THE LIMULUS AMEBOCYTE LYSATE TEST
Limulus Amebocyte Lysate (LAL) tests detect and quantify bacterial endotoxins extracted from the outer membrane of gram negative bacteria. The critical component of the LAL reagents used in endotoxin tests is derived from blood cells (amebocytes) of the horseshoe crab, Limulus Polyphemus. It contains the proteins of the blood clotting mechanism, which is triggered by endotoxins. LAL reagents are primarily used to test for endotoxins in injectable pharmaceuticals, biological products, and medical devices. They are also used in renal dialysis centers and a wide range of other applications.
Principle: In invertebrates, the amebocytes fulfil the function of white blood cells in vertebrates. They defend the organism against pathogens; therefore, they respond to the influx of endotoxins from the bacteria by releasing a series of enzymes. The amebocytes contain procoagulant enzymes (LAL enzymes ) that trigger a chain of reactions. The final product of these chain reactions is a gel comprised by coagulated proteins . If a lysate of the amebocytes extracted from the Horseshoe Crab in a watery environment is diluted, this could serve to detect very small quantities of endotoxins.
One of the main advantages of the LAL test that the crabs, from which the hemolymph is extracted to prepare the LAL reagent, remain alive. The LAL test can be conducted by using different methods to measure the process of gelation that occurs as a response from the amebocytes against the endotoxins. These methods are the so-called Gel-Clot method, turbidimetric and chromogenic methods. These test is approved for endotoxin testing of multiple therapeutic types including cell therapy, recombinant protein, monoclonal antibody, vaccines and gene therapy. The choice comes down to whether you need quantitative results or simply to detect endotoxin in your drug.
GEL CLOT ASSAY
The Gel-Clot method is based on the presence or absence of a gel clot in your sample tube. The gelation occurs when proteins are coagulated due to the presence of endotoxins. The detection limit is normally between 0.01 and 0.03 endotoxin units per one millilitre of the solution used in the test. TURBIDIMETRIC TESTING
Microtest turbidimetric testing aims to detect endotoxin found on medical devices or in drugs. Turbidity testing determines the cloudiness of a solution by measuring the loss of intensity in a light beam passing through that solution. Turbidimetric testing’s lowest limit of detection (LLD) is 0.005 EU/mL
CHROMOGENIC LIMULUS AMEBOCYTE LYSATE
The Chromogenic Limulus Amebocyte Lysate (LAL) Test is a quantitative test for gram-negative bacterial endotoxin. The natural substrate, coagulogen, is replaced by a chromogenic substrate. On cleavage of this substrate a chromophore is released from the chromogenic peptide and is measured by spectrophotometry. at 405-410 nm. If endotoxin is present in the sample, a yellow color will develop . The sensitivity of this assay is 0.10 EU/ml.
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-Dixy