Development of a VHH-nanobodies based ELISA test for human Norovirus detection

L .BRACCO; I. IBAÑEZ; K. GOMES; V. PARREÑO; M. BOK

Paris

Conferencia; Single Domain Antibody Meeting; 2023

Human noroviruses (HuNoVs) are the cause of ~95% of epidemic non-bacterial gastroenteritis worldwide. NoV infection is usually diagnosed based on qPCR, since ELISAs and strip tests that are available have low sensitivity and can only detect a few genotypes. In undeveloped countries molecular methods are only available at reference centers or universities, only with epidemiological purposes. Thus, sentinel units and first-aid rooms do not have a quick test for early outbreak detection.The aim of this study was to develop a nanobodies (Nb)-based diagnostic kit for GI, GII and GIV HuNoV detection, with high sensitivity and specificity.HuNoV-nanobodies against GI, GII and GIV genotypes were selected from llama-derived immune libraries. Bivalent versions of three clones (GI-specific N1 Nb, GII-specific M5 Nb and GIV-specific A12 Nb) were produced and used for ELISA plate coating. Other clones (GI-specific N2 Nb, GII-specific C8.5 Nb and GIV-specific B8 Nb) were fused to HRP using two different strategies: covalent labeling of Nb expressed in E. coli and expression of fusion proteins in mammalian cells. VLPs from GI, GII and GIV were expressed in baculovirus system, to use as positive controls for ELISA optimization. Positive and negative fecal samples were obtained from Instituto Malbrán, Argentina, during 2018 and stored at -80°C.From the two labeling strategies, the HRP-Nb fusion protein gave the best results reducing the background of the assay. As preliminary results, the three bivalent Nb (500 ng/well each) combined with the three Nb-HRP detected 1-10 ng of each tested VLP (GI.1 Norwalk, GII.4 MD-2004 and GIV St. Cloud). Also, 2 out of 8 GI-qPCR-positive fecal samples and 4 out of 5 GII-qPCR-positive fecal samples were diagnosed positive using our ELISA. A GI positive sample was typified as GI.5 [P4] and GII positive samples were typified as GII.4 [P16]. GIV fecal samples were not available.We successfully developed an Nb-based ELISA assay to detect HuNoV in fecal samples. Sample storing conditions and freezing-thaw cycles might be critical factors to increase detection rates. New fresh samples will be tested, and analytical and diagnosis validation of the assay will be conducted.