Noncompetitive Detection of Low Molecular Weight Peptides by Open Sandwich Immunoassay

摘要

Small peptides with less than 1000 in molecular weightare 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 enzymelinked immunosorbent assay (OS-ELISA) utilizing the antigen-induced enhancement of antibody V_(H)/V_(L) 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 V_(H) and V_(L) 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 V_(L) fragment and phage-displayed V_(H) fragment, enhanced V_(H)/V_(L) interaction upon BGP addition was observed. Especially the clone for the C-terminal fragment showed a superior detection limit as well as a wider working range than those of competitive assay. The result was reproduced with purified V_(H)-alkaline phosphatase and MBP-V_(L) fusion proteins, where the latter was directly immobilized onto the microplate wells. The minimum detectable fragment was the hexamer including the C-terminus. This simple approach with a single monoclonal antibody with a short measurement time may prove a useful tool in immunodiagnostics as well as in proteomics research.