Endotoxin Testing by Gel-clot Assay

Limulus Amoebocyte Lysate (LAL)

Limulus amoebocyte lysate (LAL) is widely relied on for the detection of bacterial endotoxin. LAL is an aqueous extract of amoebocyte cells found in the Atlantic horseshoe crab from Limulus polyphemus. Gel-clot, chromogenic and turbidimetric tests are the main methods of LAL used. 

Gel-clot test

LAL gel clot assay is the simplest form of LAL test and is widely used to detect the presence or absence of endotoxin in the prepared sample. [1] It initiates a series of enzymatic reactions, when endotoxin encounters LAL. The combination of endotoxin and zymogen Factor C initiates the protease cascade in the LAL test. The Factor B, which stimulated by activated Factor C, converts the proclotting enzyme to the clotting enzyme. The clotting enzyme alters the amoebocyte coagulogen that presents in LAL to form a gel-clot, which can be observed. [2] [3]

The basic principle flow is as follows:

Endotoxin testing By Gel-clot Assay

Method of Gel-clot test

The testing should always be performed according to the manufacturer's recommendations. The endotoxin concentration is approximated by continuously applying a dilution of sample containing endotoxin to an LAL gel of different sensitivities (critical LPS amounts were known) until a negative reaction (no observable clot) is obtained. The process can take up to several hours. [4]

Endotoxin testing By Gel-clot Assay

Advantages of gel-clot test:

By more than 20 years of experience in bio-manufacture, Creative BioMart has developed a comprehensive LAL gel clot system to meet all kinds to your samples.

Creative BioMart has a team of professional and well-equipped scientists dedicated to work with researchers around the world, to meet their requirements and ensure high quality of custom service. If you are interested in our services, please contact us for more details.

References

  1. Ong, K.; et al. A Rapid Highly-Sensitive Endotoxin Detection System. Biosens. Bioelectron. 2006, 21, 2270-2274.
  2. Muta, T.; et al. Horseshoe Crab Coagulation Factor B. J. Biol. Chem. 1993, 268, 21384–21388.
  3. T, Sandle, Pyrogens. Endotoxin and the LAL Test: An Introduction in Relation to Pharmaceutical Processing, Global BioPharmaceutical Resources Newsletter,2012, 1-16.
  4. Wenqiong Song, Xianting Ding. Methods of Endotoxin Detection, Journal of Laboratary Automation, 2015, Vol.20(4) 354-364.
  5. Alex Wheeler. Comparing Endotoxin Detection Methods, Pharmaceutical Technology, 2017 Vol.41(11), 58-62

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