An ELISA uses antigens that are adsorbed to wells of plastic microtiter plates, which are then blocked using an agent to cover any open surface left in the well. The tester adds a primary antibody to specifically adhere to the antigen and then adds a secondary antibody, combined to a reactive enzyme, to bind to the primary antibody. The tester then adds a color-changing substrate to visualize the concentration of the antigen. The color of the substrate increases along with the concentration of the antigen.
Protocols described in "Methods in Molecular Biology" require the following reagents for the either type of ELISA: bicarbonate coating buffer; PBS containing bovine serum albumin and Tween 20; phosphate buffer; citrate buffer; stop solution; PBS washing solution and hydrogen peroxide. The protocols further describe specific compositions and percentages for each reagent.
Add carbonate buffer to each well of a microtiter plate followed by diluted antigen in a bicarbonate coating buffer. Incubate at room temperature for two hours or overnight. Wash the plate and allow to dry at room temperature. Add horse-radish peroxidase conjugate to each well, diluted to appropriate concentration, and allow to incubate at room temperature for two additional hours. Add blocking buffer and wash each well with washing solution. Add citrate buffer to each well and allow for color development. After 10 minutes, stop the reaction with stop buffer. Measure absorbance using a spectrophotometer.
Based on the work of Munro and Lasley (1988), the Sandwich ELISA uses a horseradish peroxidase ligand, antiserum and commercial standards in a 96-well microtiter plate. The wells of the plate are coated with diluted antiserum in bicarbonate coating buffer. Seal plates tightly and incubate for 12 hours at 4 degrees Celsius. Dilute the enzyme conjugate in phosphate buffer. Remove the excess antibody with wash solution and allow to dry at room temperature. Dispense phosphate buffer into each well followed by conjugate solution containing standards or samples. Cover the plates for two hours. Wash the plates and allow to dry at room temperature. Add citrate buffer to wells and allow color to develop. After 60 minutes, stop the reaction with stop solution. Measure absorbance with spectrophotometer.