title: Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.
abstract: 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 (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.
authors.affiliation: National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kumano-Kuramochi
authors.affiliation: National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Fujimura
authors.affiliation: National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Komba
authors.affiliation: National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Maeda-Yamamoto
authors.affiliation: National Food Research Institute, NARO, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan. Electronic address: lili@affrc.go.jp.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Machida
mesh_terms: D001435:Bacteriophages; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006801:Humans; D002479:Inclusion Bodies / Q000276:immunology / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000276:immunology*; D058849:Protein Refolding; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013154:Spleen / Q000378:metabolism
chemical_list: D008077:Lipoproteins, LDL; D057127:Single-Chain Antibodies; C032629:acetyl-LDL; C099252:oxidized low density lipoprotein
keywords: Enzyme-linked immunosorbent assay; Low-density lipoprotein; Oxidized low-density lipoprotein; Phage display library; Protein refolding; Single-chain variable fragment