immunospot

Immunospot refers to a class of immunological assays designed to quantify secreted proteins from individual immune cells. The most widely used form is the ELISPOT (enzyme-linked immunospot) assay, in which secreted cytokines or other molecules are captured on a membrane by immobilized antibodies, forming discrete spots that can be counted to reflect the number of responsive cells.

Mechanism and procedure: Plates are coated with capture antibodies specific to the molecule of interest. Isolated immune cells are added and stimulated with antigen or polyclonal activators. Secreted proteins are captured around the secreting cell, and after incubation the cells are removed. A detection antibody is applied, followed by an enzyme substrate that develops a visible spot. Each spot corresponds approximatively to a single secreting cell, allowing quantitative measurement of the frequency of responding cells within a sample.

Variants and multiplexing: FluoroSpot is a related approach that uses fluorescent detection to visualize multiple cytokines in the same well, enabling multiplex analysis. B cell ELISPOT assays detect antibody-secreting cells. Other formats include high-throughput adaptations and automated plate readers for counting spots.

Applications: Immunospot-based assays are used to assess cellular immune responses in infectious disease research, vaccine development, cancer immunotherapy, autoimmune conditions, and transplant medicine. They are employed to monitor vaccine efficacy, characterize T cell or B cell responses, and support immunomonitoring in clinical trials.

Advantages and limitations: The method offers high sensitivity and single-cell resolution with relatively low cell numbers and straightforward readouts. Limitations include technical complexity, the need for fresh or well-preserved samples, potential non-specific binding, and limited throughput compared with some other assays.