Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

In Gram-negative bacteria, lipopolysaccharide endotoxins are vital components of the bacterial outer membrane.

Structurally, an endotoxin is comprised of the O-antigen - outermost polysaccharide chain connected via a central core to lipid A, an immunostimulatory glycolipid crucial for endotoxin activity.

To investigate endotoxin activity, take a test tube with a suitable buffer containing anti-lipid A antibodies, zymosan - an exogenous neutrophil activator, and luminol - a chemiluminescent agent.

Suspend an optimum volume of an infected blood sample comprising endotoxins and blood cells, including neutrophils. Incubate the tube under physiological conditions.

During incubation, anti-lipid A antibody recognizes the lipid-A component of the endotoxin and binds to it via antigen-binding domains, resulting in the formation of an antibody-endotoxin complex.

The endotoxin-bound antibody's FC domain binds to the neutrophils' complementary receptor, presenting the attached endotoxins to neutrophils. This binding acts as a priming stimulus for neutrophils, triggering their activation.

Activated neutrophils engulf the zymosan and initiate an immune response, resulting in a rapid release of reactive oxygen species, ROS, from neutrophils. These ROS facilitate luminol oxidation, resulting in light emission by chemiluminescence.

The chemiluminescence signal intensity corresponds to the endotoxin activity in the blood sample.