Rapid Detection of Avian Influenza Virus by Fluorescent Diagnostic Assay using an Epitope-Derived Peptide

Abstract

Currently, the point of care testing (POCT) is not fully developed for subtype-specific avian influenza virus detection. In this study, an H5N1 hemaglutinin 1 (HA1) epitope (P0: KPNDAINF) and three modified peptides (P1: KPNTAINF, P2: KPNGAINF, P3: KPNDAINDAINF) were evaluated as POCT elements for rapid detection of avian influenza virus. Based on modeling predictions by Autodock Vina, binding affinity varied depending on alteration of one amino acid in these peptides. The binding energy of P2 indicated its potential for a strong interaction with HA. Fluorescence-linked immunosorbent assay experimentally demonstrated the interaction between these peptides and virus. The four peptides interacted with HA1 of H5N3 with different binding affinities with P2 showing the strongest binding affinity. When P0 and P2 peptides were used in rapid fluorescent immunochromatographic test (FICT) as detection elements, the inter-assay coefficients of variation (CV) indicated that P2-linked FICT was more acceptable than the P0-linked FICT in the presence of human specimens. Antibody pair-linked FICT was influenced by clinical samples more than the P2-linked FICT assay, which showed a 4-fold improvement in the detection limit of H5N3 and maintained H5 subtype-specificity. Compared to the rapid diagnostic test (RDT) which is not specific for influenza subtypes, P2-linked FICT could increase virus detection. In conclusion, results of this study suggest that HA epitope-derived peptides can be used as alternatives to antibodies for a rapid fluorescent diagnostic assay to detect avian influenza virus.

Development of a novel peptide aptamer-based immunoassay to detect Zika virus in serum and urine

“…Previously, we showed that peptide aptamers rather than antibodies could be used in diagnostic applications 21 . In the current study, peptide specificity was improved by modifying the binding sites in addition to enhancing the binding affinity.…”

“…To evaluate the interaction of candidate peptides with antigen and virus, Eu NPs were conjugated with peptides by forming a stable amide bond, and a FLISA was conducted as previously described 21 . The ZIKV envelope protein (amino acid 1-504) was cloned, and the recombinant antigen was produced in an Escherichia coli expression system; this antigen was used to make mAbs, and the reactivity of antibody to virus was confirmed via enzyme-linked immunoassay (ELISA).…”

“…Conjugation of Eu NPs was conducted as previously described 21 . Antibody and peptides were conjugated with 10 µL Eu NPs based on the carboxylate amine condensation method in 500 µL 2-(N-morpholino)ethanesulfonic acid buffer (MES, 0.05 M, pH 6.1), including 13 µL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 5 mM) and 100 µL N-hydroxysuccinimide (NHS, 50 mM).…”

“…Previously, we identified an epitope-derived peptide conjugate that was used to detect influenza virus in an immunoassay 21 . In this study, a novel peptide sequence from the ZIKV epitope pool was developed using in silico modeling tools.…”

Recent Advances in AIV Biosensors Composed of Nanobio Hybrid Material

“…The operation time is around 15 min, which demonstrates the potential of rapid field-ready biosensor on site. This group went on to develop an epitope-derived peptide with europium nanoparticle to detect AIV H5N1 using the fluorescence-linked immunosorbent assay in the point-of-care testing (POCT) system [84]. This novel peptide can be used as the fluorescence signal reporter in the proposed detection system that can reduce the antibody size in the interface.…”