Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen.
1) coat the microtiter plate wells with antigen;
2) block all unbound sites to prevent false positive results;
3) add primary antibody (e.g. rabbit monoclonal antibody) to the wells;
4) add secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG);
5) reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction.