Norovirus Antigen Detection with a Combination of Monoclonal and Single-Chain Antibodies

Abstract

The performance of a norovirus antigen detection assay was assessed using monoclonal antibody NV23 and single-chain antibody HJT-R3-A9 to identify both virus-like particles and virus-containing fecal samples. The detection of 25 different norovirus genotypes as recombinant virus-like particles or in clinical samples was dependent on virus or antigen concentration. N oroviruses are a major cause of acute nonbacterial gastroenteritis in humans (1). Noroviruses are genetically highly diverse and have been classified into seven genogroups, three of which contain human strains. These genogroups are further subdivided into at least 41 genotypes based upon the amino acid sequence of the major capsid protein, VP1 (2). The diversity of genotypes has been a barrier to the development of sensitive, broadly reactive antigen detection assays. The development of monoclonal (3) and human single-chain (4) antibodies (MAbs and single-chain variable fragment [scFv], respectively) that recognize many different norovirus strains led us to assess a combination of these reagents for identification of noroviruses in fecal samples.Recombinant norovirus virus-like particles (VLPs) served as the antigen in initial studies and were generated using a baculovirus expression system, as previously described (3). Recombinant clones were generated either by amplification of viral genomes from clinical samples or by in vitro synthesis of viral genes from GenBank sequences (Epoch Life Science, Ltd., Missouri City, TX). Expressed VLPs were purified using cesium chloride gradients. Adequacy of the VLP structure was confirmed by electron microscopy, and VLP protein concentrations were determined with a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL) using bovine serum albumin as a reference standard. Fecal samples collected in previous clinical studies (5, 6) were characterized for the presence of noroviruses by conventional reverse transcription (RT)-PCR and were used in the current study under protocols approved by the Baylor College of Medicine Institutional Review Board. Each sample was from a unique individual; samples were selected to represent an array of different genotypes from available specimens. Genotype was assigned by sequencing of the 5= end of the VP1 gene, and virus concentration was determined semiquantitatively by real-time RT-quantitative PCR (qPCR) with a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) using Cog1F/Cog1R/Ring1C and Cog2F/Cog2R/Ring2 primers and probes for genogroup I (GI) and GII strains, respectively (7, 8). Viral RNA was isolated with a QIAamp viral RNA minikit (Qiagen, Mainz, Germany). A sample with a cycle threshold (C T ) value under 40 was considered positive. Negative samples were assigned a C T value of 40 for graphic display.We previously generated and characterized a panel of MAbs and scFvs that recognize norovirus antigens (3-5). For this study, we selected MAb NV23, which recognizes an epitope on the VP1 protruding domain of GI, GII, and GIV n...

The Antigenic Topology of Norovirus as Defined by B and T Cell Epitope Mapping: Implications for Universal Vaccines and Therapeutics

“…Though less conserved than S domain sequences, residues that make up the P1 domain are more readily accessible due to capsid flexibility [98]. Cross-reactive mAbs that map to linear epitopes have proven useful in diagnostic assays, but their protective capacity appears limited [132,133,134].…”

“…Several IgGs and scFvs have been generated that may expand the possible genotypes that can be detected in immunoassays, including NS14, NS22, and HJT-R3-A9 (Table S3). In fact, a combination of NS14 and HJT-R3-A9 was used to create a diagnostic platform that detected 25 genotypes of HuNoV in patient stool samples [134]. Nano-85 has also been used to create a nanobody-based lateral flow immunoassay (Nano-IC), which recognizes both outbreak genotypes GII.4 and GII.17 [128].…”

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

“…Since these biopanning experiments selected for binding with the GII.4 Houston virus (HOV), however, the binding interactions with GII are more sensitive and better characterized than for GI genotypes. Amongst the clones identified, the one that binds GI VLPs best—HJT-R3-A9—had very low signal for GI.7 and did not produce a binding signal with GI.1 VLPs in SPR analyses, though it worked in a sandwich ELISA format [30]. Alternately, HJT-R3-F7, the scFv with the strongest GI.1 binding affinity based on SPR from that study, did not produce significant signals to any of the GI VLPs in the ELISA format, and its clinical utility for binding GI.1 virus in stool was not evaluated.…”

Molybdenum Trioxide Nanocubes Aligned on a Graphene Oxide Substrate for the Detection of Norovirus by Surface-Enhanced Raman Scattering

“…Biological Materials. Monoclonal antibodies (NS14, isotype-IgG acquired from the spleen cells of orally immunized mice), which are specific and broadly reactive to genogroup II of NoV, 24 were used in this work for the sandwich-type immunoassay. Following the standard protocol for virus-like particle preparation, 25 GII.…”