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

J Immunol Methods. 2000 Jun 23;240(1-2):143-55. doi: 10.1016/s0022-1759(00)00191-5.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • CHO Cells
  • Cell Line
  • Chromium
  • Cloning, Molecular
  • Cricetinae
  • Cytotoxicity Tests, Immunologic / methods*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Flow Cytometry
  • Granzymes
  • HIV Antigens / immunology
  • Humans
  • Immunologic Memory
  • Lymphocyte Activation
  • Molecular Sequence Data
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism*
  • T-Lymphocytes, Cytotoxic / cytology
  • T-Lymphocytes, Cytotoxic / immunology*


  • HIV Antigens
  • Chromium
  • GZMB protein, human
  • Granzymes
  • Serine Endopeptidases