ELISpot

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Schematic principle of the ELISPOT

The ELISpot Assay ( Enzyme Linked Immuno Spot Assay ) is used to detect secreted cytokines or antibodies that are secreted by individual immune cells after stimulation with antigens and immobilized on a membrane .

stimulation

The selective binding of the secreted proteins takes place in the same way as ELISA , but with antibodies ( coating antibody or capture antibody ) immobilized on an opaque membrane (mostly made of PVDF ). After coating the membranes in microtiter plate format with the first antibody, the remaining unspecific protein binding sites on the membranes are blocked with sterile FCS or BSA solutions. The coated plates are then provided with the secreting cells and a stimulus to be examined (e.g. an antigen or a peptide derived therefrom ). The secreted cytokines or antibodies bind to the immobilized primary antibody. After a vibration-free incubation period, usually between 16 hours and 48 hours, the cells are removed and the detection can take place under non-sterile conditions. In the case of vibrations, non-adherent cells (synonym: suspension cells ) experience lateral movements and an incorrectly increased number of spots.

Detection

After the incubation period, the cells are removed and the secreted cytokines or antibodies are immunostained . The detection takes place via the selective binding of a second, mostly biotinylated antibody against another epitope of the cytokine to be detected, mostly followed by the binding of a streptavidin with coupled reporter enzyme and the precipitation of a dye at the secretion site. Interferon-γ is often measured as the cytokine to measure the reaction of cytotoxic T cells . By a dye substrate -converting enzyme , which to the second antibody, or to another Signalamplifikator (z. B. biotinylated detection antibody and avidin-conjugates or detection antibody and polyclonal anti-F c is coupled antibody conjugates), the dye falls on the Sekretionsstelle from . Depending on the dye, this allows the cytokines to be displayed as small red ( Naphthol-AS-MX-Phosphate / Fast Red TR or H 2 O 2 / AEC ), brown (with H 2 O 2 and DAB or TMB ) or blue dots (with NBT / BCIP ), with a diameter of 75 to 400 µm. A reaction can be detected by comparison with the corresponding negative controls (with an irrelevant antigen or peptide). A positive control with a mitogen such as Pokeweed Mitogen (PWM) or phorbol-12-myristate-13-acetate (PMA) shows that the ELISpot has been carried out successfully, for example whether the cells used were still alive. The mitogen produces an antigen-independent activation of the cells and a subsequent secretion of the sought-after immune messenger substance. In the case of the stained and dried plates, the automatic counting by means of a camera and software determines how many of the cells used reacted in each batch and with what intensity (spot area, spot color saturation) they reacted.

This method is used in the diagnosis of autoimmune diseases , transplant risks , allergies and infectious diseases or in vaccine development. For T cells and B cells , ELISpots are used to measure cytokine reactions after peptide stimulation in order to identify immunogenic T and B cell epitopes ( epitope mapping for ' epitope mapping '). Epitopes identified by ELISPOT are e.g. B. registered in the IEDB .

Individual evidence

  1. Czerkinsky C, Nilsson L, Nygren H, Ouchterlony O, Tarkowski A: A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells . In: J Immunol Methods . 65, No. 1-2, 1983, pp. 109-121. doi : 10.1016 / 0022-1759 (83) 90308-3 . PMID 6361139 .

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