Coating plates: Select assay plates that have been certified for use in ELISA applications. Dilute the coating protein (generally an antibody, however some competitive assay formats may require the antigen to be immobilized on the plate) to the desired concentration in coating buffer. For monoclonal antibodies and affinity purified polyclonal antibodies, we recommend a coating concentration of 10 micrograms per mL. For non-affinity purified polyclonals, we recommend a coating concentration of 25 micrograms per mL. Add the coating solution to the assay plate (0.1 mL per well is standard practice), cover the plate and allow it to incubate for 2 hours at room temperature, or overnight (approximately 16 hours) at 4° C.
Blocking plates: Remove the coating solution from the plate and fill the wells to 90 % of their capacity (approximately 400 microliters for a standard 96 well plate) with blocking buffer. Incubate the plate for 2 hours at room temperature, or overnight (approximately 16 hours) at 4° C.
Standards: Standard curves are typically generated by diluting the standard to 100 nanograms per mL in assay buffer, and then serially diluting to make eight standard concentrations in the range of 100 to 0.78 nanograms per mL. Apply 0.1 mls of each dilution to appropriate wells in the plate. The standard is typically run in duplicate.
Samples: Samples are diluted in assay buffer so that they will fall within the working range of the standard curve (0.78 to 100 nanograms per mL). Apply 0.1 mls of each sample to the appropriate wells in the plate. The samples are typically run in duplicate at two or more dilutions.
Incubation times: Primary incubations (i.e., those involving application of the standard or samples to the plate) are generally performed for either 2 hours at room temperature or overnight (16 hours) at 4° C. Secondary incubations those involving the application of secondary antibodies or antibody conjugates are generally done for 1 hour at room temperature. Incubation times may be further shortened by performing steps at 37° C when possible. A dilution of 5 micrograms per mL for monoclonals and 10 micrograms per mL for polyclonals is recommended as a starting point. Further optimization may be necessary depending on your assay.
Development: The two most common enzymes utilized for detection are horseradish peroxidase and alkaline phosphatase. For horseradish peroxidase the most common substrates are tetramethylbenzidine (TMB) and o-phenylenediamine (OPD). The common substrate for alkaline phosphatase is p-nitrophenyl phosphate (PNPP). We recommend following the manufacturers instructions for the use of these substrates.
Optimization: Developing a sensitive and reproducible ELISA requires optimization of many conditions including choice of antibodies, antibody concentrations, incubation times, incubation temperatures, wash conditions, etc. The choice of antibody and the concentration of antibody used may affect both sensitivity and background noise. In addition, more (or less) stringent buffer conditions may be required to achieve optimal results.