Novel single-chain antibody recognises complex glycan/alpha helix epitope in glycated haemoglobin using a subset of its complementarity determining regions

Understanding the molecular interactions that govern antibody recognition of glycated epitopes is crucial for developing advanced diagnostics and therapeutics. This requires identification of the region of the antigen that is recognized (the epitope) and the regions of the antibody that bind the antigen (the paratope). Recently, we developed a single-chain variable fragment antibody (scFv) with high affinity and specificity for a complex, non-A1c epitope of haemoglobin that is both glycated and conformational. Here, we have used immunochemical and biophysical methods, including carbene footprinting mass spectrometry, to map the scFv's epitope in human glycated haemoglobin. The epitope is composed of a glycation site at Lys-66 on the β globin chain and two α-helical regions spanning residues 9-17 and 67-72 that form a contiguous binding site on the protein's surface. The identity of the paratope within the scFv was also determined, and we found that only a subset of the predicted complementarity determining regions (CDRs) participate in interactions with the antigen. A computational model of the scFv-HbA1c complex was created and used to indicate key residues in the CDRs and framework regions that mediate paratope-epitope interaction. This work advances our understanding of the molecular basis for high-affinity binding antibodies to complex glycated epitopes and provides a foundation upon which to develop innovative diagnostics that can detect multiple glycated species in parallel.