Method for generating high affinity, bivalent binding agents for sandwich assays

A method for generating a high affinity, bivalent binding agent for a sandwich assay comprising:

• (a) amplifying a first and second library of nucleic acids, each library encoding a population of binding agents that bind to different epitopes on a target molecule, to generate a first and second pool of megaprimers;
• (b) annealing the first and second pool of megaprimers of (b) to a single-stranded, uracilated phage-display vector comprising a first binding agent coding region and second binding agent coding region each capable of hybridizing to the first or second pool of megaprimers, wherein the first and second binding agent coding regions are in tandem and linked via a linker;
• (c) primer extending the first and second pool of megaprimers of (b) to generate a phage-display library of bivalent phage clones;
• (d) screening the phage-display library to identify a bivalent binding agent comprising a first binding agent and second binding agent, each of which binds to a different epitope on the target molecule;
• and
• (e) conjugating the first or second binding agent to a member of a sandwich assay.