Methods using a modified bacteriophage for the detection of target molecules

A method for detecting a target molecule, comprising:

• a) contacting an immobilized target molecule with a first affinity ligand that specifically binds the target molecule, under conditions that promote target molecule-affinity ligand binding, to form a target molecule-first affinity ligand complex, and optionally contacting the target molecule-first affinity ligand complex with one or more additional affinity ligands under conditions that promote affinity ligand-affinity ligand binding, to produce a target molecule-first affinity ligand-additional affinity ligand complex, wherein the one or more affinity ligands are added sequentially, and wherein each successive affinity ligand specifically binds to the affinity ligand added immediately previous;
• b) contacting the target molecule-first affinity ligand complex or the target molecule-first affinity ligand-additional affinity ligand complex with a modified bacteriophage comprising SEQ ID NO:
• 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein the bacteriophage binds to the first affinity ligand, or if one or more additional affinity ligands are used, binds to the ultimate affinity ligand, to form a target molecule-affinity ligand-bacteriophage complex;
• c) contacting the target molecule-affinity ligand-bacteriophage complex with a bacterial strain susceptible to infection by the bacteriophage;
• d) incubating the target molecule-affinity ligand-bacteriophage complex with the bacterial strain under conditions that promote the propagation of the bacteriophage in the bacteria and expression of the luciferase, to produce a bacteriophage-infected culture;
• e) adding to the bacteriophage-infected culture a substrate for the luciferase;
• and
• f) measuring the presence and/or magnitude of fluorescence produced from the action of the luciferase on its substrate.