Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies
immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy
chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative
to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S)
with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent
RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation.
Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A
HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and
FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a
biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S,
RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies
support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding
ability, stability and affinity of engineered antibodies recognizing linear epitope.
Keywords: Biotin, HIS tag, rabies virus, truncated RV glycoprotein, VH and VL, scFv.
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