Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

Abstract

Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.

Antibody Phage Display Technology for Sensor-Based Virus Detection: Current Status and Future Prospects

“…SPR biosensors are also used for virus detection. Phage antibodies were used as part of the SPR biosensor for the detection of norovirus [ 154 ], porcine circovirus [ 159 ], cowpea mosaic virus [ 213 ], SARS-CoV-2 [ 214 ], etc.…”

Protein Modelling Insight to the Poor Sensitivity of Chikungunya Diagnostics on Indonesia’s Chikungunya Virus

“…In addition, Holstein et al [17] also suggested that a recombinant antibody fragment could be used as a functional protein for a paper-based diagnostic test. ScFv is widely used for diagnostic purposes in many fields, and there are to detect diseases in humans or plants [33][34][35].…”

A Survey of Analytical Techniques for Noroviruses

“…In particular, another study conducted by Kou et al [56] combining mAb NV23 and single-chain variable fragments (scFv) (HJT-R3-A9 antibody) for NoV capture and detection by a sandwich ELISA assay showed that all 25 NoV genotypes from stool samples could be identified with a Ct value of <31 and a limit of detection (LOD) of 1~10 ng/well for 8 GI and 13 GII genotypes, except 50 ng/well for GII.7 strains. In addition, Hurwitz et al [57] identified two scFvs (NJT-R3-A2 and NJT-R3-A3) that could detect NoV from infected clinical stool samples [57]. The LOD of NJT-R3-A2 scFv for the detection of GI.1 and GI.7 VLPs were 0.1 and 0.2 ng, respectively.…”

“…Shang et al [41] utilized SPR imaging to show that human milk glycan motif specifically binds to NoV and competitively inhibits viral capsid binding to host receptors. By SPR, Hurwitz et al [57] identified two scFvs (NJT-R3-A2 and NJT-R3-A3) that could bind to GI.1 and GI.7 VLPs, with Kd values of 27 nM and 49 nM, respectively. Similarly, Kim et al [137] demonstrated that concanavalin A (Con A) can bind with HuNoV (GII.4), with the metal coordination region of Con A identified as a major [129] with permission from the publisher).…”