Small peptides with less than 1000 in molecular weight are not considered amenable to sandwich immunoassays due to their difficulty of simultaneous recognition by two antibodies. As an alternative, we attempted noncompetitive detection of small peptides by open sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing the antigen-induced enhancement of antibody VH/VL interaction. Taking fragments of human osteocalcin (BGP), a major non-collagen peptide produced in bone, as model peptides, OS immunoassay was performed using the cloned VH and VL cDNAs from two anti-BGP monoclonal antibodies either recognizing the N- or C-terminal fragment, respectively. When the clones were used for OS-ELISA with immobilized VL fragment and phage-displayed VH fragment, enhanced VH / VL interaction upon BGP addition was observed. Especially the clone for the C-terminal fragment showed superior detection limit as well as a wider working range than those of competitive assay. The result was reproducible with either purified VH-alkaline phosphatase or peroxidaseconjugated MBP-VH, together with immobilized MBP-VL fusion proteins, and in the latter case the assay can be performed on microplate wells and also in microfluidics. The minimum detectable fragment was the hexamer including the C-terminus, implying hapten-like terminal recognition. To further improve the sensitivity of the assay, a phage-displayed PCR-randomized VH library was subjected to repeated selections on MBP-VL in the presence of reduced amount of peptide (OS selection). The selection successfully gave a clone with ten-fold lower detection limit, which was well below the serum peptide level in healthy human. This simple approach with a single antibody with a short measurement time may prove a useful tool in immunodiagnostics as well as in proteomics research.