Solid phase assay for detection of ligands

A method of detecting the presence or level of an analyte in a sample, the method comprising steps of:

• (a) contacting said sample with:
• (i) a solid phase having bound thereto a first ligand that binds said analyte when incubated therewith;
• and
• (ii) a second ligand that binds said analyte when incubated therewith, the result of the two contacting steps being that a first ligand/analyte/second ligand complex is formed on said solid phase,
• (b) limiting the contact time between said second ligand and said solid phase so that:
• (i) non-specific binding between said second ligand and said solid phase is not allowed to reach equilibrium;
• and
• (ii) a first binding curve, in which formation of a non-specific second ligand/solid phase complex is plotted versus time, does not level off;
• (c) binding a detectable tag to said second ligand either prior to or after formation of said first ligand/analyte/second ligand complex so that a portion of said tag is retained on said solid phase upon formation of said first ligand/analyte/second ligand complex;
• (d) detecting said retained tag to determine the presence or level of said analyte in said sample.

A method of detecting formation of a first ligand/second ligand complex on a solid phase, the method comprising steps of:

• (a) contacting an analyte that binds said second ligand with said second ligand so that analyte/second ligand complexes formed;
• (b) providing a solid phase having bound thereto a first ligand, which first ligand is characterized by an ability to bind to said second ligand in such a way that, were said first ligand and analyte exposed simultaneously to unbound second ligand, said first ligand would compete with said analyte for binding to said second ligand;
• (c) contacting said analyte/second ligand complex with said solid phase so that a first ligand/second ligand complex is formed;
• (d) limiting the time of contact between said analyte/second ligand complex and said solid phase so that substantially all of the second ligand participating in analyte/second ligand complexes remains in analyte/second ligand complexes and does not participate in interactions with said first ligand;
• (e) binding a detectable tag to said second ligand either prior to or after step (a), step (b), step (c), or step (d) so that a portion of said tag is retained on said solid phase upon formation of said first ligand/second ligand complex;
• and
• (f) detecting said portion of said tag and thereby detecting the formation of said first ligand/second ligand complex on said solid phase.

A method of detecting the presence or level of an analyte in a sample, the method comprising steps of:

• (a) contacting said sample with:
• (i) a solid phase having bound thereto a first ligand, which first ligand is characterized in that it binds to said analyte when incubated therewith, the contacting being performed so that a first ligand/analyte complex is formed on said solid phase;
• and
• (ii) a second ligand that binds said first ligand/analyte complex when incubated therewith, so that a first ligand/analyte/second ligand complex is formed on said solid phase, said contacting being performed under conditions and for a time sufficiently limited that substantially no non-specific binding between said second ligand and said solid phase occurs;
• (b) binding a detectable tag to said second ligand either prior to or after formation of said first ligand/analyte/second ligand complex so that a portion of said tag is retained on said solid phase upon formation of said first ligand/analyte/second ligand complex;
• (c) detecting said retained tag to determine the presence or level of said analyte in said sample.