Cascade immunoelectrophoresis: combined electrophoretic and solid-phase processing of immunoreactive protein by zonal...

A new approach to immunochemical analysis of complex mixtures of proteins has been devised through development of a system for (1) sequentially absorbing, desorbing, and cascading antibodies in a profile indicative of both the quantity and electrophoretic characteristics of the antigen, and (2) for carrying out multiple tests of antigenic determinants at sub-picomole levels regardless of the solubility or precipitin forming characteristics of the immunoreactive protein. The procedure employs zonal immobilization in which a novel, aldehyde-rich gel (glyoxyl agarose) is used interchangeably as an inert support for electrophoresis and as an immobilizing matrix to fix the protein for analysis by solid-phase techniques. Proteins may be separated on the gel as with ordinary agarose, and then driven to combine covalently with the gel upon exposure to NaCNBH3. After removing NaCNBH3, the distribution of specified antigens is then established by exposing the gel to antibody an profiling the pattern of antibody uptake by a cross-electrophoretic technique in which the absorbed antibody is (1) desorbed with dodecyl sulfate, then (2) transferred through a gel containing potassium ion to halt migration of the detergent, and (3) displayed by immunoprecipitation with anti-IgG antibodies. To cascade the process further, the IgG may be immobilized and used as a surrogate antigen to bind additional antibody protein. In addition to enhancing sensitivity, the cross-electrophoretic measurement of uptake of antibody by immobilized antigen eliminates dependence on direct immunoprecipitation of the antigen, and may accordingly be of particular advantage with non-precipitin forming antigens and monoclonal antibodies.