The discovery of perhaps the horseshoe crab's
most important role in human medicine was made by Frederick
in the early
Bang discovered that the blood cells (called amoebocytes)
of the horseshoe crab contain a clotting agent that attaches
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.
|W. H. Lowell first studies coagulation of L.
polyphemus blood at Johns Hopkins University.
|Bang discovers that horseshoe crab blood forms clots when bacteria (Vibrio sp.) are present.
|Levin and Bang discover that the blood clotting agent in
horseshoe crabs is an amoebocyte.
|Scientists discover that an endotoxin causes blood-clot
reaction in horseshoe crabs. The method for preparing LAL
from horseshoe crab blood is developed.
|The standard LAL assay is developed.
|The FDA replaces the standard rabbit test for endotoxins with the LAL test.
|The FDA accepts LAL as a standard test for bacterial endotoxin contamination.
|The FDA establishes guidelines for LAL testing of pharmaceuticals and medical devices.
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.
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/
Charles River Endosafe: http://www.criver.com/products/endotoxin/index.html
Alternatives to LAL are currently being investigated in India
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.