Limulus Amebocyte Lysate (LAL) Assay
Unlike RPT, LAL assay developed in the 1960s does not involve live test subjects. It does, however, rely on an extract from the blood of theLimulus polyphemus species of horseshoe crab 52,53. The extract is used in one of three ways. First and simplest, the gel-clot. This test involves mixing equal parts of extract and sample. If a gel has formed and the mixture remains intact in the bottom of the tube, the test shows positive 54. This means the sample has at least enough endotoxins to trigger a positive reaction, the limit of this being around the range of 0.03 EU/mL or 0.003 to 0.006 ng/ml. The other two methods are turbidimetric and chromogenic methods. Both are referred to as photometric tests as they require an optical reader for analysis. The chromogenic assay is performed by replacing a natural substrate, Coagulen, with a chromogenic, or colored one. The chromogenic substrate is cleaved by an endotoxin-activated enzyme coagulase, and the chromogenic molecule is released from the substrate into the suspension measured by spectrophotometry 55. The turbidimetric method is similar to the chromogenic method, but instead measures the turbidity of the solution 56. The rate of turbidity and absorbance (color change) are proportional to the endotoxin concentration. All three tests rely on the same protein, Factor C coagulation cascade found in horseshoe crabs’ blood (Figure 3 ). The endotoxin activates Factor C which goes onto activate Factor B following the formation of a clotting enzyme 57,58. In gel clot and turbidity assays, the clotting enzyme transforms Coagulen into Coagulin, creating the gel in the gel clot test, as well as the clouding agent in the turbidity test. The Chromogenic method follows the same pathway, but instead of using Coagulen, it uses a complex of amino acids and p-nitroaniline (pNA), as the chromogenic factor. The enzyme trims the pNA off of the complex, turning the suspension a yellow color. This color is too faint to discern by the naked eye so a spectrometer must be used 23. These tests are widely accepted as the official endotoxin test in the pharmaceutical community 59. Every drug and medical device that is tested by the US Food and Drug Administration (FDA) must undergo and pass a LAL test 60,61. As previously mentioned, this method is much more accurate than RPT, particularly the photometric methods. This method still has its drawbacks. For example, the protein cascade it relies on is disrupted in samples with free metal ions, and similarly to RPT, the method is subject to the same public outcry for its treatment of horseshoe crabs. While the phlebotomy itself is not fatal, an approximated 20% of the crabs fail to survive after being returned to sea 23. Following the discovery of Factor C as endotoxin-activated portion of the protein cascade, attempts have been made to replace the conventional LAL test, with one using recombinant Factor C 62. As technology improves, alternative techniques are being developed to ease the pressure on the horseshoe crab population.