Bacterial Endotoxins Test - (BET)

Useful facts about endotoxins and their detection with the Limulus Amoebocyte Lysate (LAL) assays

Endotoxin (LipoPolySaccharide, LPS) is produced exclusively by Gram-negative bacteria, such as Escherichia coli, Salmonella enteritidis, Legionella pneumophila, Campylobacter jejuni, Vibrio cholerae, Shigella dysenteriae, Pseudomonas aeruginosa and many, many more. The endotoxin concentration is expressed in EU.

1 EU = 100 pg endotoxin

1 picogram = 10^-12 gram or 0,000000000001 gram

For one specific strain of Gram-negative bacteria it has been determined that approximately 7000 bacterial cells contain 1 EU (Scan Dia Labs, unpublished data).

Mechanisms of LPS release from gram-negative bacterial cells:

Natural cell death and bacteriolysis
LPS shedding via bleb or membrane vesicle release
Serum protein and complement-mediated release
Host phagocyte ingestion with subsequent exocytosis of LPS Antibiotic mediated release

The Limulus Amoebocyte Lysate (LAL) Assays for the Detection of Endotoxin:

Gel-Clot Technique
Narrow sensitivity range and only semi-quantitative at best.
Max. sensitivity: 0.03 EU/ml.

Chromogenic and Endpoint-Turbidimetric Techniques
The chromogenic assays use a synthetic chromogenic peptide as substrate for the clotting enzyme in place of coagulogen. The chromogenic substrate is hydrolyzed by the clotting enzyme, releasing the terminal chromogenic moiety with a yellow color. Max. sensitivity >0.01 EU/ml.

Kinetic-Turbidimetric Technique

Measures the rate of the LAL-LPS reaction or the time required for the reaction to reach a specified Optical Density. Quantitative. Facilitates assessment of sample inhibition/enhancement. Max. sensitivity: >0.005 EU/ml.

ELISA Techniques and Rocket Immunoelectrophoretic Technique

The non-competitive C-peptide ELISA and the Sandwich ELISA employing monoclonal antibodies, and the rocket immunoelectrophoretic assay measuring the quantitative consumption of coagulogen using rabbit antibodies against the protein, are all elegant. However, complicated and time-consuming set-up procedures prevent their wide-spread use.

LAL Assay Interference Mechanisms:

Suboptimal pH conditions
Aggregation or adsorption of control endotoxin spikes
Unsuitable cation concentrations
Enzyme or protein modification
Non-specific LAL activation – sometimes an interference mechanism cannot be determined