The American Association of Immunologists - Blue Bloods: Bang, Levin, and the Horseshoe Crabs

Blue Bloods

by Charles Richter and John S. Emrich
October 2022

For almost half a billion years, this planet has been home to a strange creature that looks like something from another world and whose blood has become a precious commodity. Horseshoe crabs, arthropods that comprise the family Limulidae, are shallow-water animals protected by a round carapace about a foot across that covers most of their body, with a long, spiny tail dragging behind. Flip them over, and you’ll find 10 legs and a gill assembly.

Inside these odd animals is a circulatory fluid that has proven invaluable for pharmaceutical testing for decades—recently, by ensuring the purity of the COVID-19 vaccines.

First Looks at the Horseshoe Crab

At the turn of the 20th century, when scientists began investigating the blood of Limulus polyphemus, the Atlantic horseshoe crab, some of the earliest research was stymied by the blood’s propensity for quick coagulation. In 1908, Carl Alsberg and Ernest Clark decided to study the clotting mechanism itself. They found that the clot was formed of a substance they called cell fibrin, whose chemical composition was initially perplexing. It was practically insoluble in all but the most caustic alkaline solutions.

The chemical that makes horseshoe crabs’ blood blue is hemocyanin, a copper-protein, oxygen-transporting compound also found in many crustaceans and mollusks. Unlike hemoglobin, which is a component of the red blood cells of vertebrates, hemocyanin is an extracellular compound floating freely in the circulatory system.

Alsberg and Clark had access to abundant populations of Atlantic horseshoe crabs at the U.S. Bureau of Fisheries Woods Hole Marine Biological Laboratory (MBL) on Cape Cod, Massachusetts. In the early summer, typically during spring neap tide, countless horseshoe crabs crawl onto the beach to spawn and nest, leaving millions of tiny eggs in the sand. Juvenile crabs spend their first two years in the shallow intertidal waters before moving into deeper water until it is time to spawn.

The researchers could get up to 400 cc of blood out of a single crab by pumping the two halves of its body “like opening and closing a bellows.” Comparative studies quickly showed that the hemocyanin of Limulus was distinct from that of other invertebrates such as octopus. Alsberg eventually demonstrated that aside from hemocyanin and the clotting protein, there was almost no other protein matter in the blue blood.

Limulus in The JI

After Karl Landsteiner (AAI 1922, president 1927–28) described human blood groups in 1901, comparative studies of the blood of various species filled biomedical journals, including The Journal of Immunology (The JI), and Limulus blood was not left out. The first mention of horseshoe crabs in The JI appeared in 1920, when Carl Schmidt showed that hemocyanin, unlike hemoglobin, is antigenic in mammals. Although Schmidt was working with hemocyanin derived from abalones, he interpreted earlier research with an immunological perspective, identifying the Limulus hemocyanin reaction seen by Alsberg and Clark as globulin-like behavior.

Bang’s Discovery

In 1953, a researcher from Johns Hopkins University was spending a summer at the MBL studying horseshoe crabs, just as Alsberg and Clark had decades before. Frederick Bang (AAI 1953) noticed a strange reaction in the crabs’ already unusual clotting mechanism. It was known that the blood of horseshoe crabs has the ability to form a quick clot at the site of injury in response to the presence of foreign bacteria, but Bang observed an out-of-control clotting reaction that solidified nearly the entire circulatory system into a gel. He cultured a Gram-negative bacterium from the first crab and verified that it provoked the same reaction in other crabs, even when killed. His findings were quietly published in The Biological Bulletin and a few years later in The Bulletin of Johns Hopkins Hospital, but rather than continuing to pursue this line of research, Bang went back to his previous work on Rous sarcoma virus at the end of the summer.

A decade later, Bang returned to that odd coagulation he found in Limulus when a colleague at Hopkins recommended that he work with a young hematology research fellow, Jack Levin, who was at the time researching how blood clots formed in rabbits in response to bacterial endotoxins. Bang took Levin to the MBL for another summer with the horseshoe crabs.

Levin’s Development

At Woods Hole, Levin showed that in order for Limulus blood to clot from bacterial exposure, the presence of amebocytes—the only cellular component of the blood—was required. However, his samples of whole blood kept coagulating even in the absence of any known bacteria. He considered the possibility of endotoxin contamination. Levin ran his experiment again using glassware that had been sterilized at a temperature high enough to destroy endotoxins.

When the new samples did not clot, Levin had his “aha moment.” He knew that the blood of the horseshoe crab had to be particularly sensitive to endotoxins.

Further explorations confirmed the endotoxin reaction and narrowed the clotting mechanism down to specific enzymes held by granules within the amebocyte cells. Levin and Bang first published these findings in 1964. The pair also presented their findings at the 1966 Federation of American Societies for Experimental Biology meeting.

The LAL Test

Levin recognized that this discovery had enormous potential as a reliable, sensitive, and rapid test for the presence of endotoxins in pharmaceuticals. In the 1960s, the only way to test a batch of injectable drugs for endotoxin contamination was the rabbit pyrogen test, which required injecting a rabbit with a sample and waiting 4–6 hours to see if it developed a fever.

The Limulus amebocyte lysate (LAL) assay that Levin created can detect endotoxins at a concentration of one part per trillion, and in only 45 minutes. Furthermore, the rate of production of the clot is proportional to the endotoxin’s concentration, so the test can indicate to what degree a sample is contaminated. Best of all for the horseshoe crabs, the blood can be drawn non-lethally, and they can be released back into the ocean after their donation.

FDA Approval

It was not until 1983 that the LAL was fully approved by the U.S. Food and Drug Administration as a finished product test. Concerns about the sustainability of horseshoe crabs delayed approval for several years as marine biologists debated whether the species could be used responsibly as a natural resource. The potential application of the LAL prompted an increase in research on the behavior and ecology of horseshoe crabs so that scientific advances would “not endanger the elegantly adapted species and bring its long story to an end.”

When testing vaccines, LAL is used at multiple steps in the production process to ensure that the containers, stoppers, and ingredients are free of endotoxins before the finished product is tested. LAL is also instrumental in assuring the safety of injectable drugs, implantable medical devices, and IV fluids.

Conservation

Medical use of Limulus was not the first impact to the horseshoe crab population. Beginning in the 1850s and continuing into the 1940s, Americans harvested over a million horseshoe crabs per year for bait, fertilizer, and livestock feed. Although there was no baseline population data to compare, this level of predation likely had a significant impact on the overall population, prompting wildlife authorities to consider conservation measures. Horseshoe crabs can live close to 20 years and take seven to nine years to reach sexual maturity. When they are taken as they crowd the beaches during a spawn, fewer eggs may be laid, reducing the size of the next generation.

Today, around half a million horseshoe crabs are captured for blood collection and returned to their ocean habitat, with a survival rate of 85–90%. Some crabs that are harvested for bait production are bled for the biomedical industry before processing, which has effectively increased the quota of blood available for production of LAL. The Atlantic States Marine Fisheries Commission monitors the estimated population and recommends limits on capture and harvest each year, and as of 2019, has determined that biomedical use has had no impact on the population.

Bang’s Legacy

Fred Bang, who made the original discovery that led to the LAL assay, died in 1981. Five years later, his widow, Betsy Bang, bequeathed $50,000 to AAI to establish the Frederick B. Bang Scholarships. This gift provided for “support of scholarly research in the science of marine invertebrate immunology” through awards of up to $10,000 per year through 1994, when the fund was exhausted.

The LAL assay is still used today in clinical settings and to test drugs and vaccines for the presence of dangerous endotoxins before they are released. It has been an essential part of making sure that the COVID-19 vaccines are free from contamination. One manufacturer estimated that one day’s production of LAL at all U.S. facilities would be enough to test five billion doses of COVID-19 vaccine.

Bang could not have guessed the impact his discovery would have on the world when he first noticed the odd clotting in the blood of the horseshoe crab. Although he and Levin never received a major scientific award for their work, they were finally recognized in 2019 with the Golden Goose Award by the American Association for the Advancement of Science, a prize designed to encourage basic science funding by highlighting “seemingly obscure studies that have led to major breakthroughs and resulted in significant societal impact.”

References

Alsberg, Carl L. “Notes on the Properties of the Blood of Limulus polyphemus.” Journal of Biological Chemistry 19, no. 1.
Alsberg, Carl L. and Ernest D. Clark. “The Blood Clot of Limulus polyphemus.“ Journal of Biological Chemistry 5, no 4.
Alsberg, Carl L. and Ernest D. Clark. “The Haemocyanin of Limulus polyphemus.” Journal of Biological Chemistry 8, no. 1.
Alsberg, Carl L. and William M. Clark. “The Solubility of Oxygen in the Serum of Limulus polyphemus L. and in Solutions of Pure Limulus Haemocyanin.” Journal of Biological Chemistry 19, no 4.
Bang, Frederick B. “A Bacterial Disease of Limulus polyphemus.” Bulletin of Johns Hopkins Hospital 98, no. 5.
Bang, Frederick B. and J. L. Frost. “The Toxic Effect of a Marine Bacterium on Limulus and the Formation of Blood Clots.” The Biological Bulletin 105, no 2.
Food and Drug Administration. Guide for Industry—Pyrogen and Endotoxins Testing: Questions and Answers. June 2012.
Gorman, James. “Tests for Coronavirus Vaccine Need This Ingredient: Horseshoe Crabs.” New York Times. June 3, 2020.
Hoffmeister, Brett, et al. “COVID-19 and the Sustainability of the LAL Supply.” PDA Letter (Parenteral Drug Association), April 22, 2021.
Levin, Jack and Frederick B. Bang. “The role of endotoxin in the extracellular coagulation of Limulus blood.” Bulletin of the Johns Hopkins Hospital 115.
Levin, Jack and Frederick B. Bang. “Clottable Protein in Limulus: Its Localization and Kinetics of Its Coagulation by Endotoxin.” Thrombosis et Diathesis Haemorrhagica 19, no. 1.
Ravven, Wallace. “How a Study of Horseshoe Crabs Led to Safer Joint Replacements.” University of California San Francisco, accessed June 27, 2022, www.ucsf.edu/news/2020/01/416386/how-study-horseshoe-crabs-led-safer-joint-replacements.
Rudloe, Anne. “Limulus polyphemus: A review of the ecologically significant literature.” in Progress in Clinical and Biological Research, Vol. 29, Biomedical Applications of the Horseshoe Crab (Limulidae), eds. Elias Cohen, et. al. (New York: Alan R. Liss, Inc.). (This volume comprised the proceedings of a symposium held at the Marine Biological Laboratory, Woods Hole, Massachusetts, October, 1978).
Schmidt, Carl L. A. “The Antigenic Properties of Hemocyanin.” The Journal of Immunology 5, no. 3.

“2019: The Blood of the Horseshoe Crab.” American Association for the Advancement of Science, https://www.goldengooseaward.org/01awardees/horseshoe-crab-blood (accessed June 28, 2022).
“About the Golden Goose Award,” American Association for the Advancement of Science, https://www.goldengooseaward.org/history (accessed July 7, 2022).
“Conservation.” The Ecological Research & Development Group, https://www.horseshoecrab.org/info/conservation.html (accessed June 27, 2022).
“Deed of Gift.” Box 1, Folder 2, Council: Executive Director/Correspondence (Bang Scholarship). The American Association of Immunologists Collection, University of Maryland, Baltimore County; Arthur M. Silverstein to Raymond Palmer, March 23, 1994, in AAI Council Book, Spring 1994, AAI Archive, Rockville, MD.