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 noninteracting 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.Human blood contains a panoply of proteins below the detection limit of standard immunoassays, including early markers of cancer or neurodegeneration (1, 2). Immunoassays have advanced enormously over the last 60 years, opening up whole new fields of research from the ability to detect lower and lower abundance species (3). Nearly all enzyme-linked immunosorbent assays (ELISAs) 2 stop working at low picomolar concentration of target (4), but recently, methods such as nanowires (5) or the bio-barcode assay (6) have made leaps in detection sensitivity. These advances mean that amplification is not the limitation on immunoassay sensitivity any longer; it is the antibody itself that is the limiting factor (4). Antibody binding strength correlates with protection from disease agents (7, 8) and can be a limiting factor in live cell imaging, since antibody off-rates are on the order of 30 min (9 -11) but lifetimes of cell surface proteins are often ϳ1 day.Affinity-matured antibodies generally have micromolar to nanomolar monovalent affinity for their target (9). It has proved very difficult to generate antibodies of subnanomolar monovalent affinity. This may be because of a limit to the affinity of antibodies that can be selected in an immunized animal, as a result of the kinetics of B cell stimulation (11). In vitro selection methods based on phage display (12), mRNA/ribosome display (13), or yeast cell surface display (14) have sometimes managed to obtain femtomolar affinity antibodies. There are a few examples of antibodies that form covalent bonds to artificial small molecule antigens (15-17), but these strategies, such as the antibody made by Meares' group to an electrophilic metal chelator, do not enable recognition of protein antigens composed of the regular 20 amino acids. Immunizing mice with electrophilic antigens generated an antibody that formed SDS-stable complexes with its target, but the chemistry of the i...