Development of a high-capacity homogeneous fluorescent assay for the measurement of leukotriene B4.
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.
Miller
Douglas K
DK
Merck Research Labs, PO Box 2000, R80N-A32, Rahway, NJ 07065, USA. douglas_miller@merck.com
Menke
John D
JD
Hayes
Nancy S
NS
Uzieblo
Adam
A
Tew
Daniel
D
Hayashi
Yoko
Y
Guan
Yanfen
Y
Zhao
Annie
A
Cummings
Richard T
RT
Park
Young-whan
YW
Yamin
Ting-ting D
TT
D005470:Fluorometry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007975:Leukotriene B4 / Q000032:analysis* / Q000097:blood; D009504:Neutrophils / Q000378:metabolism
D007975:Leukotriene B4