Limulus lysate

Jorgensen, J.H., Smith, R.F. Preparation, sensitivity and specificity of Limulus lysate for endotoxin assay. Appl Microbiol 26 (1973) 43-48. 

McConnell JS, Cohen J. Effect of anticoagulants on the chromogenic Limulus lysate assay for endotoxin. J Qin Pathol 1985 38(4) 430-2. 

A highly sensitive test is required for the detection of endotoxic inflammatory substances to evaluate the purity of sodium hyaluronate. The Limulus-Lysate-Test, a highly sensitive test for pyrogens, was observed to be neither sensitive nor specific enough to detect impurities in sodium hyaluronate solutions. 

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. 

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. 

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. 

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. 

Toxin assay (bactaial) amoebocyte lysate/Sigma 2000 used in d ecticm and quantitatioi of aidotoxins in plasma Cdien 1979 marine hcx-seshoe crab, Limulus polyphemus (L.), Chelicerata... 

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. 

The determination of the endotoxin with limulus amebocyte lysate (LAL) is based on gel formation of a mixture consisting of a solution of endotoxin of Gram-negative bacteria with a solution of lysate. The extent and speed of the reaction depend on the endotoxin concentration, pLL, and temperature. The reaction requires the presence of certain cations, a proclotting enzymes system, and clottable protein, which are produced by lysate. 

Endotoxicity results from the interaction of a bacterial cell envelope component (e.g., LPS or PG with a cell surface receptor constituting part of the nonspecific immune system, (i.e., a toll-like receptor on white blood cells). This results in the production of cytokines [e.g., interleukin 1 (IL-1) or tumor necrosis factor (TNF)] as part of an intracellular enzyme cascade which can cause severe tissue injury. Bioassays or immunoassays can be used to detect such reactions respectively. As noted above the most widely used bioassay is the LAL assay. A lysate of amoebo-cytes of the horseshoe crab (Limulus) contains an enzymatic clotting cascade which is activated by extremely low levels of LPS (nanogram levels or lower). There are variants of this assay that can detect PG, but they are not as widely used. As noted above, other bioassays employ cultured cell lines that respond to LPS or PG, respectively. Unfortunately bioassays are highly amenable to false positives (from the presence of cross-reactive substances) or false negatives from inhibition (by contaminants present in the sample) [10]. A detailed discussion of these assays is beyond the scope of this chapter and has been reviewed elsewhere [1]. 

Saraf, A., Larsson, L., Burge, H., and Milton, D. (1997), Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry Comparison with fungal culture and determination of endotoxin by a Limulus amoebo-cyte lysate assay, Appl. Environ. Microbiol., 63, 2554-2559. 

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. 

Novitsky, TJ. (1996). Limulus amebocyte lysate (LAL) assays. In Automated Microbial Identification and Quantitation Technologies for the 2000s, W.P. Olson, ed. Inter-pharm Press, Buffalo Grove, IL, 277-298. 

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

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