Development of SNAP-Tagged Antibodies for Magnetic Bead-Based Immuno-PCR

Recombinant antibodies are frequently joined to other molecules or to surfaces via conjugation.  Many conjugation methods rely on random modification of sulfhydryl, amine, or carboxyl groups on the antibody surface, but these modifications can affect the stability, solubility, and affinity of the antibody.

SNAP-tag technology developed by the New England Biolabs (Ipswich, MA), allows the human DNA-repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) to be fused to proteins and to capture para-substituted O(6)-benzylguanine (BG) derivatives through a nucleophilic substitution reaction that releases free guanine.  By coupling AGT to antibodies it should prove possible to homogeneously label the antibodies.  This can in turn assist in developing sensitive, reproducible and accurate diagnostic assay methods.

We have successfully designed and developed SNAP-tag fusion antibodies against several cancer biomarkers such as Tumor Necrosis Factor (TNF), Granulocyte Macrophage Colony Stimulating Factor (GMCSF), and Interferon Gamma (IFNg). Using BG functionalized beads and oligonucleotides, we have produced covalently-modified capture and detection antibodies. In a bead-based sandwich immuno-PCR assay with optimized qPCR conditions we have detected as little as 35 attomoles of a protein target.  We are currently optimizing a panel of oligo-conjugated or aptamer-conjugated antibodies and magnetic bead conjugates to develop a generalizable method for immuno-PCR reagent production.