| |
CTL Culture |
Elispot |
Tetramer Staining |
| What it
does |
Bulk PBMC are co-cultured
with HIV-1 antigens, which clonally expands the antigen-specific CTL
in the sample. |
Anti-cytokine antibodies
on a plate reveal individual cytokine-producing cells as single spots
that are counted by eye or by computer. |
HLA/peptide complexes are organized into
a stable tetrameric structure that specifically binds to T-cells expressing
the appropriate T-cell receptor for the HLA/antigen complex. |
| What it
measures |
When the expanded CTL lyse Cr51 pulsed
target cells, radiation is released indicating the presence of functional
CTL. |
INF-gamma release by CTL in response
to antigen correlates well with CTL frequencies measured with lysis
assays. |
Tetramer staining directly marks antigen-specific
CD8 T-cells that can then be counted by flow cytometry. |
| Advantages |
Directly indicates lysis by CTL. |
Allows screening for response
to multiple epitopes in a single assay. |
Allows virus-specific analysis. Allows
sorting of antigen-specific cells for subsequent cloning. Allows analysis
of co-expressed surface antigens. |
| Disadvantages |
Only detects CTL able to expand in vitro;
undercounts exhausted, resting and terminally differentiated CTL. |
Not a direct measure of CTL function;
uses IFN-gamma release as a marker for lytic capacity. |
Not an assay of CTL function. Requires
precise knowledge of both epitope and MHC type; not suitable for screening. |
| Relation
to environment in vivo |
Extended artificial stimulation in vitro
means the frequency and phenotype of CTL are removed from conditions
under which lysis occurs in vivo. |
Detects antigen-specific
cells without in vitro expansion. |
Cells are stained directly
ex vivo. |
| Quantitative?
|
Quantitation is possible with limiting
dilution analysis (LDA). Undercounts compared to other techniques.
|
Indirect counting. Undercounts
compared to tetramer staining. |
Direct counting of CTL |
| Sensitivity
|
LDA can be a highly sensitive technique
for detecting low frequency CTL. |
More sensitive than tetramer
staining. |
Least sensitive technique
for detecting low frequency CTL. |
| Time required |
Several days |
Overnight |
Hours |
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