title: Electrophilic affibodies forming covalent bonds to protein targets.
abstract: Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target. The proximity between cysteine, lysine, or histidine on the target protein drove covalent bond formation to the electrophile on the affibody. Covalent bonds did not form to a non-interacting point mutant of the target, and there was minimal cross-reactivity with serum, cell lysate, or when imaging at the cell surface. Electrophilic affibodies showed more stable protein imaging at the surface of mammalian cells, and the sensitivity of protein detection in an immunoassay improved by two orders of magnitude. Thus electrophilic affibodies combined good specificity with improved detection of protein targets.
authors.affiliation: Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Lotta
authors.initials: L
authors.lastname: Holm
authors.forename: Paul
authors.initials: P
authors.lastname: Moody
authors.forename: Mark
authors.initials: M
authors.lastname: Howarth
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013205:Staphylococcal Protein A / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001798:Blood Proteins; D011506:Proteins; D013205:Staphylococcal Protein A