Development of a high-capacity homogeneous fluorescent assay for the measurement of leukotriene B4

Current immunoassays for the measurement of leukotriene B4 (LTB4) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB4 antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB4 ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647–LTB4, the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z = 0.8; S/N = 250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<$0.10/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB4 giving 50% inhibition (IC50) was ca. 55 pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB4 and 12-epi LTB4. Measurements of LTB4 determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.