ELISpot Analysis

ELISpot (enzyme-linked immunosorbent spot) assays are highly sensitive immunoassays that measure the frequency of antigen-specific T cells by measuring the number of cytokine-secreting cells within the blood at a single cell level. ELISpot testing can also be used to quantify the number of antigen-specific B cells that have recently been activated in vivo or to quantify memory B cells which have been activated with a specific stimulus ex vivo. Key applications of ELISpot analysis include monitoring immune responses during preclinical testing and clinical trials, ex vivo stimulation of immune cells to measure frequency of cells specific to the antigen of interest, and vaccine efficacy.

 

Antigen Specific T Cell ELISpot Assay Workflow

  • For the cytokine(s) of interest, fresh or thawed peripheral blood mononuclear cells (PBMCs) or other relevant tissue cells are cultured on a membrane surface coated with a specific capture antibody in the presence or absence of antigenic stimuli.
  • The cytokine(s) secreted by the cells after the stimulation is thus captured onto the membrane surface.
  • After an appropriate incubation time, cells are removed, and the secreted cytokine(s) are detected using a detection antibody.
  • The binding is captured using enzymatic (horseradish peroxidase (HRP) or alkaline phosphatase (ALP) or fluorescent readout, where spot become visible.
  • The plate is then washed to stop the reaction, and the membrane is allowed to dry before analysis.
  • ELISpot analysis can be performed for a single cytokine or a combinations of two cytokines.

 

ELISpot Assay Validation

Prior to ELISpot analysis of preclinical and clinical samples, a ‘fit-for-purpose’ validation is designed based on the logistics of each trial, along with the intended purpose of the data. For clinical trial analysis, blood is drawn from subjects and is then transported to either one of our laboratories or a local laboratory for PBMC isolation within an appropriate timeframe. PBMCs may be analyzed immediately or frozen and stored in liquid nitrogen until ELISpot testing.

Clinical Sample Workflow

A flow diagram demonstrating the clinical sample workflow from blood draw to sample transport through to cell isolation and cell storage and finally to cytokine analysis by ELISpot testing.

Validation of key elements of pre-analytical sample preparation and ELISpot setup is critical prior to analysis of clinical samples for each cytokine or assay. This includes criteria like critical reagents, validating operators, and testing parameters, such as intra-assay and inter-analyst/assay precision, linearity, stability, and LOD/cut-point calculations.

Representative data is shown below from a validation study to assess IFN-γ secreting cells in response to the CEF-X, a positive control composed of a pool of viral peptides including CMV, EBV, and influenza virus.

Representative Images from Healthy Donor PBMCs

Triplicate wells showing IFN-γ spot forming units from frozen PBMCs from a healthy single donor. PBMCs were either unstimulated (media only) or analyzed by ELISpot for activation by a negative control peptide pool (Actin) or a positive control peptide pool (CEF-X).

PBMCs from healthy donors were isolated and frozen down to mimic clinical trial logistics. The healthy donor PBMCs were then established in culture alongside commercially available frozen quality control (QC) PBMCs and challenged with media (unstimulated), a negative control peptide pool (Actin), and a positive control peptide pool (CEF-X).

Intra- and Inter-Donor Variability Determined through ELISpot Analysis

Graph displaying Spot Forming Units (SFU) per 1 x 106 PBMCs in response to CEF-X antigen over five runs for four donors. The QC donor PBMCs were from a donor known to respond to re-stimulation with CEF-X peptides and some intra-donor variability was observed through ELISpot.

Some variation is also expected in the magnitude of the response between different donors. Once it has been deemed fit for the intended purpose, it can be applied to the investigation of samples from clinical trial subjects.

ELISpot testing can be deployed as an exploratory endpoint in early-phase clinical trials by obtaining blood samples from subjects before and after dosing. ELISpot assays can be used to determine how antigen-specific cell frequencies have changed in response to the therapeutic intervention and can be a key readout in many vaccine trials to determine the elicitation of a T cell response or B cell response against the antigens contained in the vaccine.

Our ELISpot experts are available to discuss your project needs and help you figure out an effective application for the features of this platform.

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Frequently Asked Questions (FAQs) About ELISpot Assays

  • What are the benefits of using ELISpot assays?

    One of the main benefits of ELISpot assays is that they are extremely sensitive for the detection, measurement, and analysis of rare, antigen-specific T cells and B cells and because of its high sensitivity, it can detect and enumerate a single cell that secrets a protein of interest such as a cytokine. It also provides an indication of the type of cytokine response that has been elicited and hence the type of immune response.

  • What are some of the factors that can influence ELISpot assay performance?

    ELISpot assay performance can be influenced by several different factors, including cell recovery and viability, operator technique and proficiency, and result variability.