Limulus amebocyte 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. 

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

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. 

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. 

CDER/CBER/CDRH/CVR-FDA, Guidelines on validation of the limulus amebocyte lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices, December 1987. 

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... 

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. 

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. 

The USP now recognizes two tests—the pyrogen test conducted with rabbits and the bacterial endotoxins test, also termed the limulus amebocyte lysate... 

Pyrogenic contamination is detected using two tests. In the older method, rabbits are injected with product samples, and rectal temperature is measured. Compendial limits are established with respect to how much temperature increase is permitted before the product is judged to be free or contaminated with pyrogens. The newer method involves a relatively simple in vitro technique called the Limulus Amebocyte Lysate (LAL) test. It is based on the high senstivity of amebocytes of the horseshoe crab (Limulus) to the lipopolysaccharide component of endotoxins originating from Gramnegative bacteria. The LAL test is now the USP method of choice with endotoxin limits established for most SVIs. ... 

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

The word "pyrogen" means "fever-producing" and has been used to cover any substance which causes a body temperature increase on injection. In fact, the first compendial pyrogen test was published in the 1942 edition of the United States Pharmacopeia. It involved the measurement of the rise of body temperature in rabbits upon intravenous injection of a test product. More recently (1977), the FDA approved the Limulus amebocyte lysate (LAL) test which can be run for less than 10% of the cost of a rabbit test. 

Limulus amebocyte lysate (LAL) test In vioo/In vitro... 

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