Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein

Abstract

Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.

Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage‐display

“…DNA aptamers for the purification of Influenza A viruses displayed a K a in the order of 10 7 M ‐1 ) or the results are obtained using the virus protein and not the whole virus as the target (e.g. an antibody with affinity towards a rabies virus glycoprotein displayed a K a of 10 6 M ‐1 ). The maximum capacity of the support is not usually determined for virus purification schemes which does not allow a fair comparison with the current work.…”

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT

“…In this study, we developed a new mAb that reacts specifically with the RABV P protein and confirmed its applicability to the RFFIT. Numerous previous studies have reported antibodies developed as diagnostic reagents for rabies detection that target the N protein [ 12 , 13 ] or G protein [ 14 , 15 ]. N protein is related to the immune response and pathogenicity in the host.…”

“…For this reason, most of the commercial antibodies available for rabies diagnosis are designed to detect rabies-related lyssaviruses along with RABV most of the cases [ 25 ]. The majority of diagnostic laboratories utilize commercial reagents [ 26 ], whereas some utilize in-house products [ 15 , 27 , 28 ]. Both polyclonal antibodies and mAbs, which are specific to the entire virus or to the nucleocapsid protein and are conjugated with a fluorophore, can be used for rabies diagnosis.…”

Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in <i> Escherichia coli </i>

“…Bên cạnh đó, kháng thể đơn chuỗi có thể được 47 sử dụng cho mục đích điều chỉnh và phát hiện sự hoạt động của các protein trong tế bào, như vậy phù hợp cho việc sử dụng làm vaccine (Alvarez-Rueda et al, 2009). Ngoài ra, kháng thể đơn chuỗi còn được ứng dụng trong điều trị ung thư (Chester et al, 2004) và chống lại virus bệnh dại glycoprotein (Yuan et al, 2013).…”