Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity

A major goal in immunodiagnostics has been the development of assay systems that can measure CD8 + T cell immunity in humans, directly ex vivo, at high resolution, and with high throughput. We established granzyme B (grB) enzyme-linked immunospot assay (ELISPOT) in conjunction with image analysis to...

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Bibliographic Details
Published in:Journal of immunological methods 2000-06, Vol.240 (1), p.143-155
Main Authors: Rininsland, Frauke H, Helms, Thomas, Asaad, Robert J, Boehm, Bernhard O, Tary-Lehmann, Magdalena
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
CD8 antigen
Cell Line
CHO Cells
Chromium
Cloning, Molecular
Cricetinae
Cytotoxic T cells
Cytotoxicity Tests, Immunologic - methods
ELISPOT
Enzyme-Linked Immunosorbent Assay - methods
Flow Cytometry
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Granzyme B
Granzymes
HIV Antigens - immunology
Humans
Immunologic Memory
Lymphocyte Activation
Memory
Molecular immunology
Molecular Sequence Data
Peptide-specificity
Serine Endopeptidases - genetics
Serine Endopeptidases - metabolism
T-Lymphocytes, Cytotoxic - cytology
T-Lymphocytes, Cytotoxic - immunology
Techniques
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Summary:A major goal in immunodiagnostics has been the development of assay systems that can measure CD8 + T cell immunity in humans, directly ex vivo, at high resolution, and with high throughput. We established granzyme B (grB) enzyme-linked immunospot assay (ELISPOT) in conjunction with image analysis to this end. Using grB transfected and untransfected Chinese hamster ovary (CHO) cells and T cell lines, we show that the antibody pair utilized was grB-specific and that only activated T cells secrete grB. GrB release began within 4 h after antigen stimulation and stopped within 40 h. Side-by-side comparison showed grB ELISPOT assays to have a higher resolution than classic chromium-release assays in terms of signal-to-noise ratio. The linearity of the relation of the number of CD8 + effector T cells plated to grB spots detected suggests that grB ELISPOT assays measure the frequencies of grB-secreting cells directly. Reactivity to HIV peptides was seen in grB ELISPOT assays of freshly isolated PBMC from HIV patients, consistent with the detection of peptide-specific memory cells. The higher resolution and lower labor and material investment should make grB ELISPOT assays an attractive alternative to chromium-release assays in monitoring the clonal sizes of specific CD8 memory cells in vivo.
ISSN:0022-1759
1872-7905
DOI:10.1016/S0022-1759(00)00191-5