Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples.
A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the 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 technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify IL-8 in the cell samples.
Burestedt
E
E
Pharmacia & Upjohn, Consumer Healthcare, Helsingborg, S-25109, Sweden.
Kjellström
S
S
Emnéus
J
J
Marko-Varga
G
G
D000906:Antibodies; D002232:Carbocyanines; D004847:Epithelial Cells / Q000276:immunology; D005069:Evaluation Studies as Topic; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D006735:Horseradish Peroxidase; D006801:Humans; D016209:Interleukin-8 / Q000032:analysis*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016209:Interleukin-8; C097499:fluorescein carboxylate; D006735:Horseradish Peroxidase