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

摘要

The invention is a procedure for measuring the binding of an entity (ligand) to a surface by using a hapten-conjugated version of the ligand (hapten-ligand). An excess of the hapten-ligand is presented to the binding surface and excess (unbound) hapten-ligand is washed off. Bound hapten-ligand is then solubilized (removed) and applied to a membrane support or separated by electrophoresis and applied to a membrane support. Standard amounts of hapten-ligand are similarly applied to the membrane. The membrane-bound hapten-ligand is detected by application of an enzyme-conjugated antibody to the hapten; or by application of an antibody to the hapten followed by application of an enzyme-conjugated antibody to the anti-hapten antibody. The resultant membrane-associated enzyme is detected and quantitated by the application of a color or light-producing substrate which reacts with the enzyme. A combination of the use of anti-hapten antibodies along with membrane-blotting technologies to assess hapten-ligand binding to surfaces is not found in the scientific or patent literature, particularly in regards to assessing protein binding to cell surfaces.