The full name of ELISPOT is enzyme-linked immunospot assay, which combines cell culture technology and enzyme-linked immunosorbent assay (ie ELISA technology) to detect the secretion of cytokines at the single cell level. The technical principle is summarized in one sentence: the cytokines secreted by cells in culture are captured with antibodies and displayed in the form of enzyme-linked dot coloration (Sedgwick JD 2005).
The detection of cytokines by this technique has three major advantages: First, high sensitivity. Only one cytokine-secreting positive cell in one million negative cells can be detected. This is by far the most sensitive detection technology, and the sensitivity is 2-3 orders of magnitude higher than the traditional ELISA method. Second, single-cell level, live cell function detection. ELISPOT measures secretion from a single cell rather than the average secretion of a population of cells. In the process of detection, there are live cell culture and antigen stimulation stages, and the function of living cells is detected, not the remnants of dead cells. Third, the operation is simple and economical, and high-volume screening can be performed. ELISPOT has no complicated in vitro cell expansion process, does not use isotopes, and does not require large-scale, specialized experimental equipment. According to standardized experimental operation, one experimenter can process hundreds of samples at the same time, and the efficiency is much higher than other detection methods (Kalyuzhny AE 2005).
The experimental design was carried out on a 96-well culture plate, directly using the plastic bottom of the culture plate or PVDF membrane and nitrocellulose membrane as the matrix, and coated with specific monoclonal antibodies to capture cytokines secreted by cells. (Due to the process involved in cell culture, the requirements for monoclonal antibodies are much higher than the capture antibodies in ELISA. The antibodies need to be non-toxic, endotoxin-free, and have high affinity.) After that, add them to the wells of the culture plate. Cell culture medium (now that serum-free ELISPOT technology has matured, and the culture medium can no longer contain serum), cells to be tested, and antigenic stimuli for culture.
Under the stimulation of specific antigens or non-specific mitogens, within a few hours, T cells will begin to secrete various cytokines. Cytokines are immediately captured by monoclonal antibodies on the membrane beneath the cells. After washing off the cells, the captured cytokines can be combined with the biotin-labeled secondary antibody, and then combined with the enzyme-labeled avidin and biotin for chemical enzyme-linked color development, which can form a localized membrane on the membrane. round spots. Each spot corresponds to a cell that secreted cytokines, and these cells are called Spots forming cells (SFCs). The frequency of positive cells can be calculated by counting the number of spots on the membrane and dividing by the total number of cells initially added to the well.
