To provide an improved simultaneous sandwich immunoassay by leaving a polychronal antibody to be refined on an antigen using affinity chromatography.

Polychronal antibody is refined to the affinity chromatography method using an antigen corresponding to an antibofy which is in covalent bond with segregation gel. The refining of the polychronal antibody is made using a chromatographic column filled with a gel with which an antigen protein complementary to the specific antibody to be refined is in covalent bond. Only the antibody peculiar to this protein is bonded to the column, and all nonpeculiar antibodies are eluded away from the column with no hindrance. The peculiar antibody bonded to the column can be eluded from the column, for example by changing pH.