There are three analytical methods for the determination of bacterial endotoxins through the Limulus Amebocyte Lysate test, also known as the LAL test by its acronym. These methods are the Gel-Clot method, the turbidimetric method and the colorimetric method, which can be carried out in a qualitative or quantitative manner.
The Gel-Clot method or gelation measures the quantity of gel formed as a result of the reaction that occurs in the Amebocyte Lysate in the presence of endotoxins. In the hemolymph of the Limulus Polyphemus crab, a series of chain reactions take place as a response to endotoxins, concluding with the coagulation of the coagulant proteins. This is a reaction that can easily be observed with the formation of gel in the test tube. In order to be able to say that the LAL test has given a positive result through the Gel Clot method, the tube where the reaction has occurred is turned upside down and it is checked if the formed gel keeps separate from the mixture after this process. This way of using the Gel Clot method is qualitative and it is very useful for fast, in-situ tests, where it is necessary to know if a sample is contaminated by Gram-negative bacteria or not. There is also a possibility to apply this method in a semi-quantitative manner. Measuring the quantity of the gel formed in the reaction tube, it is possible to calculate the endotoxins in the sample.
As we have mentioned before, the reaction caused by the bacterial endotoxins in the hemolymph of the crab produces the appearance of solid proteins. Therefore, turbidity is generated in the sample. We take advantage of this fact to detect the presence of endotoxins with turbidimetry, a spectrophotometric technique with which we can obtain measurement data through the end point method or the kinetic method. The kinetic turbidimetric method is the most commonly used method in the industry to control the quality of raw materials and finished products as it is the method for the control of pyrogens currently recommended by international bodies. This method can be used in a wide variety of matrices and has the advantage of being able to measure the kinetics of the reaction. Therefore, this method is more precise than the Gel Clot method. Using the accesories and reagents marketed by the brand PYROSTAR™, we can measure various samples at the same time. The Toxinometer® ET-6000 Series allows us to read on multi-well plates in a very wide range of endotoxin concentrations, controlling the temperature at which the test is performed.
The chromogenic method applied to the Limulus Amebocyte Lysate (LAL) test is comprised of the addition of a stain developing reagent, which allows us to perform the quantification of endotoxins by measuring the absorbency of the sample. The chromophore reagent used in colorimetric LAL tests is the p-nitroaniline, which is first found in a colourless form as it is bound to a peptide. As a product of endotoxin reactions with the amebocyte lysate, the p-nitroaniline is released in a way that is proportional to the quantity of endotoxins found in the mixture. We take advantage of these processes to use the absorbency data, making a calibration curve for the calculation of endotoxin concentration. As with the turbidimetric method, the analyses can be performed with the end point method or the kinetic method, where the absorbency data of the sample within different time periods after the addition of the lysate are taken into account. As with any chromogenic method, you should be careful that the sample in the study does not present interferences in the measuring area. Any molecule that absorbs wavelengths that are close to the maximum absorption of the p-nitroaniline could modify the results of the analysis. The Limulus Color KY Test of the brand PYROSTAR™ allows for the quantitative detection of endotoxins through the colorimetric method.
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