Rapid, convenient, and highly sensitive detection of human hemoglobin in serum using a high-affinity bivalent antibody-enzyme complex

Human hemoglobin (Hb) is a biomarker of several diseases, and monitoring of Hb levels is required during emergent surgery. However, rapid and sensitive Hb detection methods are yet to be developed. The present study established a rapid, convenient, and highly sensitive detection method for Hb in human serum using a bivalent antibody-enzyme complex (AEC). AECs are promising sensing elements because of their ability to bind specific targets and their catalytic activity that produce signals. We recently reported a convenient and universal method to fabricate bivalent AECs with two antibody fragments, using the SpyCatcher/SpyTag system. The present study applied a bivalent AEC for highly sensitive and quantitative detection of human Hb. The bivalent anti-Hb AEC was successfully prepared by incubating both N- and C-terminus SpyCatcher-fused glucose dehydrogenase and SpyTag-fused anti-Hb single-chain variable fragments at 4 degrees C. As expected, the bivalent AEC for Hb with a multimeric structure showed higher affinity than the monovalent AEC, by means of avidity effects, unlike that for soluble epidermal growth factor receptor with a monomeric structure; this contributed to a great improvement in sensitivity. Finally, we established a rapid and wash-free homogeneous electrochemical detection system for Hb by integrating magnetic beads. The linear range of the system completely covered the clinically required Hb levels, even in human serum. This technology provides an ideal point-of-care test for Hb and other multimeric biomarkers.