Potential applications of the Horseshoe Crab in the development of health products

The Horseshoe Crab from the Atlantic, commonly known as Limulus polyphemus, has existed for more than 200 million years. It has been classified under the category Arthropods since the 19th century, and not under Crustaceans as it was first thought. This close relative of Arachnids has historically been harvested for its use as a fertilizer or bait, and more recently it is used for the extraction of blood for the Limulus Amebocyte Lysate (LAL) Test, used for the detection of bacterial endotoxins in pharmaceutical products and other medical applications.

Limulus polyphemus plays a vital role in human medicine. It is a model for the study of the innate immune system. As an invertebrate that does not have immunoglobulins, the horseshoe crab has developed a unique mechanism for detecting and responding to the antigens from the microbial surface, such as Lipopolysaccharides (LPS), lipoteichoic acids, lipoproteins, peptidoglycans (PGN) and 1,3-β-glucans. This mechanism is the coagulation of the hemolymph. The blood cells of Limulus, called amebocytes, clot in the presence of antigens from the microbial surfaces mentioned above. This reaction involving the clotting of the hemolymph is precisely the biochemical principle of the Limulus Amebocyte Lysate (LAL) Test.

Though the main use of the LAL test is for the detection of LPS in pharmaceutical products, its use extends to the diagnosis of endotoxemy associated with cirrhosis, cancer, meningitis, ocular diseases, infections of the urinary tract, as well as the analysis of water quality. Today, we continue to find new applications for the LAL test, such as its use in the detection of bacterial contamination in food, including frozen products.

Wako Chemicals, a distributor of research reagents, has a blood collection installation for Limulus polyphemus, authorized by the FDA of the USAfor the unique formulation of a LAL reagentspecific to bacterial endotoxins without the interference of beta glucans in the samples. This world-renowned company includes a series of reagents and LAL accessories in its catalogue, which comply with all the norms, regulations and quality standards established by FDA under the brand PYROSTAR™.

Reagents of the PYROSTAR™ brand, such as the Limulus PS Simple Test, allow the detection and quantification of endotoxins without the interference of other coexisting substances with the use of an affinity adsorbent for endotoxins called Pyrosep™. The PYROSTAR™ ES-F series is a unique formulation containing carboximethylated Curdlan (CMC) coliophylized with LAL with the goal of removing the interference with β-glucans. Wako was the first company to use these findings through the development of an ES-buffer which contains high concentrations of CMC, converting the LAL reagent into an endotoxin detector.

In addition, researchers can find Endotoxin-free waterand Standard Control Endotoxin, Endotoxin Extractor Solution for LAL tests for the extraction of endotoxins that cannot be extracted by water or saline solutions, the ET-6000 Toxinometer with the Toximaster QC7 software, a kinetic reader, for efficient work and high-quality data analysis which can incorporate the 3 methods of measurement: gel clot, KTA (turbidimetric) and KCA (chromogenic), among other accessories free from endotoxin.

Bibliography

1) Iwanaga, S. (2007). Biochemical principle of Limulus test for detecting bacterial endotoxins. Proceedings of the Japan Academy , Series B, 83 (4), 110-119.

2) Kumar, V., Roy, S., Sahoo, A. K., & Kumar, V. (2016). Horseshoe crabs: biomedical importance and its potential use in developing health-care products. Indian Journal of Geo-Marine Sciences , 45 (10), 1234-1244.

3) Smith, D. R., Brockmann, H. J., Beekey, M. A., King, T. L., Millard, M. J., & Zaldivar-Rae, J. (2017). Conservation status of the American horseshoe crab,(Limulus polyphemus): a regional assessment. Reviews in Fish Biology and Fisheries , 1-41.

LAL REAGENT KIT

PYROSTAR™ ES-FSingle Test Vial	Limulus PS Single Test	Limulus Color KY Series