About Wako’s LAL Test Reagents
As a world-renowned supplier of high purity chemicals and reagents, Wako Chemicals continues to maintain our proud history of quality and service through the establishment of the LAL Division and the introduction of our new PYROSTAR™ ES-F line of LAL test reagents.
The LAL assay is the most sensitive method for the detection of bacterial endotoxins currently approved by the FDA.2 The first methodology used to determine the LAL test results was the formation of a gel-clot in the bottom of a glass reaction tube. It has also been observed that the test solution becomes turbid prior to gel-formation. The time required to produce a specified level of turbidity is inversely proportional to the amount of endotoxin in a sample.1
A photometric instrument such as the Microplate Reader (Tecan Sunrise™ Microplate Reader or equivalent) is used to measure the rate of turbidity change. This quantitative measurement procedure is often referred to as the Kinetic Turbidimetric Assay (KTA).
By utilizing these properties, Wako Chemicals USA has developed an LAL endotoxin test that can be used as a quantitative turbidimetric test.
The USP Bacterial Endotoxins Test <85>6 provides standardized procedures for validation prior to routine use.
However, the specificity of LAL is not absolute.7 It has been reported that LAL reacts not only with endotoxin by also with β-1,3-glucan. Although the cascade system activated by β-1,3-glucan has been shown to be different than the one activated by endotoxin8, the end result, gel-clot formation is indistinguishable.
The activation of LAL by glucan in a sample can be prevented by adding a large amount of carboxymethylated curdlan (CMC) to LAL. The presence of large amounts of glucan does not interfere with the quantitation of endotoxin. Wako first made use of these findings by developing an ES-buffer, which contains high concentrations of CMC. When the ES-buffer is used to reconstitute LAL, the LAL reagent becomes endotoxin-specific. PYROSTAR™ ES-F is a new preparation of LAL in which CM-curdlan is colyophilized with LAL. Reconstituting PYROSTAR™ ES-F test vials with LAL Reagent Water results in an endotoxin-specific LAL reagent.
PYROSTAR® ES-F, our formulation of Limulus Amebocyte Lysate, is unlike any other LAL product available in the market today. Our reagent is designed for the specific determination of gram negative bacterial endotoxins, without interference from the presence of beta glucans, and has the unique capability to be used in either quantitative turbidimetric (KTA) or qualitative gel-clot test methodologies.
- Kit and Reagent Approved by US-FDA.
- High Sensitive Endotoxin Specific Detection without the interference of (1 → 3)-β-D-glucan.
- Labeled Gelation Sensitivity (0.015EU/mL) measured by USP Reference Standard Endotoxin.
- Highly sensitive analysis (0.001 EU/mL) using only 50 µl of LAL and Sample.
- Firm Gel Formation
- Gel-Clot sensitivity from 0.015 to .25 EU/mL
- Pyrostar ES-F/Plate is kitted with the 0.015 EU/mL sensitivity to range down to 0.001 EU/mL KTA
|Sensitivity of Gel-Clot Reagent (EU/mL)||Range of quantitative KTA (EU/mL)|
|0.015||0.001 to 10|
|0.03 to 0.25||0.01 to 10|
Limulus PS Single Test
- Endotoxin specific measurement can be performed with small amounts, avoiding any interference from coexisting substances, by adsorbing endotoxin to Pyrosep™ in the sample and condensing it.
- Since the endotoxin is adsorbed to Pyrosep™ even in ethanol, a fat-soluble sample can be tested provided it dissolves in ethanol. Such samples are not usually processed in ordinary limulus tests. (Example: fat-soluble vitamins, oils and fats)
- No LAL reagent is wasted, since it is pre-dispensed individually in vials, and can be used in the quantity required for measurement
For details on endotoxin testing in the United States (US),
users should consult the current revision of the
United States Pharmacopia (USP), chapter <85>, “Bacterial Endotoxins Test.