A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence

Abstract

A rapid (30 min) whole blood assay for the detection of lipopolysaccharide (LPS) is described. This chemiluminescent (CL) assay utilizes the CR1 and CR3 receptor-induced oxidant production of polymorphonuclear leucocytes as a detection platform. The differential priming of neutrophils in whole blood by LPS-antibody complexes allows the specificity of the assay to be achieved. Oxidant released in response to complement opsonized zymosan results in luminol oxidation and subsequent light emission. This is dependent on heat labile putative complement proteins in the plasma. The assay consists of a control which measures baseline whole blood neutrophil oxidant production. The test assay contains murine monoclonal IgM antibody against the Lipid A epitope of LPS and measures the enhanced chemiluminescent response of the neutrophils in the presence of LPS-antibody complexes. Maximal sensitivity of the CL assay is dependent upon optimal antigen-antibody equivalence and duration of pre-incubation with the whole blood sample. The quantification of LPS is possible by inclusion of a positive control containing a maximally reactive LPS dose (800 pg/ml Escherichia coli 055:B5 LPS at an antibody concentration of 0.8 microg/assay). The CL assay is insensitive to variations in patient neutrophil concentration over a minimum range of 0.5 to 20 x 10(9) cells/l. The CL assay is widely reactive with the LPS of many strains of gram negative bacteria but not with the cell wall products of gram positive bacteria or Candida and Aspergillus. In comparison to acid extraction chromogenic LAL, the CL assay demonstrates superior recovery precision and accuracy in in vitro studies. This was reproducible over a wide range of LPS concentrations (0.017-1.6 EU/ml or 20-2000 pg/ml). This assay may be a clinically useful tool for the diagnosis of infection or endotoxin in patients.