The assays are currently performed as optimized or validated assays under Good Clinical Laboratory Practice (GCLP) guidelines. The laboratory can perform these assays in support of both pre-clinical studies and clinical trials, as well as for basic research purposes.
I. Interferon Gamma (IFN-γ) Enzyme Linked ImmunoSpot (ELISpot).
The principle of this assay is based on the detection of the IFN-γ released by the T cells upon stimulation with antigens of interest, and quantified as the formation of spot son a solid membrane. Each spot represents an antigen-specific T cell. Data are reported as Spot Forming Cells per 2x105 PBMC.
II. Granzyme B flow-based Cytotoxicity Assay.
In our assay platform, active Granzyme B (GzB) is identified by its ability to cleave a selective peptide-linked fluorogenic substrate containing a GzB recognition motif. Fluorescence is conferred upon hydrolysis of the substrate, thereby producing a signal that can be used to directly identify individual cells targeted by the cytotoxic effector cells. Enumeration of these cells by high-throughput flow cytometry provides a rapid and quantitative measure of cytotoxic activity as mediated by NK cell, T cell, and ADCC responses.
III. Luciferase- based Cytotoxicity Assay.
The principle of this assay is based on the utilization of HIV-1 Infectious Molecular Clones (IMC) that either represent a full length infectious HIV-1 isolate or designed to encode HIV-1 env genes of interest in cis within an isogenic common backbone that also expresses the Renilla luciferase reporter gene and preserves all viral open reading frames. The luciferase reporter is under the control of the HIV-1 Tat gene. Upon HIV-1 infection of any cellular subset of interest, expression of Tat during HIV-1 replication will induce expression of the luciferase and the presence of infected cells can be easily quantified as Relative Luminescence Units. In the presence of the cytotoxic cells of interest, the elimination of infected target cells can quantified after a minimum of two hours by evaluating the residual luminescence units.