Horseshoe crab

A preferred method for the detection of pyrogens is the limulus amebocyte lysate (LAL) test. A test sample is incubated with amebocyte lysate from the blood of the horseshoe crab, Limulus polyphemus. A pyrogenic substance will cause a gel to form. This is a result of the... 

Another issue of relevance is that certain biopharmaceuticals (e.g. cytokines such as 1L-1 and TNF Chapter 9) themselves induce a natural pyrogenic response. This rules out use of the rabbit-based assay for detection of exogenous pyrogens in such products. Such difficulties have led to the increased use of an in vitro assay the Limulus ameobocyte lysate (LAL) test. This is based upon endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs. This test is now the most widely used assay for the detection of endotoxins in biopharmaceutical and other pharmaceutical preparations. 

Development of the LAL assay was based upon the observation that the presence of Gramnegative bacteria in the vascular system of the American horseshoe crab, Limulus polyphemus, resulted in the clotting of its blood. Tests on fractionated blood showed that the factor responsible for coagulation resided within the crab s circulating blood cells, i.e. the amoebocytes. Further research revealed that the bacterial agent responsible of initiation of clot formation was endotoxin. 

Figure 7.8 Activation of clot formation by endotoxin. The presence of endotoxin causes stepwise, sequential activation of various clotting factors present naturally within the amoebocytes of the American horseshoe crab. The net result is the generation of the polypeptide fragment coagulin, which polymerizes, thus forming a gel or clot...

Botton, M.L., K. Johnson, and L. Helleby. 1998. Effects of copper and zinc on embryos and larvae of the horseshoe crab, Limulus polyphemus. Arch. Environ. Contam. Toxicol. 35 25-32. 

Itow, T., R.E. Loveland, and M.L. Botton. 1998. Developmental abnormalities in horseshoe crab embryos caused by exposure to heavy metals. Arch. Environ. Contam. Toxicol. 35 33-40. 

Clapper, D.L., PL. Lamothe, J.A. Davis, and D. Epel. 1985b. Sperm motility in the horseshoe crab. V zinc removal mediates chelator initiation of motility. Jour. Exper. Zool. 236 83-91. 

ARACHNOIDS Horseshoe crab, Limulus polyphemus Molting and survival adversely affected during 5-day exposure 11... 

Weis, J.S. and A. Ma. 1987. Effects of the pesticide diflubenzuron on larval horseshoe crabs, Limulus polyphemus. Bull. Environ. Contam. Toxicol. 39 224-228. 

Limulus Amebocyte Eysate (EAE) Test This test is used to detect the presence of endotoxins in the drug substance. It relies on the coagulation reaction between the endotoxin and the blood of a horseshoe crab. 

Limnlus amebocyte lysate A reagent for determining the quantity of bacterial endotoxins. It is obtained from the aqueous extracts of circulating amebocytes of the horseshoe crab. 

By far the most definitive study on an arthropod hemocyanin has been that by Volbeda and Hoi (1989b), a crystallographic tour de force. Crystals of subunit b can be formed from solutions of native hemocyanin which contain three types of subunits (a, b, and c). Two subunits (a and b) are nearly identical (3% difference in sequence), whereas subunit c differs more. Subunits a and b are glycosylated at a single residue (Asn-167). While the Panuliris form has been shown to be deoxy (Volbeda et al., 1989), unpublished observations indicate that the horseshoe crab structure Limulus) is in the oxygenated state (K. Magnus, personal communication, 1988, cited by Volbeda and Hoi, 1988). 

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