Limulus test activity

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. 

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... 

Two pharmacopoeial limit tests exist. That for pyrogens uses rabbits to assess pharmacological activity and therefore the presence of pyrogens of all kinds. The test for bacterial endotoxins uses lysed amoebocytes (blood cells) of the horseshoe crab and is therefore termed the Limulus amoebocyte lysate (LAL) test. This may be extended to many drug and device products and clearly will be developed in the future to assess the presence of endotoxins in biotechnology products. 

The quahty of the purified product is an important aspect, and must be ensured because it can influence the potency of dSLIM. Therefore, integrity is tested via exonuclease activity of T7-DNA-polymerase degrading residual DNA molecules with open ends and subsequent gel electrophoresis. Furthermore, the content of endotoxin is determined by an end-point Limulus amoebocyte lysate (LAL) test, and must meet strict standards (< 10 EU mg DNA). 

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. 

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 limulus amoebocyte lysate (LAL) test with chromogenic substrate is faster than the gelation method, and it can be automated. The chromogenic substrate is attached to p-nitroaniline, that is released when reacted with the endotoxin-activated enzymes. The free p-aniline is read at 405 nm. 

The pyrogenic activity of LPS is much higher than that of most other pyrogenic substances. This is the reason why an in-vitro limit test for LPS (the Limulus Amoebocyte Lysate, or LAL test) generally suffices for quality control piuposes of parenteral medicines and raw materials, including water for injection. 

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... 

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