Correlation of Concentration Versus Pressure: A Step in the Direction Towards a New Method for Measuring Endotoxin

Endotoxins are complex lipopolysaccharides that are found on the outer cell membrane of gram-negative bacteria.  Bacteria will secrete endotoxins in large amounts throughout their life cycle. Endotoxins have an abnormally high heat resistance making it nearly impossible to destroy them under normal sterilizing conditions.  Endotoxins affect both in vitro and in vivo cell growth and functions. In vitro, endotoxins have been known to affect cell development while in vivo endotoxins are known to elicit an inflammatory response, fevers, chills and even fatal septic shock in mammals. Endotoxins are typically measured in Endotoxin Units per milliliter (EU/mL), and the US Food and Drug Administration (FDA) sets limits on the concentration of endotoxin for medical devices and parenteral drugs.  

A common way to test for the amount of endotoxin is to perform a “gel clot” assay that utilizes a Limulus Amebocyte Lysate (LAL) obtained from horseshoe crab blood that was shown to clot in the presence of bacteria. Specifically, the use of the LAL reagent in a reaction containing endotoxins will produce a gel clot under certain reaction conditions. Ultimately, the LAL reagent  can be used to detect  endotoxin down to specific levels associated with the sensitivity of the lysate. When a clot forms, the endotoxin concentration is then known to be at a level that is equal to or higher than the sensitivity level of the LAL.  A slurry or no clot will be produced at lower levels of endotoxin for a corresponding LAL sensitivity.

The procedure to produce a gel clot takes 1 hour at 37 degree C and can become expensive.  Further, testing samples in the field can require an iterative process that involves multiple  wells, LAL, and endotoxin controls.   While chromogenic and kinetic-based assays also exist, new methods for endotoxin detection could possibly result in more cost effective, high throughput approaches.  In this work, our efforts to develop a new methodology for endotoxin testing that exploits mechanical properties of the gels will be discussed.  The research might allow for quicker in house testing in many fields ranging from processing plants, hospitals, and laboratories. Further, such methods might result in reductions in the amount of LAL needed and cut down on the destruction of horseshoe crabs population and their environment.