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

Burestedt, E LU ; Kjellström, S LU ; Emnéus, J LU and Marko-Varga, G LU (2000) In Analytical Biochemistry 279(1). p.46-54
Abstract

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... (More)

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.

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author
publishing date
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Contribution to journal
publication status
published
subject
keywords
Antibodies, Carbocyanines, Epithelial Cells/immunology, Evaluation Studies as Topic, Fluoresceins, Fluorescent Dyes, Fluoroimmunoassay/instrumentation, Horseradish Peroxidase, Humans, Interleukin-8/analysis, Sensitivity and Specificity, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
in
Analytical Biochemistry
volume
279
issue
1
pages
9 pages
publisher
Elsevier
external identifiers
  • scopus:0034059240
ISSN
0003-2697
DOI
10.1006/abio.1999.4442
language
English
LU publication?
no
id
20b9149d-6d67-44aa-82f2-a8981df912ea
date added to LUP
2018-06-05 09:13:50
date last changed
2018-06-28 10:06:29
@article{20b9149d-6d67-44aa-82f2-a8981df912ea,
  abstract     = {<p>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.</p>},
  author       = {Burestedt, E and Kjellström, S and Emnéus, J and Marko-Varga, G},
  issn         = {0003-2697},
  keyword      = {Antibodies,Carbocyanines,Epithelial Cells/immunology,Evaluation Studies as Topic,Fluoresceins,Fluorescent Dyes,Fluoroimmunoassay/instrumentation,Horseradish Peroxidase,Humans,Interleukin-8/analysis,Sensitivity and Specificity,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization},
  language     = {eng},
  month        = {03},
  number       = {1},
  pages        = {46--54},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples},
  url          = {http://dx.doi.org/10.1006/abio.1999.4442},
  volume       = {279},
  year         = {2000},
}