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Screening scFv antibodies against infectious bursal disease virus by co-expression of antigen and antibody in the bacteria display system

Author:
Guo, Xiaochen, Cao, Hongxue, Wang, Yunxin, Liu, Yang, Chen, Yanmin, Wang, Nan, Jiang, Shan, Zhang, Shengqi, Wu, Qiang, Li, Tianhe, Zhang, Yingjie, Zhou, Bing, Yin, Jiechao, Li, Deshan, Ren, Guiping
Source:
Veterinary immunology and immunopathology 2016 v.180 pp. 45-52
ISSN:
0165-2427
Subject:
Infectious bursal disease virus, Western blotting, antibody detection, antigens, bacteria, baiting, chickens, clones, enzyme-linked immunosorbent assay, flow cytometry, fluorescence, genes, neutralization, polyclonal antibodies, screening
Abstract:
We have previously reported an antigen and antibody co-expression (AAC) technology to demonstrate the interaction between a known antigen and antibody. To validate the co-expression system for screening antibody libraries, a single chain fragment variable(scFv)antibody library was constructed from chickens immunized with the VP2 protein of infectious bursal disease virus (IBDV). The genes of both VP2 and scFv antibodies were inserted into the pBFD-Ab-Ag vector. The co-expression library was subjected to three rounds of screening by flow cytometry (FCM) using a polyclonal antibody against VP2 through a bacteria display system. We achieved enrichment of scFv specific for IBDV. 110 individual clones were initially selected and sequenced. Twenty clones were selected based on fluorescence intensity by FCM. The scFv antibodies were expressed by pET-27b in E.coli and purified. The specificity and affinity of the selected scFv antibodies were confirmed by western blotting assay and ELISA analysis. What's more, the neutralizing capacity was measured with IBDV-B87(100 TCID50) in vitro. Four scFvs (clone 8(1), Y8, L10 and L7) showed significant neutralizing capacity. Two of the four scFvs (clone 8(1) and Y8) demonstrated a higher neutralizing activity to IBDV-B87 and the titers were 16,384 and 8,192, respectively. The two scFvs had higher neutralizing capacity than those obtained in previous studies. We demonstrated that the AAC technology could be applied to screen antibody libraries without baiting antigen to make antibody screening process easier and obtain scFvs with higher neutralizing capacity.
Agid:
5509184