A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus

Abstract

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5–99.5%) and 95.8% (95% CI 86.0–98.8%) for human vs. 100% (95% CI 93.5–100%) and 98.3% (95% CI 91.0–99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Iterative In Silico Screening for Optimizing Stable Conformation of Anti-SARS-CoV-2 Nanobodies

“…Moreover, nanobodies have attracted significant attention because of their small size, ability to recognize hidden epitopes, and ease of production. Several of these nanobodies have been developed for identifying or inhibiting viruses, such as hepatitis E virus (HEV) [ 30 ], influenza virus, and respiratory syncytial virus. The use of nanobodies may be one of the most effective approaches for protecting against COVID-19.…”

SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice

“…This study was approved by the Internal Committee for the Care and Use of Experimental Animals (Spanish acronym CICUAE) under protocol N° 15/2020. A Nb-library was obtained as already stated [ 30 ]. Another Nb-library, previously obtained after immunizing a llama with 4 × 10 9 FFU/dose (fluorescent focus forming units) of BCoV Mebus strain, was also used in this study.…”

“…Nanobody expression was induced with 1 mM IPTG for 16 h at 28 °C. Periplasmic extracts were obtained as described [ 30 ]. Nanobodies from PE were purified using IMAC Hi-Trap columns (GE Healthcare) and eluted using 300 nM Imidazole.…”

“…Production and purification of the selected clones (n = 43) was carried out at 37 • C in TB medium supplemented with 100 µg/mL ampicillin and 0.1% glucose. Nanobody expression was induced with 1 mM IPTG for 16 h at 28 • C. Periplasmic extracts were obtained as described [30]. Nanobodies from PE were purified using IMAC Hi-Trap columns (GE Healthcare) and eluted using 300 nM Imidazole.…”

Nanobodies in the fight against infectious diseases: repurposing nature's tiny weapons

“…Many Nbs targeted against moieties in the pathogens have been considered for diagnostics purposes. For example, the type 2 NS1 protein of the Dengue virus (Fatima et al 2014 ), ORF2 of the hepatitis E virus (Arce et al 2023 ), HIV capsid proteins (Helma et al 2012 ), and other viral targets. In addition, Nbs are also used for the diagnosis of bacterial pathogens including Acinetobacter baumannii (Rasoulinejad and Gargari 2016 ), Brucella spp.…”