Limulus-lysate-test

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. 

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. 

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. 

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). 

Keywords Endotoxin Limulus amoebocyte lysate test LAL Molecular cloning Pyrogene assay Recombinant Factor C... 

Morita, T., Nakamura, T., Miyata, T., Iwanaga, S. Biochemical characterisation of Limulus clotting factors and inhibitors which interact with bacterial endotoxin. In Cate, J.W., Buller, H.R., Sturk, A., Levin, J. (eds). Bacterial endotoxin, structure, biomedical significance, and detection with the limulus amoebocyte lysate test. Alan R Liss Inc, NY (1985a) pp. 53-64. 

Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product... 

Guideline on Validation of the Limulus Amebocyte Lysate Test as an End-Product Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices Food Drug Administration, December 1987. 

Pearson, F.C. Weary, M. The significance of limulus amebocyte lysate test specificity on the pyrogen evaluation... 

Flint, O. A timetable for replacing, reducing and refining 58. animal use with the help of in vitro tests the limulus amebocyte lysate test (LAL) as an example. In Alternatives... 

Please submit a certificate of analysis of the product. For products requiring sterility test and pyrogen test (Limulus Amebocyte Lysate test is also acceptable), recent copies of such certificates should also be submitted. 

EN 14569 2004 DIF using the limulus amoebocyte lysate test in conjunction with a gram negative bacterial count (LAL/GNB) Poultry meat... 

Limulus amebocyte lysate test (LAL) 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. 

Cooper J.F. (1985) Ideal propertie.sof a LAL reagent for pharmaceutical testing. In Bacterial endotoxins structure, biomedical significance, and detection with the Limulus amebocyte lysate test. Alan R. Liss. Inc.. New York, 241-249. 

Sonesson A., Larsson L., Schiitz A., Hagmar L. and Hallberg T. (1990) Comparison of the Limulus amebocyte lysate test and gas chromatography-mass spectrometry for measuring lipopolysaccharides (endotoxins) in airborne dust from poultry processing industries. Appl. Environ. Microbiol., 56, 1271-78. 

Rietschel E.T., Brade H., Brade L., Kaca W., Kawahara K., Lindner B., Luderitz T., Tomita T., Schade U., Seydel U. and Zahringer U. (1985) Newer aspects of the chemical structure and biological activity of bacterial endotoxins. In ten Cate J.W., Biiller H.R., Sturk A. and Levin J. (eds) Bacterial endotoxins structure, biomedical significance, and detection with the Limulus amebocyte lysate test. Alan R. Liss, New York, 31-50. 

The water used in the preparation of sterile products should be tested for bacterial endotoxin at least once per week and after any repair or disturbance to the system, using the limulus amoebocyte lysate test Sampling should include worst case situations, including start-up. 

The use of the limulus amoebocyte lysate test is valuable in monitoring bacterial endotoxin levels. 

A number of tests were also developed and further used routinely to detect other process-related impurities in clinical batches, such as a Limulus amoebocyte lysate test to quantify bacterial endotoxins (< 1 I.U./mg of BBG2Na) and a specific RP-HPLC assay to detect tetracycline (no trace detected) since this antibiotic was used for selection by drug pressure during the fermentation step. 

Lee PS. Limulus Ameboc5h e Lysate testing of medical devices. In Williams KL, editor. Endotoxins Pyrogens, LAL testing and depyrogenation. 2007. p. 366. 

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. 

Endotoxic to limulus amoebocyte lysate test 3-(2-Carboxyphenyl)-4(3//)-quinazolinone [31],... 

Potassium channel blockers Antianythmic activity Vasorelaxing activity Pollen-growth inhibitors Anti-inflammatory and antihypertensive Endotoxic to limulus amoebocyte lysate test Tremor and paralysis effects Pharmacokinetic Raising body temperature Anticoccidial... 

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). 

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. 

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