Abstract: An improved method is disclosed for determining substances in a fluid sample having a mutual specific binding affinity, such as antigens and antibodies, said method utilizing a device comprising a column containing an insoluble porous matrix with which specific binding partners to the substance being determined are immobilized, preferably by chemically coupling them to the matrix material. The method comprises allowing a fluid sample containing the substance to be determined, a reference sample containing a labeled form of either the substance under determination or a specific binding partner, and an eluting liquid to flow through the device, followed by a determination of the relative amount of the labeled component retained in or eluted from the column. Comparison to standard values affords a reading of the concentration or absolute amount of the substance being determined in the fluid sample.