Endotoxin is a toxin released from certain bacteria, gram negative organisms. It may be present as a contaminant in medical devices or pharmaceutical solutions. If present in the blood of patients with septicemia, it may cause irreversible organ damage, shock, and death. Septicemia is the 13th leading cause of death in humans and accounts for 100,000 deaths annually in the United States. The only assay currently available to measure endotoxin in industrial and biological samples, Limulus amebocyte lysate (LAL) assay, is based on the reactivity of endotoxin with an amebocyte lysate isolated from the hemolymph of horseshoe crabs, Limulus polyphemus. However it is FDA approved for industrial use only. Although it is sensitive, this assay lacks specificity and is not reliable for the detection of endotoxin in biological samples, including the plasma of patients with septicemia. Using a number of highly selective radioligands for A1 adenosine receptors and membranes expressing A1 adenosine receptors, it was discovered that endotoxin displaces radioligand binding to A1 adenosine receptors. This simple radioligand binding assay is as sensitive and more specific than the LAL assay for the detection f endotoxin in saline. Currently the industrial market for the LAL assay is estimated at $60 million annually. Therefore, with this SBIR grant, the primary aim is to develop further the radioactive ligand binding assay (LBA), develop a nonradioactive LBA, and the validation of them with the LAL to measure endotoxin in pharmaceutical and industrial solutions, as well as biological products. Successful completion of the proposed experiments in this application would support a 510 (k) application for the industrial use of the LBA and lead to a Phase II SBIR grant application to support the submission of a Premarket Approval (PMA) for the clinical use of this LBA for the measurement of endotoxin in the plasma of patients with gram negative septicemia.
According to Good Manufacturing Practice (GMP) regulations by the Food and Drug Administration (FDA), all parenteral drugs and solutions intended for medical use must have an endotoxin content below certain limits that are specific for that product. The Limulus amebocyte lysate (LAL) assay is the only FDA approved test which can be used to meet these requirements. However, it lacks specificity. The ligand binding assay (LBA) for detection and measurement of endotoxin described in this application is as sensitive as, more specific than, and cost competitive with the LAL assay. Market analysis estimates that for industrial use there is a $60 million world-wide market for endotoxin test kits.
