Limulus

Pyrogenic A fever-producing substance. The presence of these substances is determined by the Limulus Amebocyte Lysate (LAL) test and measured in EU/ml (endotoxin units per milliliter). 

Endotoxin below specified level Limulus amoebocyte lysate method (see also Chapter 18)... 

However, the establishment of a new endotoxin standard by the World Health Organization is a recent example of successful international collaboration between the World Health Organization, the United Stated Pharmacopoeia and the European Pharmacopoeia (Poole et al. 1997). Thus this standard is available from any of these organizations to be employed as a reference in the harmonized Limulus Amoebocyte Lysate test. 

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. 

Brown, J. E., Faddis, M. and Combs, A. Light does not induce an increase in cyclic-GMP content of squid or Limulus photoreceptors. Exp. Eye Res. 54 403M10,1992. 

Fein, A. Excitation and adaptation of Limulus photoreceptors by light and inositol 1,4,5-triphosphate. Trends Neurosci. 9 110-114,1986. 

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. 

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. 

Figure 7 of reference 11 compares similarities between Limulus and Octopus hemocyanins by illustrating the overlap of 30 amino acids about the CuB region. The authors find that all residues within 5.0 A of the Cu-Cu midpoint are conserved between arthropodan and molluscan hemocyanins and occupy analogous positions except for one (see the following paragraph). The three histidines about CuB overlap almost exactly in orientation and are less than 1 A apart. The histidines... 

Cooper, J.F. (1975). Principles and applications of the limulus test for pyrogen in parenteral drugs. Bull. Parenter. Drug Assoc. 3 122-130. 

Develeeshouwer, M.J., Comil, M.F. and Dony, J. (1985). Studies on the sensitivity and specificity of the limulus amebocyte lysate test and rabbit pyrogen assays. Appl. Environ. Microbiol. 50 1509-1511. 

Wearly, M. and Baker, B. (1977). Utilization of the limulus amebocyte lysage test for pyrogen testing of large-volume parenterals, administration sets and medical devices. Bull. Parenter. Drug Assoc. 31 127-133. 

Some modifications to current in vivo testing methods both can and should be adopted. A current example of this would be in medical devices where a substantial portion of the requirements under the governing regulatory (ISO 10993) can be met with in vitro alternatives (cytogenicity, muscle cell implantation, the limulus test for pyrogens, and in vitro mutagenicity assays). 

Figure 7 X-ray structure of the active site of the deoxygenated form of haemocyanin from Limulus polyphemus...

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. 

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