USE PRINCIPLES

The EndoAlert® ES Plate Kit is a quantitative version of the reaction first described by Levin and Bang in 1968. The test is based upon an enzymatic cascade where Endotoxin activates Factor C in Limulus Amebocyte Lysate (LAL) which in turn activates Factor B. Factor B activates Proclotting Enzyme which then activates Clotting Enzyme. A colorless synthetic peptide substrate is hydrolyzed by Clotting Enzyme to generate a yellow color which can be measured by a spectrophotometer at 405 nm. The degree of color resulting from the reaction is proportional to the amount of Endotoxin in the test sample and can be calculated using a standard curve.

Bacterial endotoxins, found in the outer membrane of gram-negative bacteria are members of a class of phospholipids called lipopolysaccharides (LPS). LPS are not exogenous products of gram-negative bacteria, implicated in the development of Gram-negative shock. The release of LPS from bacteria takes place after death and lysis of the cell. They can cause adverse reactions in sufficient concentrations, such as fever, hypotension, pulmonary edema and disseminated coagulation events in humans and animals, by release of inflammatory mediators, such as cytokines and interleukins. For example, endotoxin reacts with lipopolysaccharide-sensitive cells producing tumor necrosis factor alpha (TNFα). Macrophages are cells mediating the toxic activities of LPS, and TNFα is the primary mediator of the lethal action of endotoxin.

Pyrogens include endotoxins (toxins derived from gram-negative bacteria) and non-endotoxic pyrogens (substances derived from microorganisms other than gram-negative bacteria, or from chemical substances). Endotoxins from gram-negative bacteria are the most common cause of toxic reactions due to contamination of pyrogenic compounds. Good examples of pyrogen producing gram- negative bacteria are Escherichia coli, Proteus, Pseudomonas, Enterobacter and Klebsiella. Due to its heat resistance and stability, complete inactivation of endotoxin is not possible with autoclaving. Dry heat sterilization for at least 30 minutes at a temperature of 250 °C or more is required complete inactiocaiton. It exists in the environment (e.g., water, air) inhabited by gram-negative bacteria, and bacterial endotoxins (LPS) remain even after the bacteria die.