In 1970 the FDA approved LAL for testing drugs, products and devices that come in contact with the blood. Prior to that date, much slower and more expensive tests on rabbits had been used for this purpose.
Blood is removed from the horseshoe crab's pericardium; the crabs are returned to the water and recover fully. The blood cells are separated from the serum using centrifugation and are then placed in distilled water, which causes them to swell up and burst ("lyse"). This releases the chemicals from the inside of the cell (the "lysate"), which is then purified and freeze-dried. To test a sample for endotoxins, it is mixed with lysate and water; endotoxins are present if coagulation occurs.
There are three basic LAL test methodologies: gel-clot, turbidimetric, and chromogenic. The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products
The LAL cascade is also triggered by (1,3)-β-D-glucan. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns", or PAMPS, substances which elicit inflammatory responses in mammals.
One of the most time consuming aspects of endotoxin testing using LAL is pretreating samples to overcome assay inhibition and enhancement. Agents such as EDTA and heparin are known to affect the assay if they are present in sufficient concentrations. All assays, independent of methodology are standardized using endotoxin in water. Therefore, unless your sample is water, some components of the solution may interfere with the LAL test such that the recovery of endotoxin is affected. If the product being tested causes the endotoxin recovery to be less than expected, the product is inhibitory to the LAL test. Products which cause higher than expected values are enhancing. Overcoming the inhibition and enhancement properties of a product is required by the FDA as part of the validation of the LAL test for use in the final release testing of injectables and medical devices. Proper endotoxin recovery must be proven before LAL can be used to release product.