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Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.
- Source :
-
Journal of bioscience and bioengineering [J Biosci Bioeng] 2016 Sep; Vol. 122 (3), pp. 287-93. Date of Electronic Publication: 2016 Mar 30. - Publication Year :
- 2016
-
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.<br /> (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)
- Subjects :
- Bacteriophages
Cloning, Molecular
Enzyme-Linked Immunosorbent Assay
Escherichia coli genetics
Gene Library
Humans
Inclusion Bodies immunology
Inclusion Bodies metabolism
Protein Refolding
Single-Chain Antibodies chemistry
Single-Chain Antibodies genetics
Spleen metabolism
Lipoproteins, LDL immunology
Single-Chain Antibodies biosynthesis
Single-Chain Antibodies immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1347-4421
- Volume :
- 122
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Journal of bioscience and bioengineering
- Publication Type :
- Academic Journal
- Accession number :
- 27038672
- Full Text :
- https://doi.org/10.1016/j.jbiosc.2016.02.014