title: Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.
abstract: Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, Republic of Korea.
authors.forename: Jun-Hyuck
authors.initials: JH
authors.lastname: Jang
authors.affiliation: Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea.
authors.forename: Dong-Hyung
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea.
authors.forename: Se-Hwan
authors.initials: SH
authors.lastname: Paek
authors.affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong, Republic of Korea.
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009211:Myoglobin / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
chemical_list: D009211:Myoglobin; D057127:Single-Chain Antibodies
keywords: acute myocardial infaction; continuous monitoring; myoglobin; rapid reaction kinetics; single-chain variable fragment