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

被引:10
作者
Burestedt, E
Kjellström, S
Emnéus, J
Marko-Varga, G
机构
[1] AstraZeneca R&D Lund, S-22187 Lund, Sweden
[2] Pharmacia & Upjohn Inc, Consumer Healthcare, S-25109 Helsingborg, Sweden
关键词
interleukin-8; noncompetitive flow immunoassay; cell samples; FLUOS; Cy5.5; HRP; MALDI identification;
D O I
10.1006/abio.1999.4442
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A noncompetitive how 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, how 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. (C) 2000 Academic Press.
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页码:46 / 54
页数:9
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