text: #Antibody coating: Throughout the ELISpot Assay technique, different substances are added to and washed away from wells. Wells are found on a laboratory plate with tiny dishes/bowls that can be filled with a substance to be examined; the amount of wells on a plate varies, but it usually ranges from 16-100. The first substance added to the wells are cytokine specific monoclonal antibodies. These antibodies coat the walls of the wells for future binding to cytokine. The monoclonal antibodies means that the antibody is produced from a single cell lineage, and is only able to bind to one protein epitope. Polyclonal antibodies, on the other hand, are capable of binding to multiple epitopes of the same protein.
  #Cell incubation: The desired cells being observed and analyzed are added to the wells. Each well can have the presence or absence of stimuli that activate the secretion of cytokine in cells. During cell incubation, the cells are allowed to react to any present stimuli and secrete cytokine. There are many procedures and methods to follow to ensure proper cell handling. To make sure that cells are of high quality, cells in blood samples should be lightly agitated if stored for longer than 3 hours, the blood samples should be diluted in PBS (phosphate buffered saline) before being stored, and the blood samples should not have granulocytes. Any cells that have been cryopreserved and thawed should be allowed to rest for an hour or more at 37 degrees Celsius (the typical temperature of the human body). There are also many things that should be taken into consideration when incubating the cells, such as making sure that the cells do not experience sudden movements that could affect spot formation, or that the incubator's humidity is high enough to avoid excessive evaporation and drying out the wells.
  #Cytokine capture: Since the cells are surrounded by cytokine specific monoclonal antibodies that coat the walls of the wells, cytokine that has been secreted by the incubated cells will start to attach to the antibodies at a specific epitope.
  #Detection antibodies: At this point, the wells must be rinsed in order to get rid of the cells and any other undesirable substances. All that should remain are the cytokine specific monoclonal antibodies and any cytokine that bonded to the antibodies. Biotinylated cytokine-specific detection antibodies are then added to the well. These cytokine-specific detection antibodies will bind to any cytokine that is left in the well since the cytokine is still attached to the first set of antibodies used. Since the cytokine attached to the first set of antibodies coating the wells, the cytokine was not washed away when the wells were rinsed.
  #Streptavidin-enzyme conjugate: Streptavidin-enzyme conjugate is added to the wells in order to bind with the detection antibodies. The purpose of biotinylating the cytokine-specific detection antibodies added to the wells in the previous step is so that the antibody can bind to the new streptavidin-enzyme conjugate. Biotinylation basically creates a strong affinity between the biotin on the cytokine-specific antibody and the streptavidin on the conjugate.
  #Addition of substrate: A substrate is added to the wells, and is catalyzed by the enzyme conjugate added in the previous step. This reaction forms insoluble precipitate that forms spots in the wells. The substrate that you use in this step will depend on the type of enzyme used in the previous step. If streptavidin-ALP (streptavidin and alkaline phosphatase conjugate) is used, then using BCIP/NBT-plus (a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride) as a substrate will produce more distinct spots that are easier to analyze. If streptavidin-HRP (streptavidin and horseradish peroxidase conjugate) is used, then using TMB (tetramethylbenzidine) as a substrate will produce better results.
  #Analysis: The spots that are formed can then be read on an automated ELISpot reader, or counted under a dissection microscope, and further used to calculate the frequency of cytokine secretion.