Development of an offline noncompetitive flow immunoassay for the determination of interleukin-8 in cell samples
(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
- Burestedt, E LU ; Kjellström, S LU ; Emnéus, J LU and Marko-Varga, G LU
- publishing date
- 2000-03-01
- type
- 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
- pmid:10683229
- 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
- 2024-01-14 21:18:34
@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}},
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}},
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}},
doi = {{10.1006/abio.1999.4442}},
volume = {{279}},
year = {{2000}},
}