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Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2008 Apr 20; Vol. 333 (1-2), pp. 61-70. Date of Electronic Publication: 2008 Jan 29. - Publication Year :
- 2008
-
Abstract
- The identification of parameters maximizing detection sensitivity in ELISpot assays is important to transfer this technology into the clinical setting for identifying rare Ag-specific CD8(+) T cells. We have therefore considered human IFN-gamma CD8(+) T cell responses against viral epitopes to analyze different variables which could be critical during the epitope-specific stimulation period. Two parameters were found to greatly enhance detection sensitivity (i.e., to specifically increase epitope-driven signal while keeping background noise to a minimum): use of human serum-free vs. serum-supplemented culture medium (2.4-fold median increase) and addition of low dose IL-7 (1.5-fold increase). Incorporating both of these parameters into the ELISpot procedure proved capable of greatly amplifying (35.1-fold increase) the low grade CD8(+) T cell responses directed against beta-cell epitopes of type 1 diabetes patients, as compared to a previously optimized procedure using human serum-supplemented medium and low dose IL-2. Implementation of this ELISpot procedure should expedite development of "immune staging" protocols for autoimmune as well as tumor and infectious diseases.
- Subjects :
- Adult
Aged
Antibodies, Monoclonal pharmacology
CD28 Antigens immunology
Cell Culture Techniques methods
Diabetes Mellitus, Type 1 immunology
Epitopes, T-Lymphocyte analysis
Female
HLA-A2 Antigen immunology
Humans
Insulin-Secreting Cells immunology
Interferon-gamma immunology
Interleukin-2 immunology
Interleukin-2 pharmacology
Interleukin-7 immunology
Male
Middle Aged
Statistics, Nonparametric
CD8-Positive T-Lymphocytes immunology
Culture Media, Serum-Free
Enzyme-Linked Immunosorbent Assay methods
Epitopes, T-Lymphocyte immunology
Interleukin-7 pharmacology
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1759
- Volume :
- 333
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 18242633
- Full Text :
- https://doi.org/10.1016/j.jim.2008.01.003