T CELL ASSAYS
EpiVax offers a variety of T cell assays suitable for measuring the immunogenicity of experimental proteins and peptides in an ex vivo environment. T cell assay readouts described in more detail below, including ELISA to measure cytokine levels in the culture supernatants and ELISpot to quantify the frequency of cytokine-producing cells. Flow cytometry is capable of measuring a number of different T cell properties, including relative proportions of T cell subsets, expression of activation markers, intracellular cytokine detection, and proliferation.
T cell assays can be utilized to identify and measure a recall or memory response in PBMCs derived from subjects who have been exposed to a given biologic product either as therapy or within the within the context of a clinical trial. Human peripheral blood mononuclear cells (PBMCs) are isolated from whole blood. The expected yield is approximately 2-3×106 viable PBMC/ml whole blood. As the precursor frequency of the antigen-specific cells in a recall response is relatively high, whole PBMC assays are both rapid and sensitive with no pre-treatment or enrichment required. Epitope mapping of the recall response can be performed using epitope-specific peptides from the whole protein at the time of challenge.
T CELL ASSAY READOUTS
Standard ELISA to measure cytokines in serum or conditioned media
The sandwich enzyme-linked immunosorbent assay (ELISA) begins by pre-coating onto a PVDF (polyvinylidene difluoride)-backed microplate an antibody (either monoclonal or polyclonal) specific for the analyte of interest. Samples (≥50ul conditioned media or serum) are pipetted into the wells and the microplate is placed into a humidified 37°C CO2 incubator for 24-48h. While incubating, the immobilized antibody at the base of the well collects the analyte of interest. At the end of the incubation period, any unbound material is washed away and a biotinylated polyclonal antibody specific for the selected analyte is added to the wells. After this incubation period and a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is removed by washing before the TMB substrate solution is added. A colored precipitate forms that is quantified by measuring the optical density (OD) of the sample and compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule. ELISAs for IFN-g, IL-2, and IL-4 are well established, and this technique is easily expanded to measure other cytokines. Lower limits of detection are approximately 10 pg/ml and can be increased by concentrating experimental samples.
SearchLight Proteomic Array to measure up to 16 analytes in a single sample
SearchLight Proteomic Arrays are quantitative multiplexed sandwich ELISA developed by Aushon Biosystems. By spotting capture antibodies in a 3 x 3 array in each well of the 96-well plate, they allow measurement of 9 different cytokines per well simultaneously in a 50µl sample. For example, in the context of immunogenicity and T cell activation, analysis of the following cytokines would be suggested: IFN-g, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, and TNF-a. These multiplexes can also be customized and expanded to 4 x 4 arrays that will measure 16 cytokines per well with a menu of over 50 human cytokines and chemokines from which to select. The quantitative chemiluminescent detection system allows for detection of low abundance cytokines with sensitivity to 20pg/ml.
ELISpot to enumerate cytokine-producing cells
ELISpot assays are based on the same principles as the ELISA. An antibody (either monoclonal or polyclonal) specific for the analyte of interest is pre-coated onto a PVDF (polyvinylidene difluoride) -backed microplate. Human PBMCs (approximately 250,000 cells per well in triplicate with poisitive and negative control wells) are pipetted into the wells with or without antigen and the microplate is placed into a humidified 37°C CO2 incubator for 24-48h. While incubating, the immobilized antibody at the base of the well collects the analyte of interest as it secreted by each local cell. At the end of the incubation period, cells and any unbound material are washed away and a biotinylated polyclonal antibody specific for the selected analyte is added to the wells. After this incubation period and a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is removed by washing before the BCIP/NBT substrate solution is added. The colored precipitate forms, appearing as a blue-black spot at each site of cytokine production. Thus, each individual spot represents an individual analyte-secreting cell. These spots are counted with an automated ELISpot reader system. ELISpots for IFN-g and IL-4 are well established, and this technique is easily expanded to other analytes. This assay is able to detect as few as 50 cytokine-secreting cells per million.
Custom Flow Cytometry Services
The EpiVax laboratory is equipped with a BD FACSCalibur Flow cytometry system. The flow cytometer is equipped with a standard argon and optional red laser that together allow for the analysis of four different fluorochromes simultaneously. Cell samples (typically 1×106 per sample) in suspension are prepared by standard protocols whereby the cell surface or intracellular proteins of interest are labeled with fluorochrome-conjugated antibodies. Cells enter the flow cytometer in a hydrodynamically focused stream of fluid and one by one pass through the laser beams. Utilizing a specific configuration of light filters and detectors, the fluorescent light emitted from the fluorochromes is separated, collected and digitized. The data collected can be presented in one-dimension as a histogram, to demonstrate the proportions of cells expression a given marker and the intensity with which it is expressed. Data can also be presented in a two-dimensional “dot-plot” were two different markers can be examined by plotting the fluorescent intensity for each individual cell on x- and y- axes. Flow cytometry offers several unique advantages, including the rapidity with which thousands to millions of cells can be analyzed in a short period of time, high sensitivity and specificity, and the ability to define certain parameters of cell function within specific cell subsets. Applications of this technique include, but are not limited to, 4-color staining of cell surface markers, phenotypic analysis to determine relative proportions of cell subsets (CD4 vs CD8 T cells, B cells, NK cells, etc.), phenotypic analysis to determine activation of a cell subset (CD62L, CD45 RA/RO, CD154), intracellular staining for cytokines (IFN-g, IL-2, IL-4) or transcription factors (FoxP3), proliferation (CFSE), cytokine capture assays (IL-10, IL-2) and tetramer staining to quantify antigen-specific T cells.
Cell depletion or enrichment
The EpiVax laboratory is equipped to perform MACS cell separation. MACS® Technology, a product of Miltenyi Biotech, Inc., is based on MACS MicroBeads, MACS Separators, and MACS Columns. MACS MicroBeads are superparamagnetic particles of approximately 50 nm in diameter composed of a biodegradable matrix and conjugated to a monoclonal antibody with specificity to a cell surface protein. When a MACS Column is placed in a MACS Separator, a strong permanent magnet, a high-gradient magnetic field is induced on the column matrix. Cells labeled with MACS MicroBeads are retained in the magnetic field while unlabeled cells pass through and can be collected. The labeled cells are released after removal of the column from the magnet. Thus, with MACS Technology both labeled and unlabeled live cell fractions can easily be isolated with high purity and viability. This technique can be used to enrich for specific populations, as in positive selection of CD4+ T cell using anti-CD4 MicroBeads. It can also be used for negative selection, as in enrichment to pan T cell populations by depletion of B cells, monocytes, NK cells and granulocytes. The ability to enrich and/or deplete certain populations is particularly useful when examining small populations such as antigen-specific cells or regulatory T cells. Enriched cells can immediately be used for further experiments.
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