Isolation of Single-Chain Antibody Fragments Against Venezuelan Equine Encephalomyelitis Virus from Two Different Immune Sources

Abstract

Venezuelan equine encephalomyelitis (VEE) virus is an important human and veterinary pathogen of Central and South America. The virus can cause widespread epidemics, affecting hundreds of thousands of horses, and thousands of humans. Detection of the virus early in infection and in mosquito populations may allow epidemics to be predicted such that suitable prophylaxis, such as vaccination, can be used to reduce disease severity and transmission. The sensitivity and specificity of current immunoassays, based on conventional monoclonal and polyclonal antibodies, needs to be improved for the diagnosis of infection. We have examined phage display libraries expressing single-chain antibodies (scFv) produced from two different immune sources, a hybridoma cell line and an immunized mouse spleen. The libraries were panned against VEE virus to select for specific scFvs. scFvs isolated from both libraries were specific for the same epitope on the VEE virus and sequence analysis showed that the scFvs were almost identical apart from the CDR3 region of the heavy chain. The data presented in this article suggest that although scFvs may be useful tools for the detection of viruses, there are serious limitations with the use of phage display as a tool for the isolation of specific antibodies.

Human-like antibodies neutralizing Western equine encephalitis virus

“…Non-human primates (NHP) antibody gene libraries and phage display are highly suitable tools for the generation of human-like antibodies for diagnostic and therapeutic purposes 41 , 42 . Phage display has been successfully used for the generation of murine, 43 human 44 , 45 and human-like 46 antibodies against VEEV. This study concerns the generation and characterization of the first human-like neutralizing antibodies against WEEV.…”

Engineering and functional evaluation of a single-chain antibody against HIV-1 external glycoprotein gp120

“…Sequence analysis showed that the scFv gene we constructed was replicated with high fidelity and that the variable regions of the heavy and light chains were effectively rearranged with a flexible linker. This gene structure was demonstrated to resemble the published pattern of recombinant murine resource FV molecules and the conformation of this gene was shown to have better affinity than that of the V L linker and V H gene structure [26]. It is worth stressing here that the linker sequence and the metal‐binding insert locations in scFv were essential for retaining the paratope affinity.…”

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

“…To date, VEEV diagnosis is performed using monoclonal and polyclonal antibodies [41] and also scFv fragments have been analysed [42]. This study showed that scFv phage are applicable for a broad range of anti-VEEV diagnosis assays: antigen ELISA on purified virus particles, ELISA on cell lysate and immunoblot.…”

“…The panning procedure based on protocols by Hust et al [25] with numerous modifications in 96 well microtitre plates (Maxisorb, Nunc, Wiesbaden, Germany). The mAb 8747 (Chemicon, Temecula, USA; [42]) and mAb VEE-WIS1 (WIS, Munster, Germany) were incubated in concentrations of 1,5 g/mL each overnight at 4°C in microtitre wells, followed by blocking with 1% (w/v) BSA in PBST (phosphate buffered saline + 1% Tween 20; [48]) for 1 h at RT. For every panning round one well was coated for the selection and one well was coated for a preselection step.…”