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

Important note: Information in this article was accurate in 2000. The state of the art may have changed since the publication date.

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

J Immunol Methods. 2000 Jun 23;240(1-2):143-55. Unique Identifier : AIDSLINE MED/20314647
Rininsland FH; Helms T; Asaad RJ; Boehm BO; Tary-Lehmann M; Institute of Pathology, School of Medicine, Case Western Reserve; University, Cleveland, OH 44106, USA.

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.

Keywords: JOURNAL ARTICLE Animal Base Sequence Cell Line Chromium Cloning, Molecular Comparative Study Cytotoxicity Tests, Immunologic/*METHODS CHO Cells Enzyme-Linked Immunosorbent Assay/*METHODS Flow Cytometry Hamsters Human HIV Antigens/IMMUNOLOGY Immunologic Memory Lymphocyte Transformation Molecular Sequence Data Serine Endopeptidases/GENETICS/*SECRETION Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. T-Lymphocytes, Cytotoxic/CYTOLOGY/*IMMUNOLOGY
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A00A0971

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