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Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics
- Source :
- Clinical Diagnostic Laboratory Immunology. 8:776-784
- Publication Year :
- 2001
- Publisher :
- American Society for Microbiology, 2001.
-
Abstract
- The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data ( r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 ( P = 0.003) and less IFN-γ ( P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-γ than that of atopic nonasthmatic subjects ( P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.
- Subjects :
- Adult
Male
Microbiology (medical)
T-Lymphocytes
medicine.medical_treatment
Clinical Biochemistry
Immunology
Antibodies and Mediators of Immunity
Cross Reactions
Biology
Microsphere
Flow cytometry
Interferon-gamma
medicine
Humans
Immunology and Allergy
Multiplex
Whole blood
Immunoassay
Reproducibility
medicine.diagnostic_test
Flow Cytometry
Asthma
Microspheres
Cytokine
Cytokines
Female
Tumor necrosis factor alpha
Interleukin-4
Subjects
Details
- ISSN :
- 10986588 and 1071412X
- Volume :
- 8
- Database :
- OpenAIRE
- Journal :
- Clinical Diagnostic Laboratory Immunology
- Accession number :
- edsair.doi.dedup.....2b516a208ee3f86aba34227f601a0bb1