Real-time femtomolar detection of cancer biomarkers from photoconjugated antibody–phage constructs

Here we describe novel covalent conjugates of antibody–phage for the detection of multiple cancer biomarkers using real time immuno-polymerase chain reaction (immuno-PCR). While the conventional process of immuno-PCR utilizes DNA-conjugated antibodies, chemical modification of antibodies not only reduces antibody affinity but also creates a heterogeneous population of products. However, phage naturally encapsulate genomic DNA, which can be used as a PCR template. To produce covalently conjugated antibody–phage constructs without recombinant antibody expression or chemical modification of antibodies, we incorporated a photocrosslinkable non-canonical amino acid within an antibody-binding domain displayed on one of the phage coat proteins. To correlate antigen presence to a specific DNA sequence, the phage genomes were modified with domains that recognized specific sets of primers. The crosslinked antibody–phage conjugates were then tested in a sandwich-type immunoassay using real-time PCR where low pg ml⁻¹ concentrations of antigen could be detected and identified from a single solution containing a mixture of three different types of cancer biomarkers.