By Isabel C.
There exists a keystone species on the coasts of America, Mexico, and Asia that has some of the most valuable blood in the medical world. Since the late 1970s these creatures have been captured and lugged to laboratories where they are safely bled for several hundred milliliters of blood. These are horseshoe crabs, ancient and oddly-shaped creatures possessing blue blood and a unique defense against contaminants. Their diet of clams, crustaceans, and worms exposes them to heavy concentrations of bacteria containing plenty of endotoxins, common contaminants carried in bacteria that can cause a slew of health issues. Horseshoe crabs would be affected if not for the presence of a compound called Limulus Amebocyte Lysate, or LAL, in their amebocytes. It has the ability to form a clot around bacteria in the crab’s bloodstream, completely encapsulating it and protecting against the bacteria’s endotoxins. For the past four decades, this reaction to bacteria has helped manufacture safe vaccines and is continuing to be useful during the rush for COVID-19 vaccinations.
The FDA requires that every drug must be tested for contaminants before getting approved. Drugs used to be tested using live rabbits, but horseshoe crabs beat that method in speed, ease, and ethics. The endotoxin-rich habitat of the horseshoe crab is the reason for its remarkable ability, an ability that can easily be made into a purity test to ward off endotoxins from our own medicine. Endotoxins are particularly dangerous contaminants as they can cause a high fever and toxic shock if injected into someone’s bloodstream. There is no doubt that there has to be a method of detecting and removing even the smallest amount of endotoxins in an injectable drug, and LAL in horseshoe crab blood happens to be the perfect way to do this.
The science behind the coagulation involves the actions of amebocytes, cells which flow freely through a horseshoe crab’s blood and are especially sensitive to molecules present on endotoxins. Any invading bacteria in the blood is immediately surrounded by a gel-like clot from the amebocytes and blocked off from the rest of the circulatory system. The bacteria is not killed, but rather locked up in a clot and unable to do any harm. Amebocytes are so sensitive to the presence of bacteria that they form clots around even the smallest concentrations. The clots last for multiple weeks and take a short time to form in the presence of bacteria, putting an immediate stop to a risk of infection. This has formed the basis for the LAL test where a small amount of lysate from a horseshoe crab’s amebocytes is used to test for vaccine contamination. Based on whether clots form when LAL is added to a vaccine, it’s quickly known whether it can safely be injected or not.
Just as we do not want endotoxins in our bloodstream, neither do horseshoe crabs. Their exoskeleton, however, is not much of a barrier to bacteria and leaves some tissues unprotected--bacteria inevitably find a way in. This is why amebocytes had to devise a way of forming clots around harmful bacteria by sensing the presence of endotoxins. All four species of horseshoe crab are regularly captured, taken to labs, and bled for around 200 or 400 mL of their blood, a process that usually takes 10 minutes or less. From there, LAL is extracted from the blood sample for medical use. As we look forward to safe and effective COVID-19 vaccines, the horseshoe crab’s blood will be all the more valuable in making sure we can quickly produce uncontaminated vaccines.
Unfortunately, there is a downside to this collection of blood; it has whittled down the horseshoe crab population and made surviving crabs much more sluggish and less likely to reproduce relative to unbled crabs. However, recombinant factor C, or rFC, is a synthetic alternative to LAL made from the genes of horseshoe crabs. Several studies have shown that tests using rFC are just as effective as the LAL test, if not more. rFC molecules can bind to more endotoxins than LAL and are thus more sensitive. Rather than relying on clotting, rFC tests involve binding endotoxins to rFC molecules, which produces a fluorescent compound that is measured after a test is complete; final endotoxin results are measured from this fluorescence. rFC tests are also more reliable and consistent than LAL tests, since the latter can run into false-positive and false-negative results for contamination. This is because horseshoe crabs’ amebocytes are not just reactive around endotoxins, but rather a variety of things--even simple polysaccharides can trigger false-positive results. This issue has yet to be reported with rFC, which is specific to targeting endotoxins.
If rFC tests were to become the new industrial standard, it would be much cheaper and environmentally friendly to test the safety of vaccines and other drugs. There is already the allure of reducing harm to horseshoe crabs while keeping the remarkable abilities of their blood in use, and rFC tests provide just that. In the meantime, rFC has been used in tests involving COVID-19 antibodies and has proven to be just as reliable as an LAL test in checking for purity.
“What makes a horseshoe crab’s blood so special?”
Horseshoe crabs have a unique diet that requires them to wade through bacteria-ridden sludge quite often. Their exoskeletons don’t heavily protect their tissues that come into direct contact with their habitat, leaving their bloodstream wide open to endotoxin-carrying bacteria. The crabs naturally developed a way to defend themselves using amebocytes in their blood that form clots around contaminants and prevent them from causing harm in the crab’s circulatory system. This is essentially what happens during vaccine testing--a bit of LAL is used to test for endotoxins in vaccines, which will be revealed through the formation of clots.
“How does the LAL test compare to rFC tests?”
The FDA requires that all drugs go through the LAL test before getting approved since it’s such an effective way of checking for contamination. However, rFC offers an alternative way to test without having to bleed horseshoe crabs or spend as much money--it is also much better for the horseshoe crab population. rFC tests are more reliable than LAL tests and don’t carry the same false-positive and false-negative risks. The rFC molecule is specifically targeting endotoxins and works in much the same way as LAL, so there are many advocates for rFC to become the new industrial standard.