Horseshoe Crab-Living Fossil

Back in time, The earliest horseshoe crab species were crawling around coastal seas for at least 100 million years before the dinosaurs even arrived (which was about 200 million years ago). Horseshoe crabs are among the world's oldest and most fascinating creatures. They are estimated to be at least 300 million years old. Thousands of other species have evolved and gone extinct, but horseshoe crabs have survived and today remain much as they were those millions of years ago, so they are truely living fossils.

HORSESHOE CRAB BLOOD

The blood of the horseshoe crab provides a valuable medical product critical to maintaining the safety of many drugs and devices used in medical care. A protein in the blood called Limulus Amebocyte Lysate (LAL) is used by pharmaceutical and medical device manufacturers to test their products for the presence of endotoxins, bacterial substances that can cause fevers and even be fatal to humans. The LAL test is one of the most important medical products derived from a marine organism to benefit humans.

Biomedical companies obtain the LAL by bleeding horseshoe crabs in a laboratory environment. The crabs are washed to remove sand and other marine debris from their exoskeletons. Those crabs without visible injuries are placed on a bleeding rack and bled by puncturing the heart with a large gauge needle. On average, 30% of the crab’s blood is removed before the wound clots naturally. The blood is placed in a centrifuge to separate the amebocytes from the blue haemolymph that comprises the supernatant. Distilled water is then added to the separated amebocytes. The added water will eventually cause the cells to burst, or lyse. Clotting proteins inside the cells are released and separated from the rest of the solution. The collected proteins are further processed into the powdered LAL product. The Horseshoe Crabs are generally returned to the water within 72 hours of bleeding.

Discovery of LAL

The white blood cells (amebocytes)of horseshoe crab clotted when exposed to endotoxins is the basis of LAL Test (Limulus Amebocyte Lysate)

Limulus -Taxonomic name of the horseshoe crab
Amebocyt-The white blood cells of the horseshoe crab
Lysate-Describes the original process used by Bang and Levin to rupture the cell membrane

In the 1970's, the LAL test was recognized by the Food and Drug Administration (FDA) as an alternative to current methods of testing for endotoxins. LAL test became an FDA required test for the presence of endotoxins in injectable drug products and implanted medical devices.

What are Endotoxins?

Endotoxins are fever inducing bacterial toxins found only in gram-negative bacteria. They are able to resist destruction by steam sterilization and removal by filtration. Endotoxins are found within the cell wall of gram-negative bacteria. As these cells grow and die, the endotoxins are released into the surrounding aquatic environment. Lipopolysaccharide is the biologically active component of endotoxins. During the late 1800 vaccinations were being developed to combat certain diseases. In many cases, people that where given these vaccines became sick with an illness called 'injection fever'. Years later, injection fever was discovered to be an immune response to the presence of endotoxins in two types of bacteria. Gram-negative bacteria is the one type commonly found in aquatic environments. It is called gram-negative because the bacteria do not become stained during the Gram staining process.

LAL Test Methods

1.The LAL Gel Clot test is simple LAL Test. Add a small amount of the sample solution being tested to a glass test tube containing the LAL solution. Next, incubate the test tube at 37 Celsius for specified time. After time has lapsed, invert the test tube and observe the reaction.

2.Turbidity Assay is a quantitative, kinetic assay for the detection of endotoxin. Test sample mixed with the reconstituted turbidity LAL reagent and Assay is carried with help of Incubating Photometer and dedicated software. Results are measured at the 340 nm wavelength. Time taken for appearance of turbiduty is inversely proportional to the amount of endotoxin present. Turbidity Assay is perfect for processing large number of samples.

3.Kinetic Chromogenic LAL test is most sensitive of all LAL tests, its performed with help of Incubation Photometer and dedicated software. Kinetic Chromogenic LAL test is less affected by inhibitory products,it is very well suitable for testing Vaccines, Antibiotics, Biological Products.

Endotoxin Detection Kits

Gel Clot LAL Assay