A general method to select antibody fragments suitable for noncompetitive detection of monovalent antigens

Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region (VH)/light-chain variable region (VL) interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the VH/VL interaction would become fairly strong along with the addition of antigen. Here, we devised a phage-based "split-Fv system" to rapidly evaluate and select antibody variable region (Fv) fragments that are suitable to OS immunoassay. When three antibodies raised against endocrine disruptor bisphenol A were tested with this system, all were more or less suitable to OS-ELISA. Among them, the best Fv selected was used to construct fusion proteins of VH tethered to an alkaline phosphatase and a tagged VL that can be site-specifically biotinylated to perform direct OS-ELISA. The results showed that the OS-ELISA detects bisphenol A with higher sensitivity than the corresponding competitive assay, also implying that many antibodies to small haptens have suitable properties for OS-ELISA.