LAL Research

The discovery of perhaps the horseshoe crab's most important role in human medicine was made by Frederick Bang in the early 1950s. Bang discovered that the blood cells (called amoebocytes) of the horseshoe crab contain a clotting agent that attaches to dangerous endotoxins produced by gram-negative bacteria.

A Bit of History

About seventy years before his discovery, in the 1880s, scientists had recognized two major families of bacteria. They were called "gram-positive" and "gram-negative" after Dr. Hans Christian Gram, who developed the staining technique that distinguished the two groups (gram-positive bacteria turn purple under the staining technique; gram-negative bacteria don't change color).

Gram-negative bacteria naturally occur in the air we breathe and in the water we drink. They are even found in our intestines! They have existed for hundreds of millions of years, just like horseshoe crabs. People have a mechanism regulated by the liver that prevents absorption of the bacteria from the gastrointestinal tract into the blood system, so under normal, healthy circumstances, gram-negative bacteria pose no threat to people.

However, if the bacteria have an opportunity to enter the blood stream, such as through trauma, they can cause high, potentially fatal fevers. Gram-negative diseases include toxic-shock syndrome, spinal meningitis, typhoid, and gonorrhea. Scientists discovered that the fevers were caused by endotoxins which are found in the cell walls of gram-negative bacteria. The term "pyrogens," meaning "burning bodies," was given to these endotoxins. The function of endotoxins is to assist in selective transport of matter into the bacterial cell. They also help to defend the bacterial cell, creating potential damage and antibodies in its host.

Back (or Forward) to Dr. Bang

Bang's studies on horseshoe crabs revealed that the amoebocyte cells in horseshoe crab blood act like a primitive immune system. When a crab is wounded, the amoebocytes swarm to the area and coagulate, forming a viscous gel surrounding the invading bacteria. Unable to escape, the bacteria are soon devoured by defense molecules such as antimicrobial proteins and polypeptides. This blood-clotting mechanism prevents infection from occurring in the horseshoe crab. Bang realized this clotting agent could be used as a fast and accurate way to test pharmaceutical drugs for the presence of gram-negative bacteria. Up until then, drugs were tested by injecting rabbits with the drug and then waiting 48 hours to see if they developed a fever. Within a few years of his initial discovery, Dr. Bang and Dr. Levin had created Limulus amoebocyte lysate, or LAL, and a new method to test for gram-negative bacteria. It was so effective that the U.S. Food and Drug Administration (FDA) accepted it as a standard test for endotoxins in 1983. Since then, LAL has gained widespread use, replacing rabbit tests for clinical and biomedical applications.

The LAL Test

LAL provides significant benefits to each of us. Have you ever had surgery? Have you ever taken an antibiotic? The FDA now requires an LAL test for injectable and intravenous drugs as well as for screening prosthetic devices such as heart valves or hip replacements. LAL is also used to diagnose spinal meningitis and other diseases.

LAL Production

In order to produce LAL, large horseshoe crabs are caught, examined for health, and bled using a stainless steel needle that is inserted into their circulatory system. The crab's blood is centrifuged to separate the amoebocytes from the liquid plasma. The amoebocytes are then freeze-dried and processed for use in the pharmaceutical industry. Horseshoe crabs are not seriously harmed during this process, and studies have indicated that bled horseshoe crabs have a relatively low mortality rate (10%). People in the LAL business carefully monitor their methods to guard their "golden goose." One quart of LAL is worth $15,000!

Three United States companies produce nearly all Limulus Amoebocyte Lysate products used worldwide. Associates of Cape Cod: http://www.acciusa.com/
Cambrex: http://www.cambrex.com/default.asp
Charles River Endosafe: http://www.criver.com/products/endotoxin/index.html

New Discoveries

Alternatives to LAL are currently being investigated in India (http://www.nio.org) and China. TAL, or Tachypleus amoebocyte lysate, functions similarly to LAL, aiding in the detection of gram-negative bacteria.

Scientists in Singapore are working to clone the toxin-detecting gene in horseshoe crab blood. If the gene can be cloned, LAL derivatives can be prepared without the harvest of horseshoe crabs for blood extraction.