Solid phase non-separation enzyme complementation assay

A method for determining the presence of an analyte in a sample, said analyte being a member of a specific binding pair ("mip"), said method employing as reagents fragments of beta-galactosidase comprising an N-terminal enzyme donor fragment ("ED") and a C-terminal enzyme acceptor fragment ("EA"), where said acceptor and donor fragments when joined together form an active enzyme complex and said enzyme donor is conjugated to a mip to form a mip/ED-conjugate which is immunologically competitive with said analyte;

• and wherein a mip, complementary to said mip/ED conjugate and said analyte, is immobilized;
• said method comprising:
• contacting in an assay medium, said sample, said mip/ED conjugate, and immobilized mip and incubating for sufficient time for complex formation to go to substantial completion to provide a first mixture, wherein said mip/ED conjugate bound to said immobilized mip is substantially inhibited in forming an enzymatically active complex;
• adding said EA, substrate and any additional reagents necessary to produce a detectable product;
• and
• determining the enzyme activity of said assay medium by means of said detectable product in comparison to an assay medium, having a known amount of analyte.

A method for determining the presence of an analyte in a sample, said analyte being a member of a specific binding pair ("mip"), said method employing an N-terminal enzyme donor fragment ("ED") and a C-terminal enzyme acceptor fragment ("EA"), where said acceptor and donor fragments when joined together, form an active enzyme complex and said enzyme donor is conjugated to a mip to form a mip/ED conjugate which is complementary to said analyte;

• and wherein a mip complementary to said mip/ED conjugate is immobilized;
• said method comprising:
• contacting in an assay medium, said sample and said mip/ED conjugate in substantial binding excess to said analyte, and incubating for a sufficient time for complex formation to go to substantial completion to form a first assay medium;
• combining said first assay medium, with said immobilized mip, said immobilized mip being in substantial binding excess to said mip/ED conjugate to form a second assay medium, wherein said mip/ED conjugate bound to said immobilized mip is substantially inhibited in forming an enzymatically active complex;
• combining, either concurrently or consecutively with said immobilized mip, EA, substrate and any additional reagents necessary to produce a detectable product;
• and
• determining the enzyme activity of said second assay medium by means of said detectable product in comparison to an assay medium having a known amount of analyte.