Immunoassay Methods Utilizing Trapping Conjugate

Abstract

Methods are provided for determining the presence of a first ligand in a sample. In some embodiments depletion conjugates are used to deplete the ligands different from but related to the first ligands from the sample. In some embodiments, interim binding agents are used to enhance the test signal.

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         7 . A method for testing a sample for the presence of a first ligand, comprising:
 a) combining the sample with depletion molecules to obtain a mixture, said depletion molecules comprising molecules chosen to specifically bind to analyte having a second ligand different from but related to said first ligand to which said depletion molecules will not bind, thereby generating bound second ligand and a remainder;   b) applying at least the remainder to an immunoassay device; and   c) inspecting the immunnoassay device to determine the presence of the first ligand.   
     
     
         8 . A method according to  claim 7 , wherein:
 said depletion molecules comprise a conjugate of said second ligand with a larger particle.   
     
     
         9 . A method according to  claim 8 , wherein:
 said larger particle is a colored latex.   
     
     
         10 . A method according to  claim 8 , further comprising:
 generating said conjugate by obtaining freeze dried latex conjugate and a diluent and mixing them together.   
     
     
         11 . A method according to  claim 7 , further comprising:
 filtering said mixture to substantially remove said depletion molecules and bound second ligand prior to said applying.   
     
     
         12 . A method according to  claim 11 , wherein:
 said immunoassay device has a first sorbent strip having a first location for receiving a solution and defining a first migration path, a marker conjugate located on or in said first migration path, said marker conjugate adapted to bind to said first ligand, and a second sorbent strip distinct from said first sorbent strip and having a second location for receiving said remainder and defining a second migration path, and a test site located on or in at least one of said first sorbent strip and said second sorbent strip, said test site having an immobilized ligand-binding mechanism for said first ligand, and said first and second sorbent strips touching each other at the test site location, wherein said second location is removed from said test site such that remainder applied to said second location requires time to migrate to said test site and does not immediately wet said test site.   
     
     
         13 . A method according to  claim 11 , wherein:
 said immunoassay device has a first sorbent strip having a first location for receiving a solution and defining a first migration path, a marker conjugate located on or in said first migration path, said marker conjugate adapted to bind to an interim binding agent, and a second sorbent strip distinct from said first sorbent strip and having a second location for receiving said remainder and defining a second migration path, a second conjugate located on or in said second migration path, said second conjugate including a first-ligand-binding molecule and said interim binding agent, and a test site located on or in at least one of said first sorbent strip and said second sorbent strip, said test site having an immobilized ligand-binding mechanism for said first ligand, and said first and second sorbent strips touching each other at the test site location, wherein said second location is removed from said test site such that remainder applied to said second location requires time to migrate to said test site and does not immediately wet said test site.   
     
     
         14 . A method according to  claim 13 , wherein:
 said interim binding agent is biotin and said marker conjugate comprises at least one of streptavidin and an anti-biotin antibody.   
     
     
         15 . A method according to  claim 11 , wherein:
 said immunoassay device has a first sorbent strip having a first location for receiving a solution and defining a first migration path, a marker conjugate located on or in said first migration path, said marker conjugate having a first interim binding agent of a first pair adapted to bind to a second interim binding agent of said first pair, and a second sorbent strip distinct from said first sorbent strip and having a second location for receiving said remainder and defining a second migration path, second molecules and a second conjugate located on or in said second migration path, said second molecules including a first-ligand-binding molecule and a first interim binding agent of a second pair, and said second conjugate including particles having bound thereto a second interim binding agent of said second pair and a second interim binding agent of said first pair, and a test site located on or in at least one of said first sorbent strip and said second sorbent strip, said test site having an immobilized ligand-binding mechanism for said first ligand, and said first and second sorbent strips touching each other at the test site location, wherein said second location is removed from said test site such that remainder applied to said second location requires time to migrate to said test site and does not immediately wet said test site.   
     
     
         16 . A method according to  claim 15 , wherein:
 said first interim binding agent of said second pair is biotin, said second interim binding agent of said second pair is streptavidin, said first interim binding agent of said first pair is anti-FITC antibody, and said second interim binding agent of said first pair is FITC antigen.   
     
     
         17 . A method according to  claim 16 , wherein:
 said particles of said second conjugate comprise white latex particles.   
     
     
         18 . A method according to  claim 17 , wherein:
 said first ligand is HIV p24 ligand, and said first-ligand-binding molecule of said second molecules are HIV p24 molecules.   
     
     
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