Endotoxin Detection and Testing

Endotoxins are heat-stable toxins associated with the outer membranes of certain gram-negative bacteria, including Brucella, Neisseria, and Vibrio species. Endotoxins are not secreted but are released only when the cells are disrupted; they are less potent and less specific than the exotoxins; and they do not form toxoids. In large quantities they produce hemorrhagic shock and severe diarrhea; smaller amounts cause fever, altered resistance to bacterial infection, leukopenia followed by leukocytosis, and numerous other biologic effects.

In the manufacturing of pharmaceuticals, it is important to test products meant for humans for the presence of endotoxins. Testing, detection and removal of endotoxins is an important quality control step to ensure safe pharmaceuticals are delivered to patients.

Pharmaceutical Endotoxin Detection Assays

There are three principal LAL test methods: the gel-clot, turbidimetric and chromogenic methods. The latter two may be grouped together as photometric methods as they require an optical reader.

Chromogenic Method The LAL reagent is formulated with a synthetic substrate which produces a chromophore when cleaved by endotoxin activated enzyme. The test is read at 405 nm, usually in a microplate reader.

Turbidimetric Method The optical density (turbidity) increase that accompanies the clotting reaction is read in a tube reader or in an incubating microplate reader.

Gel-clot Method This is the simplest LAL test. The formation of a gel-clot indicates the presence of endotoxin in a sample. The method is performed in small test tubes and is read manually by inverting the test tubes. The maximum sensitivity is 0.03 EU/mL.