Cellular immune response such as T cell production of IFN-γ is taken as a measure of vaccine immuno-genicity in experimental vaccine trials, especially for new vaccines such as those against tuberculosis and human immunodeficiency virus (HIV). Currently, a variety of methods, including proliferation assays, cytotoxic T lymphocyte assays, tetramer staining, intracellular cytokine staining and cytokine enzyme-linked immunospot (ELISPOT) assays can be used to assess cell-mediated immune responses (1, 7). Efficient clinical trials to evaluate vaccination aimed at eliciting specific T cells require rapid, accurate, and reliable methods for detecting and quantifying these cells. The interferon (IFN)-γ ELISPOT assay is a method for determining the number of individual T cells secreting this cytokine after stimulation with a specific antigen or peptide (8, 10). The number of spots increases in proportion with the strength of the immune response. The important advantages of the IFN-γ ELISPOT assay are that it directly measures T cell-mediated immune responses, is relatively simple to perform, does not require large numbers of cells and is sensitive. Hence, it is widely used for monitoring the effec-tiveness of vaccines for inducing cell-mediated immunity (5). Several groups in China are actively involved in HIV vaccine research with the aim of eliciting both humoral and cellular immune responses. As these investigations move into preclinical and clinical trials, it will be important to objectively assess the qualitative and quantitative differences in the cellular responses elicited by the different vaccine candidates. However, since the ELISPOT is a proliferation assay for the detection of T cells, many factors including the assay format, nature of the cells being analyzed, choice of antigens and method of plate reading may affect the results. In order to assess the competency and comparability of different laboratories when performing ELISPOT assays, a quality control panel is required. Generally, the volume of a quality control panel should be sufficient for supply and its quality should be stable for a certain time period. In the present study, the possibility of using cryopreserved peripheral blood mononuclear cells (PBMCs) as a quality control panel for ELISPOT was preliminarily evaluated.
Preliminary Study on a Potential Panel for Quality Assurance of ELISPOT
- Received Date: 15 January 2008
- Accepted Date: 29 July 2008
Abstract: Abstract: The ELISpot assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories