LAL Reagents for Research Use29th April 2015
The LAL (Limulus amebocyte lysate) test is conducted in the industry for the detection of bacterial endotoxins and pyrogens in general. However, this test is not only used at the industrial level; it is also necessary to use the LAL test in research laboratories when we need to know if a sample contains bacterial endotoxins. In these cases, it is important to have reagents which can be used in different ways and which adapt to the conditions of the experiment that the researcher needs to conduct. To this end, the company "Wako" has developed a product line called "PYROSTAR™" with reagent kits and all the necessary accessories to conduct the LAL test.
The PYROSTAR™ brand includes the standard control endotoxin presented in vials which contain 500 ng of endotoxin from a strain of E.Coli each, with a defined potency of 10000 units. Once reconstituted, this endotoxin can be cold stored for a month. The standard endotoxin can be used to validate the sensitivity of the LAL reagents, as well as the detection methods which will be used in each test. Moreover, the standard control endotoxin is important in the preparation of inhibition controls.
The reagent kits of LAL division allow for endotoxin determination through the different analytical methods which are used in the detection phase of this test:
- If the detection test will be conducted through the kinetic colorimetric method, the Wako test for this purpose is called the Limulus Color KY Test, which allows for the quantitative determination of endotoxins.
- In order to use the gel-clot method as the endotoxin detection method, we have the Limulus ES-II Test of Wako. In this case, the appearance of the gel can provide a quantitative measure for the presence of endotoxins or the quantity of pyrogens present can be determined via the Turbidimetric method.
- When the Limulus ES-2 Test or the PYROSTAR™ ES-F / Plate Reagent is used, the detection of endotoxins is carried out via the kinetic turbidimetric method.
These reagent kits are sold in different formats, from 4 single-dose vials or 100 single-dose vials to multi-test vials to use in microplates and conduct the analysis of various samples at once or single-test vials, where the measurement procedure is conducted in the same tube where the reagent mix comes from. All of these include the carboxymethylated-curdlan which is added to the reagent mix to eliminate the interference of the β-1,3-glucan. β-1,3-glucan is a substance which also causes the start of the reaction chain that occurs in the hemolymph of the horseshoe crab when endotoxins are present and which makes the LAL test possible.
Furthermore, Wako offers researchers water that is free of endotoxins, pipettes free of endotoxins as well as dilution tubes. All this material is necessary to conduct the LAL test in a reliable manner. As the endotoxins could be contaminating any reusable laboratory material, the use of glassware, tubes or pipettes that are not certified to be free of endotoxins is not recommendable.
An important accessory which complements these reagent kits and laboratory material of the PYROSTAR™ line is the Toxinometer® ET-6000, which is a computerized system for reading the tubes of the LAL test. The Toxinometer® ET-6000 can be used with three detection methods: the gel-clot method, the kinetic turbidimetric test and the kinetic chromogenic method. The Gel-clot method and the kinetic turbidimetric method can offer the results within one same tube. The advantages that the use of the Toxinometer® provides is a lower risk of contamination in comparison with the microplates, the ease of software management for the technician in charge of the analysis and the fact that the Toxinometer® can be used both for single-use reagents and those that are of multiple use. Moreover, the Toxinometer® also has the possibility of starting a new test while another one is being conducted and archiving the standard curves.
1) McCullough, K. Z., Pharm. Techno., 12, 5, 1988.
2) Cohen, J., and J. S. McConnell., J. Infect. Dis., 150, 916-924, 1984.
3) Thompson, M. et al., Pure Appl. Chem., 74, 5, 835-855, 2002.
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