Development of an Immunochromatographic Strip for Rapid Detection of H9 Subtype Avian Influenza Viruses

Abstract

An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry, using two monoclonal antibodies (MAb), 4C4 for H9AIV hemagglutinin (HA) and 4D4 for nucleoprotein. The 4C4 MAb was labeled with colloidal gold as the detection reagent, and the 4D4 MAb was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the HA and HA inhibition (HI) tests, the strip was specific for the detection of H9AIV, with a sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 postinoculation, earlier than the appearance of clinical symptoms. Application of the strip for the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens that were infected with H9AIV. Further characterization of 10 positive and 30 negative randomly selected samples showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability. This finding, together with the advantages of rapid detection and easy operation and without the requirement for special skills and equipment, makes the strip suitable for onsite detection and the differentiation of H9AIVs from other viruses in poultry.

Evaluation of Diagnostic Applications of Monoclonal Antibodies against Avian Influenza H7 Viruses

“…The assay can detect H7 virus with an HA titer of Ն4, which is similar to detection by an H5-specific DAS ELISA (39). Immunochromatographic strips for rapid detection of influenza viruses using two specific MAbs have been reported for H5, H7, and H9 virus subtypes (14,32,21). The pair of MAbs (3 and 7) reacted with all 14 of the H7 strains and has the potential for use in the development of an immunochromatographic strip test for rapid detection of the H7 antigen.…”

Development and optimized pairing of mouse monoclonal antibodies for detecting hemagglutinin in novel H7 subtype influenza viruses

“…Immunochromatographic strips with mAbs have been reported for the rapid detection of influenza viruses of H9, H7, and H5 subtypes (Peng et al, 2008;Kang et al, 2014;Yeo et al, 2017;Nguyen et al, 2017;Iwatsuki-Horimoto et al, 2018). In a europium nanoparticle-based rapid diagnostic method with mAbs against avian influenza H7, the detection limits of the fluorescent immunochromatographic strip test employing novel mAbs were 31 ng mL -1 for H7N9 rHA1 protein and 40 HAU mL -1 (2 HAU/50 µL) for H7 subtype virus (Yeo et al, 2017).…”

Teste Imunocromatográfico De Fluxo Lateral: Uma Ferramenta Rápida De Diagnóstico

“…O fluxo irá continuar até a linha controle e os restantes de partículas de ouro conjugados ligam-se ao anticorpo imobilizado, originando uma linha colorida. Se o teste for negativo, apenas a linha controle apresenta a coloração, não ocorrendo ligações na linha teste (PENG et al, 2007;PENG et al, 2008).…”