Blocking Unoccupied Sites
In an ELISA, it is important to block the unoccupied sites on the surface of the well to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay. A variety of blocking buffers ranging from nonfat milk to highly purified proteins have been used to block unreacted sites. The blocking buffer should improve the sensitivity of the assay by reducing the background interference. An individual blocking buffer will not be compatible with every system; for this reason, a variety of blockers in both Tris buffered saline (TBS) and phosphate buffered saline (PBS) are available. The proper choice of blocker for a given assay depends on the antigen itself and on the type of enzyme conjugate to be used. For example, with applications using an alkaline phosphatase conjugate, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.
For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Many factors can influence nonspecific binding, including various protein:protein interactions unique to a given ELISA. The most important parameter when selecting a blocker is the signal:noise ratio, which is measured as the signal obtained with a sample containing the target analyte as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background and a reduced signal:noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the enzyme, again causing a reduction of the signal:noise ratio. When developing any new ELISA, it is important to test several different blockers for the highest signal:noise ratio in the assay. No single blocking agent is ideal for every occasion because each antibody-antigen pair has unique characteristics.