Bacterial Endotoxin Test
Nature of Pyrogens
Pyrogen (fever-inducing agent), principally known as bacterial endotoxin, is one of the most potent bacterial toxins. Its only source is Gram (-) bacteria (GNB), where endotoxin comprises about 75% of the GNB cell wall.
Bacterial contamination (bioburden) in water and on surfaces normally contains GNB, so endotoxin is ubiquitous in nature.
USP General Chapter <85> describes how to conduct the BET with LAL reagent and qualify an analyst for routine testing.
Endotoxin Detection
Endotoxins are large complexes from GNB bacterial cell wall that are constantly shed into the environment when the bacteria disintegrate or multiply. Endotoxins contain lipid, carbohydrate, protein, and are composed of a common structure of a hydrophilic polysaccharide covalently bound to a hydrophobic region known as Lipid A.
The skin and gastrointestinal tract are barriers to endotoxin. However, when endotoxin gains access to blood or tissues by injection, nanogram (parts per billion) quantities can cause fever and hypotension while large amounts can lead to irreversible shock and death.
Gel Clot Method
The traditional gel clot method is more complicated, time-consuming and technique-dependent than other methods.
In its simplest form, a gel-clot BET is conducted by mixing 100 μL (0.1 mL) of sample or positive (endotoxin) control with 100 μL of LAL reagent, using aseptic technique, and incubating the mixture undisturbed at 37°±1C for one hour (60±2 minutes).
If endotoxin is present in a concentration greater than the labeled LAL sensitivity, an opaque gel will form that remains firm when the assay tube is carefully inverted.
Gel-clot reagents are available in single and multi-test vials; single test vials eliminate handling liquid LAL reagent. The labeled sensitivity of the LAL reagent, lambda (λ), is the minimum concentration of endotoxin that will produce a gel endpoint under standardized test conditions; a 0.06 EU/mL reagent is most frequently used.
The assay is time and temperature sensitive, so attention to test conditions is important to achieve meaningful results.
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