Amebocyte Lysate assay

Limulus Amebocyte Lysate assay (Thermo Fisher, Waltham, MA). This assay is used to periodically check reagents for the presence of endotoxin. 

It is best to make certain all reagents are free of endotoxin contamination before attempting to isolate neutrophils. This can be done using the Limulus Amebocyte Lysate assay (as described by the manufacturer). 

Petsch D, Deckwer WD, Anspach FB. Proteinase K degestion of protein improves detection of bacterial endotoxins by the Limulus Amebocyte lysate assay Application for endotoxin removal from cationic proteins. Anal Biochem 1998 259 42-47. 

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. 

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. 

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. 

Remillard J.F., Case Gould M., Roslansky PF. and Novitsky T.J. (1987) Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay. In Detection of bacterial endotoxins with the Limuius amebocyte lysate test. Alan R. Liss, Inc, New York. 197-210. 

Lipopolysaccharides (LPS) or endotoxin may be introduced as the result of low level bacterial contamination of the cell culture or during the fill of the final drug product, and can be assessed by the Limulus amebocyte lysate (LAL) assay. Similarly, antifoaming agents that are introduced during cell culture are removed during purification, but verification of removal through quantitative HPLC assays is required. Lastly, purification process impurities such as debris from resin or... 

---