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Friday, July 13, 2012

Bacterial Endotoxin Test (LAL Test): Principle & Advantages

Bacterial Endotoxin Test (LAL Test)
The Limulus amebocyte lysate (LAL) test is an alternative method to the rabbit pyrogen test focussed on detection of pyrogenic substaces in sterile parenteral drugs. It is an invitro test performed to check the presence endotoxin in sample (eg: Parenteral drugs). LAL (Limulus amoebocyte lysate) test detects the presence of bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. LAL test is used for detecting and quantifying the amount of bacterial endotoxin in the sample. LAL test is FDA approved test for detecting endotoxin content in drugs.

LAL Test Principle

The sample to be analysed for bacterial endotoxin is added to the lysate, which is taken from the haemolymph cells of horse shoe crab(limulus polymhemus) (crab is not killed).
The principle of the LAL test is based on the physiological affect. The blood of horseshoe crabs contains a type of blood cell, called the amebocytes. These cells are important in exhibiting defence mechanism against pathogens. Amebocytes contain granules with a clotting factor known as coagulogen, this clotting factor is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation is thought to contain bacterial infections in the animal's semi-closed circulatory system.

Biochemical studies on the principle of Limulus test indicate that the amebocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1,3)-β-D-glucan-mediated coagulation pathway which also results in the formation of gel.The rate of reaction depends upon conc. of endotoxins, pH, temperature and presence of clotting enzyme system and clottable proteins from lysate.

Before the LAL test was developed, Pyrogens were tested on rabbits. Sample to be analyzed will be injected on rabbit and temperature elevation will be monitored.

Advantages of LAL Test

  1. Simple and Easy to perform.
  2. Results obtained in few hours.
  3. Animals (Rabbits) not used in testing.
Paper Publications  / Protocols

List of Commercial Limulus Amebocyte Lysate Quantification Kits

Pierce - LAL Chromogenic Endotoxin Quantitation Kit

Lonza - Limulus Amebocyte Lysate (LAL) QCL-1000TM

Endotoxin detection assay kits - Genscript

E-TOXATE™(Limulus Amebocyte Lysate) test kit - Sigma Aldrich

Biochemical Principles of Limulus test for detecting bacterial endotoxins