Establishment of sandwich ELISA for detecting the H7 subtype influenza A virus

Abstract

Avian H7N9 subtype influenza virus infects human with high case‐fatality rate since it emerged in 2013. Although the vaccination has been rapidly used in poultry due to the emergence of highly pathogenic strain, this virus remains prevalent in this region. Thus, rapid diagnosis both in poultry and human clinic is critically important for the control and prevention of H7N9 infection. In this study, a batch of H7 subtype‐specific monoclonal antibodies (mAbs) were developed and a pair of mAb, 2B6, and 5E9 were used to establish a double‐antibody sandwich enzyme‐linked immunosorbent assay (ELISA) to quantify H7 protein and detect influenza A virus baring H7 subtype HA. The lowest detection limit for the recombinant H7 protein was 10 ng/mL and 0.5 HAU/50 μL of A/Guangdong/17SF003/2016(H7N9), 2 HAU/50 μL of A/Netherlands/219/2003(H7N7) and A/Anhui/1/2013(H7N9) for live virus, respectively. The ELISA could not only detect the prevailing H7N9 virus, but also antigenic drift H7 subtype viruses, showing excellent sensitivity and high specificity. Hence, it could serve as a valuable approach to diagnose H7 subtype virus which showed great potential to cause pandemic, as well as antigen quantification.

Production and identification of monoclonal antibodies and development of a sandwich ELISA for detection of the H3-subtype avian influenza virus antigen

“…Thus, we attempted to use inactive whole virus to prepare and produce the H3 mAb. Whole viruses have widely been used as immunogens for the production of mAbs to develop ELISAs for the diagnosis of disease (Chen et al 2019;Huang et al 2019;Zhang et al 2018). The HA glycoprotein, the major surface protein of influenza A virus, plays a critical role in viral infection and is a primary target of neutralizing antibodies (Ito et al 2019).…”

“…mAbs have a single epitope, display high specificity and have been widely adopted for medical examination. ELISA is highly suitable for epidemiological analyses involving a large number of samples, and sandwich ELISA methods based on mAbs have been developed for other pathogens (Chen et al 2019;Huang et al 2019;Zhang et al 2018). This study had two purposes.…”

Development of a monoclonal antibody‐based antigen capture enzyme‐linked immunosorbent assay for detection of H7N9 subtype avian influenza virus

“…20 An additional sandwich ELISA was developed with a lowest detection limit of 10 ng/mL for recombinant H7 protein and 0.5 −2 HAU/50 μL for live H7 AIVs, respectively. 21 T A B L E 1 Detection sensitivity of the H7N9 strip using clinical specimens Note: AC-ELISA and virus isolation methods are highly correlated (P < .001). Abbreviation: AC-ELISA, antigen capture enzyme-linked immunosorbent assay.…”

Establishment of Sandwich ELISA for Quality Control in Rotavirus Vaccine Production

“…The mAbs were screened by enzyme-linked immunosorbent assay (ELISA) and micro-neutralization assay (ELISPOT). After three rounds of cloning, the stable clones were inoculated to mice, and the mAbs were purified from the ascites using protein A [22]. The mAb screening and preparation protocol was approved by Xiamen University Laboratory Animal Center.…”