Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1. (15th October 2016)

• Record Type: Journal Article
• Authors: Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho
• Is Part Of: Food chemistry. Volume 209(2016)
• Page Start: 312
• Page End: 317
• Publication Date: 2016-10-15
• Subjects: Single-chain variable fragment (scFv) -- Aflatoxin B1 -- Back mutation -- Affinity improvement; Food -- Analysis -- Periodicals; Food -- Composition -- Periodicals; 664
• DOI: 10.1016/j.foodchem.2016.04.085
• Languages: English
• ISSNs: 0308-8146

Abstract

Highlights: A deleterious mutation was identified in an affinity-maturated scFv against aflatoxin B1 . The back mutation of the deleterious residue resulted in a 3.2-fold affinity improvement. Specificity and structural properties were not influenced by the back mutation. 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 ( k d ) 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 .