Multilayer column chromatography specific binding assay method, test device and test kit

In a specific binding assay method for determining a ligand in, or the ligand binding capacity of, a liquid medium,

• wherein for determining said ligand, said liquid medium is combined with assay reagent means comprising (i) as labeled component, said ligand or a binding analog thereof incorporated with a label and (ii) a binding agent for said ligand;
• or wherein for determining the ligand binding capacity of said liquid medium suspected of containing a binding agent for said ligand, said liquid medium is combined with assay reagent means comprising, as labeled component, said ligand or a binding analog thereof incorporated with a label;
• thereby to form a binding reaction mixture having a bound-species as said labeled component bound to said binding agent and a free-species as said labeled component not bound to said binding agent;
• wherein said bound-species and said free-species of said labeled component are separated by contacting at least a portion of said binding reaction mixture, a predetermined time after formation thereof, with a column comprising a bed of an adsorbent material which is both (a) selective for binding one of said bound-species and fre-species and (b) capillarily absorbent relative to said reaction mixture, whereby said at least a portion of said reaction mixture is drawn into said column by capillary action and said bound-species and said free-species are separated along said column;
• and
• wherein said label is measured in one of the separated species;
• the improvement wherein said column comprises at least one additional bed of material which is capillarily absorbent relative to said reaction mixture, such additional bed being disposed in said column in contact with the end of said adsorbent bed opposite that which is contacted with said reaction mixture, and said additional bed material being substantially nonadsorbent relative to the one of said bound-species and free-species which said adsorbent bed selectively binds.