Limulus test

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

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). 

Cooper, J.F. Hochstein, D.H. Seligmann, E.B. The limulus test for endotoxin (Pyrogen) in radiopharmaceuticals and biologicals. Bull. Parent. Drug Assoc. 1972, 26, 153-162. 

Nagi N., Ohno N.. Adachi Y., Aketagawa J., Tamura H., Shibata Y., Tanaka S. and Yadomae T. (1993) Application of Limulus test (G pathway) for the detection of different conformers of (l 3)-p-l)-glu-cans. Biol. Pharm. Bull.. 16, 822-828. 

For the Limulus test, a 10-fold dilution series of the sample is prepared and equal volumes of the Limulus lysate and diluted sample are mixed in a test tube. The test tube is then incubated before being inverted and read. If the mixture remains unchanged and runs out of the tube then that dilution of the sample does not contain LPSs. If a firm opaque gel is formed that sticks to the bottom of the tube then that dilution of the sample contains LPSs. Generally, a visual reading of the tenfold or twofold dilutions gives sufficient information about the level of LPSs present in the sample. 

Brandenburg, K Howe, J., Gutsman, T, and Garidel, P. (2009) The expression of endotoxic activity in the Limulus test as compared to cytokine production in immune cells. Curr. Med. Chem., 16, 2653-2660. 

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). 

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. 

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. 

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. 

Sakai H, Hisamoto S, Fukutomi I, et al. Detection of lipopolysaccharide in hemoglobin-vesicles by Limulus amebocyte lysate test with kinetic-turbidimetric gel clotting analysis and pretreatment of surfactant. J Pharm Sci 2004 93 310. 

IL-6 induction in human peripheral whole-blood cell culture [10,11] and determination of its level using ELISA and Limulus activity measured by means of Endospecy Test (Seikagaku Co., Tokyo, Japan) were performed as described [8]. lL-6 induction in THP-1 cells was performed as reported [12]. In brief, sample solution (25 p.L) and 100 p.L of THP-1 cells (1.0 X 10 cells/mL, preincubation with 50 ng/mL vitamin D3 at 37°C in CO2 for 72 h) were incubated in RPMl-1640 medium with or without 2% fetal calf serum... 

Sialic acids may also be specifically stained by using peroxidase-labelled Limulus polyphemus agglutinin in combination with 3,3 -diaminobiphenyl or Aleian Blue, and the procedure was tested with various, mammalian tissues by light-microscopy.199... 

Limulus amebocyte lysate is obtained from a licensed manufacturer. Each lot of reagent is tested per USP for release. Endotoxin used in all bacterial endotoxin analysis testing is obtained from a licensed manufacturer and is standardized against the USP reference standard. 

U.S. Department of Health and Human Services (DHHS) (1987), FDA guidelines on validation of the Limulus amoebocyte lysate test as an end product test for human and animal parenteral drugs, biological products and medical devices, DHHS, Rockville, MD. 

Limulus Amebocyte Lysate test kit (Pyrogen Plus, BioWhittaker Inc., Walkersville, MD). 

Following the dry-heat cycle, aseptically transfer the units containing endotoxin to an aseptic area for extraction procedures, sampling, and conducting the limulus amebocyte lysate (LAL) test. 

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