Development of an off line noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples

A noncompetitive how immunoassay system (FIA) for the analysis of interleuk in-8 (IL-8) in cell samples was developed. Affinity interaction assays base d on offline incubation of excess labeled antibodies and antigen (IL-8) wer e carried out. The residual unbound labeled antibody was trapped in an immu noaffinity column with immobilized IL-8 while the immunocomplex, labeled an tibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLU OS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparis on between the two fluorescent labeled antibodies were performed with regar d to pH, antibody concentration, how rate, injection volume, and associatio n time. Additionally, a horseradish peroxidase enzyme label was used for th e conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL -8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA techniq ue was applied for the analysis of IL-8 in cell samples. Matrix-assisted la ser desorption/ionization time-of-flight mass spectrometry was used to iden tify IL-8 in the cell samples. (C) 2000 Academic Press.