FluoroSpot assays utilize the ELISpot assay principle which is based on the two-site sandwich enzyme-linked immunosorbent assay (ELISA) technique. Antibodies specific for the chosen analytes are coated by the end-user onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are then pipetted into the wells, and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibodies, in the immediate vicinity of the secreting cells, bind the secreted analytes.
After washing away any cells and unbound substances, a biotinylated detection antibody specific for the first analyte is added to the wells.
Following a wash to remove any unbound biotinylated antibody, a mixture of NorthernLights™ 557 (NL557)-conjugated Streptavidin and a Alexa Fluor® (A488)-conjugated detection antibody specific for the second analyte is added to the wells. After washing away any unbound fluorescent tags, NorthernLights Fluorescence Enhancer is added to the wells. Following incubation with the fluorescent enhancer, the solution is aspirated and the plate dried. The developed microplate can then be analyzed by counting spots using either an automated ELISpot reader system capable of detecting green and red fluorescent spots, or manually using a conventional epifluorescence microscope.
Alexa Fluor is a registered trademark of Life Technologies.