Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses

J Immunol Methods. 2002 Mar 1;261(1-2):145-56. doi: 10.1016/s0022-1759(01)00566-x.


To be able to determine the frequencies of helper-type T lymphocyte (Th) precursors specific for viral or tumor-associated antigens, an ELISPOT assay for IL-5 production was developed. The assay reproducibility was first determined using fresh or cryopreserved peripheral blood mononuclear cells (PBMC) obtained from six normal donors and tested for IL-5 production after 48 h stimulation with phytohemagglutinin (PHA). Both inter-assay (within-subject CV=1.6%) and intra-assay (within-subject CV=0.8%) variabilities were found to be acceptable, and the frequencies of IL-5 secreting cells were comparable in cryopreserved and fresh PBMC of the same donors. The presence and frequency of Th precursor cells to viral capsid L1 protein (viral-like particles, VLP-L1) of human papillomavirus type 16 (HPV-16) in PBMC obtained from seven non-immunized donors and two volunteers immunized with VLP-L1 were then evaluated. Using autologous dendritic cells as antigen-presenting cells (APC) for VLP-L1 in direct 48-h ELISPOT assays, the mean frequency of IL-5 secreting CD4+ T cells was found to be <1/10(5) (negative) in normal donors but was 1/2, 436 and 1/3678 in the two volunteers immunized with VLP-L1. The assay is applicable to monitoring of the frequency of antigen-specific T cells in the peripheral circulation of individuals immunized with HPV-derived antigens. To test the assay utility in the assessment of Th tumor-reactive lymphocytes, we also used it to determine the frequency of the wild-type sequence (wt) p53(22-36) peptide-specific, HLA-DR4-restricted T cells in the bulk CD4+ T cell line. This frequency was 1/33. The ELISPOT assay for IL-5 production can be reliably used to measure Th-type responses in a variety of experimental settings.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral
  • CD4-Positive T-Lymphocytes / immunology
  • Capsid Proteins*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • HLA-DR4 Antigen
  • Humans
  • Immunization
  • In Vitro Techniques
  • Interleukin-5 / analysis*
  • Interleukin-5 / biosynthesis
  • Oncogene Proteins, Viral / immunology
  • Papillomaviridae / immunology
  • Reproducibility of Results
  • T-Lymphocytes, Helper-Inducer / immunology*
  • Tumor Suppressor Protein p53 / immunology


  • Antigens, Viral
  • Capsid Proteins
  • HLA-DR4 Antigen
  • Interleukin-5
  • Oncogene Proteins, Viral
  • Tumor Suppressor Protein p53
  • L1 protein, Human papillomavirus type 16