Improvement in the binding specificity of anti-isomiroestrol antibodies by expression as fragments under oxidizing conditions inside the SHuffle T7 E. coli cytoplasm

Author:

Juengsanguanpornsuk Wipawee1,Kitisripanya Tharita2,Boonsnongcheep Panitch3,Yusakul Gorawit4ORCID,Srisongkram Tarapong1,Sakamoto Seiichi5ORCID,Putalun Waraporn1ORCID

Affiliation:

1. Faculty of Pharmaceutical Sciences, Khon Kaen University , Khon Kaen 40002, Thailand

2. Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University , Bangkok 10400, Thailand

3. Institute of Molecular Biosciences, Mahidol University , Nakhon Pathom 73170, Thailand

4. School of Pharmacy, Walailak University , Nakhon Si Thammarat 80160, Thailand

5. Graduate School of Pharmaceutical Sciences, Kyushu University , Fukuoka 812-8582 , Japan

Abstract

ABSTRACT Sensitive and specific analysis of isomiroestrol (Iso) is required for the quality control of Pueraria candollei, a herb used to treat menopausal disorders. The anti-isomiroestrol monoclonal antibody (Iso-mAb) exhibits cross-reactivity with miroestrol and deoxymiroestrol, which impacts the analytical results. Here, the active and soluble forms of the single-chain variable fragment (Iso-scFv) and fragment antigen-binding (Iso-Fab) against Iso were expressed using Escherichia coli SHuffle® T7 to alter the binding specificity. The Iso-scFv format exhibited a higher binding activity than the Iso-Fab format. The reactivity of Iso-scFv towards Iso was comparable with that of the parental Iso-mAb. Remarkably, the binding specificity of the scFv structure was improved and cross-reactivity against analogs was reduced from 13.3-21.0% to ˂ 1%. The structure of recombinant antibodies affects the binding characteristics. Therefore, the immunoassays should improve specificity; these findings can be useful in agricultural processes and for quality monitoring of P. candollei-related materials.

Funder

National Research Council of Thailand

Thailand Science Research and Innovation

International Research Network for Functional Food Discovery & Development

Khon Kaen University

Publisher

Oxford University Press (OUP)

Subject

Organic Chemistry,Molecular Biology,Applied Microbiology and Biotechnology,General Medicine,Biochemistry,Analytical Chemistry,Biotechnology

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