Abstract
The quantification of frequency of IFN-γ-producing T cells responding to donor alloantigen using the IFN-γ enzyme linked immunosorbent spot (ELISPOT) holds potential for pretransplant and posttransplant immunological risk stratification. The effectiveness of this assay, and the ability to compare results generated by different studies, is dependent on the utilization of a standardized operating procedure (SOP). Key factors in assay standardization include the identification of primary and secondary antibody pairs, and the reading of the ELISPOT plate with a standardized automated algorithm. Here, we describe in detail, an SOP that should provide low coefficient of variation results. For multicenter trials, it is recommended that groups perform the ELISPOT assays locally but use a centralized ELISPOT reading facility, as this has been shown to be beneficial in reducing coefficient of variation between laboratories even when the SOP is strictly adhered to.