It prevents non-specific binding of the secondary antibody, and thus reduced background.

Primary antibodies are directly targeted against the antigen of interest, while secondary antibodies are targeted against the primary antibody. Secondary antibodies are used to amplify the signal from the primary antibody in techniques such as immunohistochemistry and Western blotting.

Generally there are two antibodies used. Primary antibody which can bind specifically to the protein of interest. And a secondary antibody coupled with a detection system such as HRP that would bind the primary antibody and signals the presence of protein of interest.

Specific proetins can be detected by its specific mono clonal antibody. Primary antibodies specifically binds to the proetins on the membrane. Secondary antibody interact with primary antibody and signals its presence by chemiluminescence.

Not including the antigen will prevent the primary antibody from binding to it which will disrupt the results of the ELISA. Not including the primary antibody will prevent the secondary antibody from binding it, which will again negatively affect the results of the ELISA. All components are necessary to get an accurate ELISA.