ELISPOT

The ELISpot assay ( enzyme-linked immunosorbent spot assay ) is used for detecting secreted cytokines or antibodies are immobilized on individual immune cells by stimulation with antigens to a membrane.

Stimulation

The selective binding of the secreted proteins is analogous to ELISA, but with on an opaque membrane (mostly from PVDF) -immobilized antibodies ( engl. coating antibody or capture antibody ). After coating the membranes in microtiter plate format with the first antibody, the remaining non-specific protein binding sites of the membranes are blocked with sterile FCS or BSA solutions. The coated plates are provided in connection with the secreting cells and a stimulus to be examined (eg, an antigen or a peptide derived from it ). The secreted cytokines or antibodies bind to the immobilized first antibody. After a vibration-free incubation period, usually between 16 hours and 48 hours, the cells are removed and the detection can be carried out under sterile conditions. When shocks occur in non- adherent cells ( synonym: suspension cells) and lateral movements to a falsely elevated number of spots.

Detection

The detection is carried out on the selective binding of a second, usually biotinylated antibody to a different epitope of the protein to be detected, usually followed by the binding of a streptavidin -coupled with the reporter enzyme and precipitation of a dye to the Sekretionsstelle. By a dye substrate converting enzyme, which is coupled to the second antibody, or to another Signalamplifikator (for example, biotinylated detection antibody and avidin - conjugates or detection antibody and polyclonal anti-Fc antibody conjugates ), the dye of the Sekretionsstelle precipitates. This allowed depending on the dye a representation of the cytokines as small red ( Naphthol-AS-Mx-Phosphate/Fast Red TR or H2O2/AEC ), brown ( with H2O2 and DAB or TMB), or blue spots ( with NBT / BCIP ), with to make a diameter of 75-400 microns visible. By comparison with the corresponding negative controls ( with an irrelevant antigen or peptide), a reaction can be detected. A positive control shows the successful implementation of the ELISPOTS, such as whether the cells used were still alive. The automatic counting using a camera and software determined at the stained and dried plates, how many of the introduced cells responded in each approach, and with what intensity ( spot area, spot color saturation) they responded.

This method is inter alia in the diagnosis of autoimmune diseases, transplant risks, allergies and infectious diseases, or in the vaccine research application. T- cells and B- cells ELISPOTs are performed for the measurement of cytokine responses by peptide stimulation to identify immunogenic T- and B -cell epitopes (English epitope mapping for, epitope mapping "). By ELISPOT epitopes identified are registered in the IEDB for example.