Target cell lysis is the hallmark of immune effector responses of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and monocytes. The most commonly used assay to measure target cell lysis is the 51Cr release assay and is considered the ‘gold standard’. However, this assay has many disadvantages that limit its use by most laboratories. Thus, several alternative assays have been developed. Some of these alternative assays are more sensitive, easy to perform and do not use radioactive elements. In this study, four of these assays were evaluated for their ability to detect antigen- specific CTL responses in equine blood. Three long-term equine arteritis virus (EAV) carrier stallions, two vaccinated stallions and one naïve stallion were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected of these stallions to be used as effector cells. The PBMCs were stimulated with EAV in vitro for 7-10 days to generate antigen-specific effector cells. The granzyme B assay, the Carboxyfluorescein succinimidyl ester (CFSE)/7-Aminoactinomycin D (7AAD) assay and the Lactate dehydrogenase (LDH) assay were performed using these effector cells and autologous equine dermal cells (isolated from each stallion) as target cells. The first two assays (i.e., granzyme B and CFSE/7AAD assays) were difficult to optimize for this study because they work well with non-adherent targets and require immediate flow cytometry analysis. The LDH assay, however detected CTL lysis in one of the two vaccinated stallions at day 99 post vaccination and no response was detected in PBMCs isolated from carrier stallions and control stallion. Based on these findings, the LDH assay is the most suitable assay since it works well with adherent target cells, it produces quantitative data, and is ideal for high-throughput screening.

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