The Limulus Amebocyte Lysate Test (LAL) is a method for the detection and quantification of bacterial endotoxins that is widely used by the pharmaceutical and biotechnology industry. The biochemical principle of the test is based on the clotting reaction of the hemolymph of the Limulus polyphemus, commonly known as the Atlantic Horseshoe Crab, in the presence of antigens with microbial surface, such as Lipopolysaccharides or bacterial endotoxins.
The company “Wako Chemicals” is among the companies that manufacture LAL reagents at the international level due to its unique formula of a LAL reagent specific to bacterial endotoxins without the interference of beta glucans in the samples, thanks to the addition of carboximethylated Curdlan (CMC) coliophylized with LAL. Under the brand PYROSTAR™, this distributor company of research reagents manufactures and markets LAL reagents and accessories that comply with all the norms, regulations and quality standards established by the FDA from the USA.
We are now going to talk about the different reagents and accessories with which Wako contributes to the study of bacterial endotoxins:
First, we have the Limulus PS Simple Test kit, which allows for the detection and quantification of endotoxins without the interference of other coexisting substances with the use of an affinity adsorbent for endotoxins called Pyrosep™. With this method, we can measure the endotoxin in a lipophilic sample provided that it can be dissolved in ethanol, such as liposoluble vitamins, oils and fats, albumins, etc. which cannot normally be measured with a conventional Limulus test. Moreover, no LAL reagent is wasted as it is applied individually in vials of the simple test and can be used in the exact quantity required for the measurement.
The PYROSTAR™ ES-F series is a unique formulation containing carboximethylated Curdlan (CMC) coliophylized with LAL with the goal of removing the interference with β-glucans. The ES-buffer developed by Wako contains high CMC concentrations, turning into an endotoxin-specific LAL reagent. This reagent is designed to be used both in the qualitative detection of endotoxins through the gel-clot method and for their quantitative detection through the kinetic turbidimetric methods (KTA). A photometric instrument called the Toxinometer®, developed by Wako Pure Chemical Industries, Ltd. is used for measuring the speed of turbidity change. The kits PYROSTAR™ ES-F are available with and without the Standard Control Endotoxin (SCE): a lyophilized reagent refined from E.coli and used to confirm the sensitivity of the LAL reagent, validate the product test methods and prepare inhibition controls.
To support these reagents, Wako is also developing an Endotoxin Extracting Solution capable of extracting endotoxins from surfaces where they cannot be extracted with water or salt. Its use is recommended in endotoxin samples on equipment and devices that could be in contact with liquids containing blood or proteins.
Toxinometer® ET-6000 is a computerized kinetic incubation reader which can incorporate 3 test methods: gel-clot, KTA and KCA (kinetic chromogenic test). This equipment allows us to obtain gel-clot and KTA results from the same sample. It simultaneously measures up to 16 samples with a contamination risk that is much lower than the microplate and allows us to archive the standard curves. Toximaster® QC7 is the software that provides all the functions for data processing and report preparation for the LAL test with the Toxinometer® ET-6000.
Moreover, among the accessories from Wako’s LAL catalogues, we also see test tubes with and without aluminium lids. These tubes are produced free from endotoxins, made of borosilicate glass and designed for the gel-clot method. The glass thickness can be specifically calibrated to the Toxinometer for stable light intensity and transmittance.
Another LAL reagent marketed by Wako is the KY Color Limulus kit. This reagent has been designed for the KCA method and can be used both in the kinetic reader and in a microplate reader. It includes a multi-test kit and a simple test kit, each designed for a colorimetric analysis based on time, using a synthetic substrate which generates a yellow color and which can specifically detect the endotoxin with high sensitivity.
1) Iwanaga, S. Biochemical principle of Limulus test for detecting bacterial endotoxins. Proceedings of the Japan Academy, Series B, 83(4), 110-119. (2007).
2) Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test of Human and Animal Parenteral Drugs, Biological Products and Medical Devices. U.S. Dept. of Health & Human Services, FDA, December (1987).
3) Katsuhito Inoue, Aya Takaoka, Masakazu Tsuchiya, Shuji Matsuura: Excerpt 110 (1992), 6th Annual Meeting of the Japanese Society for Alternatives to Animal Experiments.
4) Katsuhito Inoue, Tomoaki Harada, Masakazu Tsuchiya, Shuji Matsuura: Excerpt 44 (1994), 21st Annual Meeting of the Society for Antibacterial and Antifungal Agents.
5) Tsuchiya, M.; Oishi, H.; Takaoka, A.; Fusamoto, M. y Matsuura, S. Discrimination between endotoxin and (1–3) beta-D-glucan using turbidimetric kinetic assay with Limulus amebocyte lysate. Chem Pahrm Bull (Tokyo), 38(9). 2523. (1990).
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