title: Granzyme B ELISPOT assay for ex vivo measurements of T cell immunity.
abstract: 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.
authors.affiliation: Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: F H
authors.initials: FH
authors.lastname: Rininsland
authors.forename: T
authors.initials: T
authors.lastname: Helms
authors.forename: R J
authors.initials: RJ
authors.lastname: Asaad
authors.forename: B O
authors.initials: BO
authors.lastname: Boehm
authors.forename: M
authors.initials: M
authors.lastname: Tary-Lehmann
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002857:Chromium; D003001:Cloning, Molecular; D006224:Cricetinae; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D053804:Granzymes; D015488:HIV Antigens / Q000276:immunology; D006801:Humans; D007156:Immunologic Memory; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*
chemical_list: D015488:HIV Antigens; D002857:Chromium; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases