Pyrogen Testing
Pyrogens are fever-inducing substances usually derived from microorganisms or chemicals that cause severe biological reactions. Examples include bacterial endotoxin, peptidoglycan (PG) and (1 → 3)-β-D-glucan (BDG).
Products contaminated with pyrogens can cause severe consequences as they activate a strong inflammatory immune response, even when a small amount enters the bloodstream.
Applications
Pyrogen testing is vital for the detection of pyrogenic contamination in research, quality control and development of pharmaceuticals, medical devices and laboratory consumables for use in the healthcare field. Including its use for the validation of depyrogenation processes, as sterility does not necessarily imply free of pyrogens or endotoxins.
Bacterial Endotoxin Testing (BET) uses Limulus Amoebocyte Lysate (LAL) reagents for the high sensitivity and specificity detection of endotoxins, complex lipopolysaccharides (LPS) found in the cell wall of gram-negative bacteria. They are a highly heat resistant toxin that exhibits various biological activities such as pyrogenicity (fever). LAL reagents utilise the LAL clotting cascade triggered as self defence mechanism of the Atlantic horseshoe crab (Limulus polyphemus).
FUJIFILM Wako provides a lineup of reagents formulated to perform pyrogen detection using the activation of the LAL cascade:
Gel Clot Assay (GCA) - Presence of gel formation
Kinetic Turbidimetric Assay (KTA) - Measurement of time for change in turbidity
Kinetic Colorimetric Assay (KCA) - Measurement of time for colour change
GCA
Easy to use
Low equipment cost
Reference method USP
Qualitative
KTA
Real time measurement
Automated readings
Less dilution than GCA
Over-coming interference
Works well with Coloured samples
Fast and more sensitive
Quantitative
KCA
Real time measurement
Automated readings
Less dilution than GCA
Over-coming interference
Works well with Turbid samples
Fastest and most sensitive
Quantitative
Product Selection Flow Chart
Featured Products
PYROSTAR™ ES-F
The PYROSTAR™ ES-F kit series are designed for both qualitative GCA which uses gel formation or quantitative KTA which uses the change in gel turbidity to detect the activation of LAL. They are endotoxin specific and specifically formulated to not generate false positives for (1 → 3)-β-D-glucan, because of the addition of carboxymethylated curdlan (CMC). All reagent kits are matched with a specific Control Standard Endotoxin (CSE).
The single-test vial configuration is ideal for a smaller number of samples or for a less experienced user. It can be ideal to use to ease the transition from GCA to KTA methodology as it designed for the Toxinometer®. The multi-test vial is ideal for testing a larger number of samples.
The ES-F/Plate reagent is designed to be specifically used for 96-well microplates by using an absorbance microplate reader, simultaneous measurement of multiple samples is easily made possible.
Key Features
Dual-purpose: qualitative GCA or quantitative KTA
Can be used with water bath, heat block, microplate reader or Toxinometer®
Works well with coloured samples
GCA sensitivities range: 0.015 - 0.25 EU/mL, KTA quantitative range: 0.001 to 10 EU/Ml
Endotoxin specific testing
FDA approved
Limulus Color KY
The Limulus Color KY kit series are quantitative KCA that are endotoxin-specific and unreactive to (1 → 3)-β-D-glucan. This series includes both a multi-test kit and a single-test kit depending on sample size, user expertise and equipment available, that are matched with a CSE. It is great option for turbid samples and facilitates high sensitivity endotoxin specific testing.
The reagent is based on the colour development mechanism, which is activated by the presence of endotoxin. First, a series of activations of serine protease precursors in the reagent occur, consequently triggering the final reaction, in which a clotting enzyme hydrolyses the chromogenic substrate to release a yellow chromogen (pNA), producing the yellow colouration.
As a result, these reagents work well with samples that are very turbid or show difficulty in quantification by the KTA method.
Key Features
Quantitative KCA
Can be used with microplate reader or Toxinometer®
Works well with turbid samples
Detection limit: 0.0002- 2 EU/mL (Single) / 0.0005 - 5 EU/mL (Multi)
Reduces the chance of interfering factors due to high sensitivity
Endotoxin specific testing
Toxinometer®
The Toxinometer® ET-7000 from FUJIFILM Wako is a computer-operated kinetic incubating tube reader, designed to be exceptionally user-friendly and easily expandable.
Depending on the number of samples to be processed, expansion modules can be connected to allow for endotoxin testing in a wide range of fields and sample quantities.
Performs BET for all LAL testing methodologies (GCA, KTA and KCA). The instrument has 16 wells for single-test configuration to easily detect erroneous readings or contamination associated with each well.
The temperature setting can be switched between 30 and 37 ℃, also facilitating (1 → 3)-β-D-glucan and peptidoglycan testing using the SLP reagent for research projects.
Principle of Measurement
Light from an LED goes through the reaction tube filled with reaction mixture via aperture diaphragm 1. The light passes through the reaction mixture and then, while coming through aperture diaphragm 2, is detected by the silicon photocell.
Determination of Kinetic Measurement
The Toxinometer® reports your Reaction Time as either Gelation Time (Tg for Turbidimetric technique) or Activation Time (Ta for Chromogenic technique) based on the methodology used. The higher the concentration of endotoxin present, the shorter the reaction time. The Toxinometer measures the Transmittance Ratio of each well independently and simultaneously. The Reaction Time is determined when the Transmittance Ratio falls below the Threshold value.
Toximaster®
The Toximaster® QC8 software from FUJIFILM Wako allows you to measure endotoxin levels, through use with the Toxinometer ET-7000, with vastly improved efficiency and ease-of-use. Using the Toximaster QC8 software set you can conduct a wide variety of tasks, including designing and running of protocols, analysis of results numerically and visually and generation and processing of standard curves. The QC8 software, when used with the Toxinometer ET-7000 complies with FDA 21 CFR. Exclusive software for efficient routine work & high-quality analysis.
Benefits
Our Pyrogen Testing range is perfect for those looking to enhance their methods or to take control of their testing by moving it in house. The features of our large product range provide many advantages:
High Sensitivity and Specificity – Improve your current methodology for specific bacterial endotoxin detection.
Selection of Methodologies – GCA, KTA and KCA all available to meet your specific endotoxin/LAL test requirements for different applications.
Testing that Fits Around You – All assays are available in different formats and pack sizes at a range of specificities allowing you to tailor your testing and workflow to your needs.
GCA/KTA Assay – PYROSTAR™ ES-F is available for both methods giving you a qualitative and a quantitative option for endotoxin detection.
KCA Assay – Limulus Color KY is the most sensitive at detecting the presence of endotoxins, providing swift and accurate results.
Synthetic LAL Alternative – PYROSTAR™ Neo+ is a recombinant endotoxin detection reagent, which is a non-animal derived LAL substitute with the same high sensitivity as PYROSTAR™ ES-F.
SLP Assay – SLP-HS Single Reagent Set II is a highly sensitive test to detect the pyrogens; peptidoglycan (PG) found in all bacterial cell walls and (1->3)-β-D-glucan (BDG) in most fungi.
Greater Control – Control every aspect of your testing process allowing you to ensure that it is carried out to your standards.
Quicker Results – In-house testing, results are available as soon as the assay finishes, no more waiting for external laboratories.
Improved Troubleshooting – Fix issues with testing and products as soon as they occur, don’t rely on other laboratories to alert you to or fix problems. Track product results over time to identify potential problems before they affect quality.
FDA Approved – GCA/KTA endotoxin testing methods have been approved for use by the FDA when used with the Part 11 software, PC and testing module set.
Testing that Grows with You – With the modular nature of the Toxinometer® ET-7000 your testing capabilities can grow as you need them. Each testing module can process 16 per run with up to 11 modules connected to a single PC. This allows you to test up to 176 samples per run for each PC.
