Saving the Horseshoe Crab Blood: Alternative Testing Methods for Endotoxin Detection

The horseshoe crab plays an important role in safely manufacturing vaccines and other injectable medications. However, extensive use of horseshoe crabs in biomedical testing has caused a decline in the populations of all four species of these animals around the world.

Importance of Horseshoe Crab in the Biomedical Industry

In the U.S., every drug must be tested for bacterial contaminants like endotoxins to be approved by the Food and Drug Industry. Endotoxins, the potentially dangerous contaminants in the cell wall of gram-negative bacteria, can cause life-threatening conditions if introduced into the human body. This makes endotoxin test an essential step in safely manufacturing injectable medications.

From the 1940s to 1970s, the pharmaceutical industry relied on rabbits for endotoxin detection, causing thousands of rabbits to be euthanized yearly. By 1970, experts had developed a new technique using horseshoe crab blood, which was found to have a highly sensitive immune response to endotoxin contamination.

The primitive animal has a defense system expressed in the amebocyte cells, which are extremely sensitive to the lipopolysaccharide (LPS) found in endotoxins. The reaction between the amebocyte and endotoxins is the basis for the Limulus amebocyte lysate (LAL) test, the current standard for endotoxin testing worldwide.

In carrying out the LAL test, horseshoe crabs are captured and bled. Their blood cells undergo centrifugation and are lysed to release the enzymes responsible for endotoxin recognition. To test a sample for the presence of endotoxins, it is mixed with the lysate at a specified ratio to create a gel clot.

This test can be carried out using any of the four extant species of horseshoe crabs: Tachypleus tridentatus, Tachypleus gigas, Carcinoscorpius rotundicauda, and Limulus polyphemus. Hundreds of thousands of horseshoe crabs are captured each year to drain as much as a third of their blood. The bled crabs are sold as bait for other fisheries while the rest are returned to the ocean.

However, being removed from the water and undergoing the bleeding process cause injury and potential death on horseshoe crabs. The declining population of these animals can also affect a subspecies of birds called the red knot, which rely on their eggs for food.

Alternative Endotoxin Testing Methods

Companies willing to practice sustainable, animal-friendly approaches in endotoxin detection can use alternative methods. One of these methods is recombinant factor C (rFC) derived from the brackish water arthropod.

In this method, the DNA for one of the clotting factors, Factor C, is cloned and manufactured recombinantly or synthetically. Just like the native Factor C, the recombinant factor C is activated by endotoxin.

Another technique that does not need horseshoe crab blood is the Monocyte Activation Test (MAT) or the In Vitro Pyrogen test. In this method, the cytokines released from blood cells due to the presence of pyrogenic substances like endotoxins are measured in the test sample. It mimics what occurs in our bloodstream upon exposure to pyrogens.