Method for the detection and measurement of Hapten-conjugated biological binding entities by western and dot-blot using anti-hapten antibodies

A method for the quantification of ligand binding to a surface, using hapten-conjugated ligands, comprising:

• (a) applying a hapten-ligand, comprising a ligand possessing an antibody-recognizable hapten, to said surface, and,
• (b) waiting for a period of time, so as to allow a binding of said hapten-ligand to said surface, thereby producing bound ligand, and,
• (c) removing any unbound hapten-ligand, from said surface, and,
• (d) solubilizing said bound ligand, thereby producing a lysate, and,
• (e) applying separately onto a membrane,
• (1) said lysate, and,
• (2) a plurality of standards, comprising solutions containing increasing levels of known amounts of the hapten-ligand,
• thereby producing membrane-bound hapten-ligand and,
• (f) applying to said membrane,
• (1) an enzyme-conjugated antibody to specific for said hapten, and,
• (2) a color or light-producing substrate that contacts the enzyme on said enzyme-conjugated antibody,
• thereby producing a signal, and,
• (g) comparing said signal arising from said enzyme associated with said membrane-bound hapten-ligand arising from said standards, to the known amount of hapten-ligand contained in said standards, thereby producing a standard curve, and,
• (h) quantifying the amount of said hapten-ligand originally bound to said surface, by quantifying the amount of the hapten-ligand contained in said membrane-bound hapten-ligand arising from said lysate, by comparing said signal arising thereof to said standard curve and,
• whereby the use of radio-labeled ligand is avoided.