Development of a rapid immunoassay for human pathogenic markers

Abstract

A demand exists for a fast, sensitive, reliable and economical test for pyogenic sepsis that provides a “real time” bed-side assessment. Detection of significant intra-abdominal sepsis can be particularly problematic in the ICU setting and in patients with multi-system organ failure. Lysozyme, first reported by Fleming (1922), is a bacteriolytic enzyme released during phagocytosis. Previous studies have shown significant correlation between lysozyme levels and the presence of intra-abdominal abscess in both animals and humans. A method which determines and quantifies lysozyme as part of an assessment of an acutely ill patient in whom major sepsis is suspected; would significantly aid diagnosis and prescription of the most effective form of treatment. To date measurement of lysozyme has been by turbidometry, with consequent poor sensitivity and reliability. Other methods of assay include fluorescence, radial immunodiffussion and enzyme linked immunosorbent assay (ELISA). This study reports a modified ELISA technique which provides a cheap, sensitive and reliable method of lysozyme determination, producing results in <100 minutes. The ELISA has been tested with ~200 clinical samples provided by the patients at the Great Western Hospital, Swindon. Two ELIFA techniques were also developed for lysozyme and E.coli detection. These techniques also provide a cheap and rapid alternative to the more traditional immunoassays. Results from the ELIFA and mini-ELIFA were obtained qualitatively after only 10 minutes. An SPR detection technique was also devised. The BIAcore 3000 was used to create a biosensor for serum lysozyme using an artificial receptor in the form of an aptamer. This system was tested with clinical serum samples, is reusable and took <80 minutes to immobilise a ligand on a blank sensor and analyse a serum sample for lysozyme. Although further research and development is required on the mini-ELIFA and lysozyme biosensor, the ELISA detection system may prove a useful tool in the diagnosis of sepsis in critically ill patients.