title: Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.
abstract: Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1.
authors.affiliation: Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Won-Ki
authors.initials: WK
authors.lastname: Min
authors.affiliation: Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Kang-In
authors.initials: KI
authors.lastname: Na
authors.affiliation: Department of Biomedical Science, Youngdong University, Chungbuk 370-701, Republic of Korea.
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Yoon
authors.affiliation: Department of Biomedical Science, Youngdong University, Chungbuk 370-701, Republic of Korea.
authors.forename: Yoon-Jee
authors.initials: YJ
authors.lastname: Heo
authors.affiliation: The 5th R&D Institute-3, Agency for Defense Development, Daejeon 305-152, Republic of Korea.
authors.forename: Daesang
authors.initials: D
authors.lastname: Lee
authors.affiliation: Department of Biomedical Science, Youngdong University, Chungbuk 370-701, Republic of Korea. Electronic address: sgkim@yd.ac.kr.
authors.forename: Sung-Gun
authors.initials: SG
authors.lastname: Kim
authors.affiliation: Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-742, Republic of Korea. Electronic address: jhseo94@snu.ac.kr.
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D016604:Aflatoxin B1 / Q000032:analysis* / Q000276:immunology; D000915:Antibody Affinity; D001231:Aspergillus flavus / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D020349:Surface Plasmon Resonance
chemical_list: D007128:Immunoglobulin Fragments; D057127:Single-Chain Antibodies; C055966:immunoglobulin Fv; D016604:Aflatoxin B1
keywords: Affinity improvement; Aflatoxin B(1); Back mutation; Single-chain variable fragment (scFv)