・25 tests + 1 vial CSE (500ng/vial)

PYROSTAR™ ES-F MULTI KIT

0.001-10 EU/mL

・80 tests+ 1 vial CSE (500ng/vial)・200 tests+ 1 vial CSE (500ng/vial)

PYROSTAR™ ES-F MULTI KIT, BULK

0.001-10 EU/mL

・2,000 tests
・5,000 tests

PYROSTAR™ ES-F/Plate with CSE

0.01-10 EU/mL

・160 tests + 1 vial CSE (500ng/vial)

PYROSTAR™ ES-F/Plate without CSE

0.01-10 EU/mL

・200 tests

Page

6～8p

9p

LAL Chromogenic Reagents
Bland NameTypeSINGLE TESTFor Toxinometer

Color KY

Product Name
Limulus Color KY Single Test wako

MULTI TESTFor Microplate Reader Limulus Color KY Test Wako
and Toxinometer

Quantitative Range
0.0002-5 EU/ｍL

QuantityPage

・25 tests + 1 vial CSE (500ng/vial)
10p

0.0005-5 EU/mL

・60 tests※ + 1 vial CSE (500ng/vial)

Sensitivity

Quantity※120 tests with microplate reader

SLP Reagent
Bland Name
SLP

Type
SINGLE TESTFor Toxinometer

Product Name
SLP-HS Single Reagents Set Ⅱ

10 pg/mL (PG),
・20 tests + 2 vial (5.0mL) Diluent
1 pg/mL (β-glucan)+ 1 vial (0.5mL) Standard

Page
11～12p1

Basics of Bacterial Endotoxins Test
1. What is an endotoxin?
An endotoxin is a lipopolysaccharide (LPS) found in the cell wall of gram-negative bacteria. It is a typicalpyrogen, which induces various biological reactions when even a small amount of pg (10-12 g) or ng (10-9 g)enters the bloodstream. Due to its heat resistance and stability, complete inactivation of endotoxin is not possible
with autoclaving and dry heat sterilization for at least 30 minutes is required, at a temperature of 250 °C or
more. It exists in the environment (e.g. water, air) inhabited by gram-negative bacteria, and bacterial endotoxins(LPS) remain even after the bacteria die.
Figure 1 shows the LPS structure schematic, which illustrates lipid A as the component responsible for thebioactivity. The molecular weight of this portion is approx. 2000. The entire molecular weight, including thesugar chain moiety, is usually approx. 5000 to 8000. However, since an LPS consists of a hydrophilic region(sugar chain) and hydrophobic region (lipid A), it associates in an aqueous solution to form a micellar structurewith apparent molecular weight of hundreds of thousands to several millions. A change in the micellar structurereportedly influences the strength of bioactivity.
Figure 2 illustrates the structures of Salmonella-type and E. coli-type lipid A, which indicate that the basic
structure of the lipid A is maintained fairly well, regardless of the strain variation.

Figure 2. Lipid A Structure（E.coli and Salmonella types)

Figure 1: Lipopolysaccharide Structure Schematic Diagram

2. Various Endotoxin Test Methods using LAL Reagents
A lysate reagent prepared from the amebocytes of horseshoe crab (Limulus polyphemus) is used to detectbacterial endotoxins. As shown in Figure 3, the cascade reactions start by the presence of an endotoxin, whereby
Factor C, a serine protease precursor, is initially activated. There follows the subsequential activation of Factor
B, also a serine protease precursor and a proclotting enzyme, which hydrolyzes coagulogen into coagulin,
forming an insoluble gel. In LAL tests, endotoxin can be quantified in three ways: measurement of gel
formation, increased turbidity, or a yellow chromogen released due to the synthetic substrate cleavage.
Endotoxin (LPS)
Factor C(1 → 3)-β-D-glucan

Factor C

Factor B

Factor B

Factor G

Proclotting enzyme
Coagulogen

Peptidyl-pNA synthetic substrate

Factor G

Clotting enzyme

Coagulin(gel formation and increase in turbidity)
Peptide + pNA (yellow chromogen release): activated factors
Figure 3: LAL Reagent Reaction Mechanism

2Ordinary LAL reagents react not only with the endotoxin but also (1→3)-β-D-glucan (a fungal cell wallcomponent), since the Factor G pathway can be activated in the reagents. To eliminate this (1→3)-β-D-glucan
activation, various endotoxin-specific reagents are being developed in industry by removing Factor G or
inhibiting its activation.
Various LAL reagents are commercially available, as well as measuring systems based on the Figure 3 reaction
mechanism. It is essential to select the most appropriate product depending on the required accuracy, test
frequencies, number of samples and other relevant factors.
Pharmacopoeias in the U.S., Europe and Japan refer to three means of endotoxin detection, namely the gel-clot
technique, chromogenic and turbidimetric techniques, which are detailed in the following sections while
introducing the characteristics and application examples of our relevant LAL reagents.

(1) Gel-clot TechniqueMix a sample with LAL reagent in a test tube and incubate it using a block heater at 37 ± 1 °C, 60 ± 2 minutes,
without subjecting to vibration. Upon completion of heating, immediately but slowly tilt the tube through 180°.
If a gel has formed and maintains its integrity without deformation or collapse, the result can be determined
positive, while it is negative if no gel has formed. During the test, a series of samples is diluted multiple times(usually 2-fold) to check if the result is positive in each sample. The maximum valid dilution or the minimum
concentration determined positive is referred to as the endpoint.
Invert the tube through about 180̊.
(Turn the tube upside down.)

180°

Figure 4. Gel-clot Technigue

Positive ( + )Negative ( ー )Corresponding Reagent:
The reagent is available in a single-type kit with reaction vials containing pre-dispensed reagent for a single
measurement, and a multi-type kit for dispensing the required amount of the dissolved reagent into reaction
vials. The single-type kit is ideal for an assay with a few samples, and the multi-type kit for a larger number ofsamples.
A multi-type kit is used by dispensing 0.1 mL of dissolved LAL reagent into reaction tubes, which is then mixed
after having 0.1 mL of the sample added. A single-type kit can be used by adding 0.2 mL of the sample to thereaction vial with pre-dispensed, lyophilized LAL reagent.

ES-F Series
Endotoxin-specific LAL reagents (not activated by (1→3)-β-D-glucan), compatible with the BET (USP)
compliance tests U.S Food and Drug Administration (FDA) approved. They have various gelation sensitivities,
and are available in both single- and multi- type kits.

*BET: Bacterial Endotoxins Test3

(2) Chromogenic TechniqueThis technique uses synthetic chromogenic substrate cleavage to detect the activation of LAL reagent induced
by endotoxin. Since the yellow color of p-nitroaniline is measured by absorbance at approx. 405 nm,
the technique is not applicable if the sample has considerable absorbance at approx. 405 nm.

Corresponding Reagent:
Color KY Series
Endotoxin-specific chromogenic technique LAL reagents, compatible with the BET* (JP) compliance testing.
A single-type kit combined with a Toxinometer® and a multi-type kit for use in combination with a microplate
reader and Toxinometer® are available for kinetic chromogenic testing. These series also feature measurements
at the lowest concentration (highest sensitivity) among our reagent products: detection limit of 0.0002 EU/mL(single-type) and 0.0005 EU/mL (multi-type).

(3)Turbidimetric TechniqueThis technique uses the change in gel turbidity to detect the activation of LAL reagent induced by endotoxin.
It cannot be applied to samples with considerable turbidity.

Corresponding Reagent:
ES-F Series
Kinetic turbidimetric measurement is available by combining the ES-F series and Toxinometer®
or a microplate reader.These reagent kits can provide gel-clot results while obtaining kinetic turbidimetric data,with the measurement time specified as 60 minutes.

3. Tools Used for Testing
All tools used for endotoxin detection must be free from endotoxin and β-glucan. Dry-heating at 250 °C for
more than 30 minutes is required to deactivate endotoxin. The use of glassware treated by dry heat sterilizationis recommended. Avoid metal tools, since even a small amount of eluted metal ions (e.g. Fe, Al, Ga, Cr) may
affect testing. When using disposable plastic tools (not guaranteed by its manufacturer for use for testing
purposes), check if they satisfy the requirements: 1) Not contaminated by endotoxin; 2) No adsorption ofendotoxin; and 3) No eluted substance; in comparison with glassware.

4. Reference Standard Endotoxin
Use the appropriate type of standard endotoxin based on the testing purpose.
• Tests in compliance with BET (USP/EP/JP) such as final product inspection of pharmaceuticals and medical
devices→ Reference Standard Endotoxin of USP, EP or JP must be used.
• Tests for inspecting materials, processes and other related subjects→ Control Standard Endotoxin (CSE) can be used.

*BET: Bacterial Endotoxins Test

4

5. Interference of Sample
Precautions are required against the potential impact (reaction interferences) of samples on endotoxin tests.
These interferences are categorized into the following two types:❶ Influence on LAL Reagent• Protein denaturants (e.g. acids, alkalis, urea, surfactants, organic solvents)• Protease and protease inhibitors• Chelating agents (whereby Ca and Mg required for reactions are scavenged)• For the chromogenic technique: coloring matter (substance with considerable absorbance at approx. 405 nm)• For the turbidimetric technique: turbidity❷ Influence on Endotoxin• Metal ions (e.g. Fe, Al, Ga and Cr ions. Influential even at a micromole level)• Surfactants
The effect of samples can be judged by a test referred to as the test for interference factors by Pharmacopoeias:
namely, it is conducted by measuring a sample which has been spiked with a known amount of endotoxin andobtaining the recovery of the spiked endotoxin. If the recovery is within the 50 to 200 % range, then the sample
is determined as not influential, in other words, the measured endotoxin concentration is correct. If any sample
influence is found, it can be reduced by diluting the sample solution for measurement. However, dilution of thesample solution raises the endotoxin concentration value obtained through the conversion to the concentration of
the original solution (pre-dilution solution). The possible dilution multiple (maximum valid dilution) is
determined based on the desired endotoxin concentration to be detected and the detection sensitivity of the LAL
reagent to be used (refer to the Bacterial Endotoxins Test by the Pharmacopoeia for details of the reaction
interference factors and maximum valid dilution).

6. Principle of Limulus ES (Endotoxin Specific Reagent)
The reaction cascade mechanism of the LAL reagent and endotoxin isshown in Figure 5. If (1 → 3)-β-D-glucan* exists in the reaction system, it
activates Factor G, causing a false positive reaction in which gelation
occurs. This happens regardless of the presence of endotoxin, meaning
endotoxin speciﬁc detection is not available. Wako's Limulus ES has beendeveloped to inhibit the interference of (1 → 3)-β-D-glucan, by making an
excessive amount of (1 → 3)-β-D-glucan (carboxymethylated curdlan)
coexistent in the reaction system. Thus, the activation of LAL reagent by
β-glucan is inhibited, enabling endotoxin speciﬁc detection.
The reason why an excessive amount of β-glucan can inhibit its own
reaction is shown in Figure 6: the reaction range between the β-glucan
and LAL reagent is too narrow for reaction. On the other hand, the reactionof endotoxin and Limulus occurs over a wide range of concentration and is
not subject to any interference from the large amount of coexisting
β-glucan. Wako's Limulus ES use this principle (see Figure 7).

Figure 5: Limulus Test Reaction Cascade*(1 → 3)-β-D-glucan derived from mold (fungus) or cellulosic membrane ﬁlter.

Measured by using a Toxinometer® (with themeasurement time set to 99 minutes) and Limulus HS○ : Gelation was not determined within 99 minutes● : Gelation was determined

A: Limulus HS
B: Limulus ES○ : Endotoxin dilution series▲ : Endotoxin dilution series containing 1 μg/mL CM-curdlan

Figure 6: CM-curdlan and LAL Reaction

Figure 7: Eﬀect of CM-curdlan on Endotoxin Measurement Using
Limulus HS and Limulus ES

5LAL Reagent Kit

PYROSTAR™ ES-F Series
Turbidimetric TechniqueEndotoxin Speciﬁc

FDA approved

Intended Use: Limulus amebocyte lysate (LAL) is intended for the detection of gram-negative bacterial endotoxins.
PYROSTAR™ ES-F is intended for the qualitative detection of endotoxins by gel-clot or quantitative detection by kinetic
turbidimetric methods.
● Endotoxin-speciﬁc lysate, avoids false positive results from glucans● Available in multi-tests vials or single-test vials● Can be used as either a gel-clot or Kinetic-Turbidimetric Assay (KTA) reagent● (KTA) assays can be performed in tube reader or microplate reader● Gel-Clot lysate sensitivities range from 0.015 to 0.25 EU/mL● Available with matched control standard endotoxin (CSE)● PYROSTAR™ ES-F reagents are available with a KTA quantitative range of either 0.001 EU/mL to 10 EU/mL.
The KTA quantitative range is related to the Gel-Clot sensitivity.
● 100uL sample size when used with tube reader; 50uL sample size when used with microplate reader

PYROSTAR™ ES-F Single Test Vial

6

Gel-clot Sensitivity(EU/mL)KTA Quantitative Range(EU/mL)

0.015

0.001 to 10

0.03

0.01 to 10

Catalog Number

Product Name

Quantity

with CSE

295-72301

Limulus Amebocyte Lysate PYROSTAR™ ES-F SINGLE TEST 0.015

25 tests

with CSE

292-81601

Limulus Amebocyte Lysate PYROSTAR™ ES-F SINGLE TEST 0.03

25 tests

PYROSTAR™ ES-F (2 ML) Multi Test Vials with CSE

Gel-clot Sensitivity(EU/mL)KTA Quantitative Range(EU/mL)

0.015

0.001 to 10

0.03 to 0.25

0.01 to 10

Catalog Number

Code

Product Namewith CSE

548-10141

WPEK4-20015

PYROSTAR™ ES-F MULTI KIT (2ML), 0.015 EU/ML

2mL x 4vials

with CSE

541-10131

WPEK4-20003

PYROSTAR™ ES-F MULTI KIT (2ML), 0.03 EU/ML

2mL x 4vials

with CSE

545-10151

WPEK4-20006

PYROSTAR™ ES-F MULTI KIT (2ML), 0.06 EU/ML

2mL x 4vials

with CSE

542-10161

WPEK4-20125

PYROSTAR™ ES-F MULTI KIT (2ML), 0.125 EU/ML

2mL x 4vials

with CSE

549-10171

WPEK4-20025

PYROSTAR™ ES-F MULTI KIT (2ML), 0.25 EU/ML

2mL x 4vials

Quantity

CSE：1 vial(500ng/vial)

PYROSTAR™ ES-F (2 ML) Multi Test Vials without CSE

Gel-clot Sensitivity(EU/mL)KTA Quantitative Range(EU/mL)

0.015

0.001 to 10

0.03 to 0.25

0.01 to 10

Catalog Number

Code

Product Name

Quantity

546-10201

WPEM-20015

PYROSTAR™ ES-F MULTI KIT (2ML), BULK, 0.015 EU/ML 2mL x 100vials

546-10181

WPEM-20003

PYROSTAR™ ES-F MULTI KIT (2ML), BULK, 0.03 EU/ML

2mL x 100vials

543-10191

WPEM-20006

PYROSTAR™ ES-F MULTI KIT (2ML), BULK, 0.06 EU/ML

2mL x 100vials

540-10221

WPEM-20125

PYROSTAR™ ES-F MULTI KIT (2ML), BULK, 0.125 EU/ML 2mL x 100vials

543-10211

WPEM-20025

PYROSTAR™ ES-F MULTI KIT (2ML), BULK, 0.25 EU/ML

2mL x 100vials

7

PYROSTAR™ ES-F (5.2 ML) with CSE

Catalog Number

Gel-clot Sensitivity(EU/mL)KTA Quantitative Range(EU/mL)

0.015

0.001 to 10

0.03 to 0.25

0.01 to 10

Code

Product Name

Quantity

with CSE

543-10071

WPEK4-50015 PYROSTAR™ ES-F MULTI KIT (5.2ML), 0.015 EU/ML

5.2mL x 4vials

with CSE

542-10041

WPEK4-50003 PYROSTAR™ ES-F MULTI KIT (5.2ML), 0.03 EU/ML

5.2mL x 4vials

with CSE

547-10111

WPEK4-50006 PYROSTAR™ ES-F MULTI KIT (5.2ML), 0.06 EU/ML

5.2mL x 4vials

with CSE

544-10125

WPEK4-50125 PYROSTAR™ ES-F MULTI KIT (5.2ML), 0.125 EU/ML

5.2mL x 4vials

with CSE

549-10025

WPEK4-50025 PYROSTAR™ ES-F MULTI KIT (5.2ML), 0.25 EU/ML

5.2mL x 4vials

CSE：1 vial(500ng/vial)

PYROSTAR™ ES-F (5.2 ML) without CSE

Catalog Number

8

Code

Gel-clot Sensitivity(EU/mL)KTA Quantitative Range(EU/mL)

0.015

0.001 to 10

0.03 to 0.25

0.01 to 10

Product Name

Quantity

547-10231

WPEM-50015 PYROSTAR™ ES-F MULTI KIT (5.2ML), BULK, 0.015 EU/ML 5.2mL x 100vials

544-10241

WPEM-50003 PYROSTAR™ ES-F MULTI KIT (5.2ML), BULK, 0.03 EU/ML

5.2mL x 100vials

541-10251

WPEM-50006 PYROSTAR™ ES-F MULTI KIT (5.2ML), BULK, 0.06 EU/ML

5.2mL x 100vials

548-10261

WPEM-50125 PYROSTAR™ ES-F MULTI KIT (5.2ML), BULK, 0.125 EU/ML 5.2mL x 100vials

545-10271

WPEM-50025 PYROSTAR™ ES-F MULTI KIT (5.2ML), BULK, 0.25 EU/ML

5.2mL x 100vials

Endotoxin Detection ReagentsPYROSTAR™ ES-F/Plate
Turbidimetric TechniqueEndotoxin Speciﬁc

FDA approved

PYROSTAR ™ ES-F / Plate is a speciﬁc Turbidimetric lysate reagent speciﬁcally for 96-well microplates.
By using an absorbance microplate reader, simultaneous measurement of multiple samples is easily made possible.
The quantitative range is wide, from 0.01 to 10 EU / mL, and it can be applied to water type tests, as well as to the testing ofname drugs that require dilution.
■ Product Features● It is possible to speciﬁcally detect endotoxin without it being aﬀected by (1 → 3) - β-D - Glucan in the sample.
● US Food and Drug Administration (FDA) approved .
● Regarding U.S. exports, it can also be applied to the ﬁnal product testing.
● Since it can be measured with a sample volume of 50 μL per well, even a precious sample can be measured in a small amount.
● Stable endotoxin measurement is possible, due to the minor pH impact of the measurement
sample on testing.

Multi-test
Catalog Number
with CSE

Product Name

Quantitative Range(EU/mL)

Quantity

293-75401

Limulus Amebocyte Lysate
PYROSTAR™ ES-F/Plate with CSE

0.01-10160 tests(2.0mL×4vials)

297-75301

Limulus Amebocyte Lysate
PYROSTAR™ ES-F/Plate without CSE

0.01-10200 tests(2.0mL ×5vials)
CSE：1 vial(500ng/vial)

9Chromogenic Technique (Kinetic Yellow Synthetic Substrate Method)

The Limulus Color KY Series Endotoxin Speciﬁc Limulus Reagent
Chromogenic TechniqueEndotoxin Speciﬁc

The Limulus Color KY Series includes both a multi-test kit and a single-test kit, each designed for time-based
chromogenic analysis, using a synthetic substrate which produces a yellow color and can speciﬁcally detect
endotoxin with high sensitivity.

■Product features● Endotoxin-speciﬁc lysate, avoids false positive results from glucans● Available in multi-tests vials or single-test vials● Quantitative Kinetic-Chromogenic Assay (KCA) reagent● KCA assays can be performed in tube reader or microplate reader● KCA quantitative range detection limit of 0.0002 EU/mL (single-type)and 0.0005 EU/mL (multi-type).
● Available with matched control standard endotoxin (CSE※)● 100uL sample size when used with tube reader; 50uL sample size whenused with microplate reader※CSE：1 vial(500ng/vial)

Single-test
Catalog Number
with CSE

291-53601

Product Name
Limulus Color KY Single Test Wako

Quantity
25 tests
CSE：1 vial(500ng/vial)

Multi-test
Catalog Number
with CSE

291-53101

Product Name
Limulus Color KY Test Wako

Quantity
60 tests
CSE：1 vial(500ng/vial)

10Peptidoglycan and β- glucan Detection

SLP-HS Single Reagent SetⅡ Silkworm Larvae Plasma Reagent

Research Use only, Not for use in diagnostic procedures■ Principle
The hemolymph of the silkworm (Bombyx mori) contains a self-defense mechanism termed "theprophenoloxidase 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 melaninpigment. 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 ofgram-negative bacteria.
(1→3)-β-D-Glucan

Peptidoglycan

β-GRP1 β-GRP2

PGRP-S1

β-GRP：(1→3)-β-D-glucan recognition protein
PGRP ：peptidoglycan recognition protein
GNBP ：gram-negative bacteria-binding protein
SPH ：serine protease H
CSPS ：clip-domain serine protease S
PPAE ：phenoloxidase activating enzymePO：phenoloxidase
DOPA ：L-3,4-dihydroxyphenylalanine

GNBP
ProSP HSP H

ProcSP S

cSP S

ProPPAE

PPAE
ProPO

PO

DOPA

DopachromemelaninFigure 1. Phenoloxidase precursor cascade of Bombyx mori blood

Since PG is found not only in gram-positive but also gram-negative bacteria, the SLP reagent responds to awide 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 typeof microorganism in a sample to be predicted.

11

Silkworm Larvae Plasma Reagent■ Product Features
1.High sensitive detection of PG and β-glucan
2.Accurate and sensitive quantiﬁcation of PG and β-glucan with Toxinometer■ Applications
1.Study of the structure-activity relationship, biosynthesis, metabolism and etiological signiﬁcance of PG
2.Investigation of water pollution
3.Microbial contamination testing of the dialysate
4.Detection for fungal compounds in pharmaceuticals and medical devices, biologics andgenetically-engineered products
5.Elucidation of the biological defense mechanism of insects■ Kit Contents

1.SLP-HS Reagent Ⅱ

for 0.1mL x 20 vials(Lyophilized reagent containing Silkworm Larvae Plasma and DOPA)
2.SLP Diluent

for 5mL x 2 vials

3.Standard

for 0.5mL x 1 vial

(Digested Peptidoglycan from Staphylococcus aureus)

Activation time (min)Transmission light ratio (%)■ Measurement Method
PG and β-glucan can be detected using SLP-HS Single Reagent Set and an instrument of Toxinometer.
The measurement principle is the same with endotoxin detection method using Toxinometer (Figure 2).
The absorbance change (melanin production) generated by the activation are detected using Toxinometer.
The correlation between the PG(or β-glucan) concentration and Ta is obtained (Figure 3), and the PG(or β-glucan) concentration is calculated from Ta of each sample.

Gel time (min)Figure 2. Measuring principles:
It measures time (Ta) of index moving from
R(t) to threshold (Rth). (Th: decision threshold)

Catalog Number
296-81001

Product Name
SLP-HS Single Reagent Set Ⅱ

Concentration (ng/mL)
Figure 3. Measuring example using Toxinometer®

Quantity
20 tests

Related Products
Catalog Number

12

Product Name

Quantity

030-09903

Curdlan1g

162-18101

Peptidoglycan, from Micrococcus luteus

2 mL

Endotoxin Measurement System

Toxinometer® ET-7000
The Toxinometer® ET-7000 is our computer-operated kinetic incubating tube reader, which is
exceptionally user-friendly and easily expandable. Depending on the number of samples to beprocessed, our state of the art expansion modules can be connected to allow for endotoxin
testing in a wide range of ﬁelds and sample quantities.
■ Product Features● Kinetic Incubating Tube Reader● Single-test conﬁguration avoids “Hot Wells”● For use in Kinetic-Turbidimetric, Kinetic-Chromogenic, and Gel-Clot assays● A single assay module can simultaneously measure up to 16 samples● Expansion modules are available to extend number of samples in multiples of 16● Temperature settings at both 30℃ and 37℃● Endotoxin determination in compliance with FDA guidelines as well as withPharmacopeal(USP/EP/JP) BET monographs

Catalog Number
Part 11 System

Model
Power Source※The system complies with FDA21 CFR, Part 11.

294-35871

Toxinometer® ET-7000/U
Part11 Set

100-120 +/- 10% VAC(USA)299-35821

Toxinometer® ET-7000/E
Part11 Set

220-240 +/- 10% VAC(Europe)

Non-Part 11 System

Contents

1 Toxinometer® ET-7000
Toximaster® QC8 Software w/5User Licenses
1 Personal Computer
System Validation Documents（in CD）※The system doesn’t comply with FDA21 CFR, Part 11.

291-35881

Toxinometer® ET-7000/U
Non-Part11 Set

100-120 +/- 10% VAC(USA)296-35831

Toxinometer® ET-7000/E
Non-Part11 Set

220-240 +/- 10% VAC(Europe)1 Toxinometer® ET-7000
Toximaster® QC7 Software w/1User Licenses
1 Personal Computer

Expansion Module ※Additional Module for Part 11 Set or Non-Part 11 Set.
297-35861
292-35811

Toxinometer® ET-7000/U

100-120 +/- 10% VAC(USA)Toxinometer® ET-7000/E

220-240 +/- 10% VAC(Europe)

16 well expansion unit

13

The "Welcome Screen" helps navigate you where you need to go!

Toximaster® Software
Exclusive software for Eﬃcient routine work &High quality analysis.
■ Protocol Settings

Easy to edit! Once you create a protocol, you can start ameasurement immediately.

■ Time Course Graph■ Standard Curve

Enables visual conﬁrmation of measurement status.
You can predict results and prepare the next steps.

Conveniently monitored!
All information can be seen on one screen.

Part 11 Functions ※Ensure data integrity■ Electronic Signature■ Operation Authorities
Each account belongs to a speciﬁc group and eachgroup can deﬁne its own authorities for operation.
・Modify System Preferences
・Register Instrument
・Register Protocol

All measurement records are linked to signatures.
Never allows for manipulation and falsiﬁcation.

■ Audit Trail

・Register Reagent
・Register Accessory
・Register Standard Curve Data
・Register Product・Load Protocol into Test Record
・Star Measurement
・Load Test Record
・Review Test Plan
・Conﬁrm Test Record
・Approve Test Record

Major procedures are recorded automatically.
History of operation can be conﬁrmed as a log ﬁle.

14

・Submit Test Record

Etc ..

Endotoxin Measurement System

MPR Endotoxin Measurement System for BT
lt is a system dedicated to endotoxin measurement using absorbance Microplate Reader ELx808IU andstandard equipment“Toximaster® MPR” software. lt can be used for various endotoxin tests such as
quality control tests on pharmaceuticals and medical equipment. lt enables you to arrange samples andinput sample information on one software screen,and supports accurate multi-sample processing withsimple and small software operation.
■ Product Features● Combined with the appropriate LAL reagent, two testing techniques: turbidimetric and chromogenic
are usable with a single system.
● Simultaneous measurement of up to 96 samples is possible with 96 well plate.
● Systems comply with the FDA21 CFR Part 11 are also available.
● The software supports to BET (USP/EP/JP).
● Prepare dedicated software “Toximaster® MPR Part11” or “Toximaster® MPR Non- Part11”

Catalog Number
Part 11 system292-35931

Product Name

Toximaster®

Contents

QC8 MPR Part 11 PC Set

Toximaster® QC8 MPR Part 11 Software
Personal Computer
System Validation Documemt(in CD)

QC7 MPR Non-Part 11 PC Set

Toximaster® QC7 MPR Non-Part 11 Software
Personal Computer

Non-Part 11 system-

Toximaster®

15

Endotoxin Test Related Equipment

Endotoxin Test Related Accessories
BioClean® Series■ Product Features● Endotoxin-free pipette tip (<0.005 EU/tip)● Individually packaged, ideal for use in clean rooms● Packaging ﬁlm features dust-free protection● Sterilized by gamma irradiation (25kg ray)
● Tips ﬁt Finnpipette digital micro pipetters(20-200uL or 100-1000uL sizes)● Endotoxin-free tubes and caps sterilized at 250 ℃● For use with Toxinometer® measurement system

Bio Clean Tip Wako®

Bio Clean Plate Wako®

Bio Clean Tip Wako® Extend SⅡ

Bio Clean Tip Wako® 200Ⅱ

Bio Clean Tip Wako® 1000Ⅱ

Endotoxin-free Pipette Tips and Microplates
Catalog Number

16

Product Name

Volume Size

Quantity294-35011

BioClean Tip Wako® Extend S Ⅱ

200 μL

100 tips

291-35021

BioClean Tip Wako® 200 Ⅱ

200 μL

100 tips

298-35031

BioClean Tip Wako® 1000Ⅱ

1000 μL

100 tips

293-35221

Bio Clean Plate Wako® Microplate

96 well

50 pcs/pk

Limulus Test Tube-S with Aluminum Cap

Endotoxin-free Test Tubes and Caps/ Gel-clot Reaction Tubes
Catalog Number

Product Name

Volume Size

Quantity

292-32751

Limulus Test Tube-S with Aluminum Cap

12 x 75mm

80 pcs293-26551

Limulus Test Tube-S

12 x 75mm

100 pcs

293-28251

Aluminum Cap-S

14.7 x 18mm

100 pcs

Volume Size

Quantity

Gel-clot Reaction Tubes■ Product Features● Endotoxin-free test tubes (<0.001 EU/tube)● Designed for Gel-clot testing● Made with Borosilicate glass
Made with borosilicate glass

Catalog Number

Product Name

CT-1075

Gel-clot Reaction Tube

10×75mm

200 tubes/pk

CT-1075B

Gel-clot Reaction tube,Bulk

10×75mm

1250 tubes/pk

542-10281

Depyrogenated Dilution Tube

13 x 100mm

50 pcs/pk

17

Endotoxin Test Related Accessories
Control Standard Endotoxin■ Product Features● Endotoxin derived from E.Coli UKT-B● Can be used to prepare controls and standard curves● RSE/CSE ratios supplied to match speciﬁc lots of LAL● Reconstituted CSE can be stored at 2-10℃ for 1 month

Catalog Number

Code

Product Name

Quantity

638-01021-

Japanese Pharmacopoeia Endotoxin Reference Standard

1 vial (10,000~
25,000 EU/vials)

546-10061

CSE 4037-5006

Control Standard Endotoxin

6 vials (500 ng/vial)

LAL Reagent Water
Wako proudly provides high-quality, endotoxin free water ideal for all of your LAL testing needs.
■ Product Features● Endotoxin-free water (<0.001 EU/mL)● Steam sterilized by USP standards● Derived from water for injection, USP● Non LAL reactive

18

Catalog Number

Code540-10081

LRW-12100

Lysate Reagent Water, 100mL

12 x 100mL vials

547-10091

LRW-2030

Lysate Reagent Water, 30mL

20 x 30mL vials

Product Name

Quantity

Endotoxin Extracting Solution
Endotoxin Extracting Solution for LAL Test
Traditionally, water or saline solution has been used to extract endotoxin in tests on medical devices andequipment; however, the eﬃcacy of this extraction method has recently come into question. In order toprovide our customers with a more reliable method for medical device testing,
FUJIFILM Wako has developed an endotoxin-free extracting solution containing human serum albumin(HSA) that is capable of extracting endotoxins which cannot be extracted in water or saline solution.
■ Product Features● Capable of extracting endotoxins from surfaces which cannot be extracted in water or saline● Recommended for use in endotoxin testing on equipment and devices which may come into
contact with ﬂuids containing blood or protein.

Catalog Number
293-51601

Product Name
Endotoxin Extracting Solution for LAL Test

Quantity
4 x 10 mL vials

19Endotoxin Indicator
Endotoxin Indicator Vial (>1,000 EU/vial)■ Product Features
Intended Use: The Endotoxin indicator is intended for performing validation for depyrogenation
processes of dry heat oven cycles. To ensure that a cycle is eﬀectively inactivating and/or
destroying endotoxin, an analysis comparing the endotoxin concentration before and after the
depyrogenation process is completed. The United States Pharmacopeia (USP) describes preparing
endotoxin indicators of a suﬃcient concentration to allow recovery of a minimum 1,000 Endotoxin
Units (EU) in order to accurately demonstrate at least a 3-log reduction in endotoxin levels during
the depyrogenation process challenge. The Endotoxin indicator is intended to demonstrate aminimum 3-log reduction after depyrogenation when used with PYROSTAR™ ES-F series by gel
clot and kinetic methods

20

Catalog Number

Code549-10291

EIV-025

Product Name
Endotoxin Indicators, >1,000 EU/vial

Quantity
25 vials

Contribution to Measurement Technology- Acquisition of FDA approval
Endotoxin is a potent toxin contained in cell wall components of Gram-negative bacteria. Since it brings about the feveror lethal shock when entering the blood of a person, it is necessary to conﬁrm that medicines are not contaminated withendotoxin. We have strived to evolve the endotoxin measurement technology by manufacturing lysate reagents anddeveloping the measurement system"Toxinometer", and supported medicines from behind for over 30 years.

From sales of lysate reagents to the development of endotoxin measurement system
IInitially, the rabbit pyrogen test was used for theendotoxin test method. After injecting the sample into
the rabbit, the amount of endotoxin contamination wasmeasured from the increasing degree of its bodytemperature. However, with this test method, there
were drawbacks such as taking time to get results,
being unable to maintain the reliable results because ofusing animals.
In 1956 in the United States, F.B.Bang discovered aphenomenon that the blood of horseshoe crabs solidiﬁed
Our US Plant (Location: Richmond, Virginia)
in a gel state with endotoxin, and then the endotoxin
measurement reagent (Limulus reagent, renamed as lysate reagent later) prepared from the blood extract of horseshoe
crabs and "Gel-clot technique" using this were developed. Gel-clot technique were listed in the US Pharmacopoeia in
1980, but we also began importing and selling the reagent prior to this. However, in the gel-clot technique, problems
remained such as being the semi-quantitative method and the judgment accuracy depending on the proﬁciency ofthe measurer.
We challenged to solve these problems, then in 1985, developed the measurement method "Turbidimetric time analysis
method" based on the diﬀerent principle from the conventional one and released its measurement system "Toxinometer"
for the ﬁrst time in the world. At the same time, in the United States, ACC company (Associates of Cape Cod, .INC)
developed a system using the same measurement principle. In response to this situation, the turbidimetric time analysis
method was listed in the US FDA Guideline in 1987.
Currently, Toxinometers corresponding to the turbidimetric, chromogenic and gel-clot technique are provided along withnumerous reagents. Meanwhile, also oﬀering "MPR Endotoxin Measurement System" corresponding to the chromogenic
and turbidimetric techniques using plate readers, we are contributing to the safety of pharmaceutical production andquality control of pharmaceuticals as a pioneer of endotoxin measurement.

Establishment of the FDA approved plant and development of the world's only speciﬁc reagent for endotoxin
The world's ﬁrst lysate reagent was developed in the United States, and we initially handled only imported items from theUnited States. As the market expanded and the needs of reagents increased, we established a plant in the United States.
First of all, we bought Hemakem Inc. in St. Louis in 2003 and made it a foothold for entering the United States.
After that, we built a new plant in Richmond, received FDA approval, and began shipping reagents. Incidentally, theendotoxin speciﬁc reagents with FDA approval are only our products worldwide.
Also, there is a convenient single type reagent that is lyophilized in a test tube beforehand and can be measured simply byputting the sample. Even in this category, the company having the endotoxin speciﬁc reagents with FDA approval is only us
in the world.
As described above, our company, which has been involved in endotoxin reagents for more than 30 years and making
eﬀorts on the improvement of measurement methods and the development of "Toxinometer", has tried hard to develop thepharmaceutical industry and others, as one of pioneers of endotoxin measurement.
21

Wako LAL System
Web Site for Endotoxin Analysis

http://www.wako-chem.co.jp/lal/en/index.html
Contact
http://ffwk.fujifilm.co.jp/en/contact/index.html
http://ffwk.fujifilm.co.jp/
1-2,Doshomachi 3-Chome
Chuo-Ku,Osaka 540-8605,Japan

www.wako-chemicals.deE-mail：info_wkeu@fujifilm.com

www.wakousa.com
E-mail：wkuspyrostarinfo@fujifilm.com