Original Research ARTICLE
Screening of a scFv antibody with high affinity for application in human IFN-γ immunoassay
- 1School of Life Sciences, Fujian Agriculture and Forestry University, China
Interferon gama (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on scFv antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 2.6×109 L/mol. Maltose binding protein(MBP) was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag, and the resulted fusion protein (MBP-LK-scFv) has high solubility and antigen biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive ELISA (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6-60 pg/mL with IC50 of 25 pg/mL. The limit of detection was 2 pg/mL (1.3 fm), and the average recovery was 85.05%, further demonstrating that the detection method based on scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis.
Keywords: Interferon gama (IFN-γ);, scFv, phage display, Affinity, ic-ELISA, detection
Received: 16 Nov 2017;
Accepted: 02 Feb 2018.
Edited by:José R. Mineo, Federal University of Uberlandia, Brazil
Reviewed by:Ana Afonso, Universidade de São Paulo, Brazil
Maryam Dadar, Razi Vaccine and Serum Research Institute, Iran
Copyright: © 2018 Yang, Zhong, Wang, Zhang, Li, Ling, Wang and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Prof. Shihua Wang, Fujian Agriculture and Forestry University, School of Life Sciences, Fuzhou, China, email@example.com
Prof. Rongzhi Wang, Fujian Agriculture and Forestry University, School of Life Sciences, Fuzhou, China, firstname.lastname@example.org