Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (gamma + kappa and mu + lambda) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The gamma kappa 5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). gamma kappa 5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of gamma kappa 5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The gamma kappa 5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of gamma kappa 5 to low concentrations (1-2 mu g/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using gamma kappa 5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 mu g/mL. Our results indicated that the gamma kappa 5 scFv was a promising molecule for the detection of modified LDL at very low concentrations. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.