title: Development of a high-capacity homogeneous fluorescent assay for the measurement of leukotriene B4.
abstract: Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) 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-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), 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 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.
authors.affiliation: Merck Research Labs, PO Box 2000, R80N-A32, Rahway, NJ 07065, USA. douglas_miller@merck.com
authors.forename: Douglas K
authors.initials: DK
authors.lastname: Miller
authors.forename: John D
authors.initials: JD
authors.lastname: Menke
authors.forename: Nancy S
authors.initials: NS
authors.lastname: Hayes
authors.forename: Adam
authors.initials: A
authors.lastname: Uzieblo
authors.forename: Daniel
authors.initials: D
authors.lastname: Tew
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Guan
authors.forename: Annie
authors.initials: A
authors.lastname: Zhao
authors.forename: Richard T
authors.initials: RT
authors.lastname: Cummings
authors.forename: Young-whan
authors.initials: YW
authors.lastname: Park
authors.forename: Ting-ting D
authors.initials: TT
authors.lastname: Yamin
mesh_terms: D005470:Fluorometry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007975:Leukotriene B4 / Q000032:analysis* / Q000097:blood; D009504:Neutrophils / Q000378:metabolism
chemical_list: D007975:Leukotriene B4