Establishment of an indirect ELISA for detecting H7N9 subtype avian influenza virus antibodies applied with hemagglutinin containing a replaced transmembrane domain

Abstract:

[Objective] H7N9 avian influenza virus can infect chickens, and high pathogenic avian influenza (HPAI) H7N9 strains have appeared after natural mutation. Thus, H7N9 vaccines immunization in chickens would be a tendency, and developing an antibody detection method for immunization is a need. To establish a sensitive, rapid, high-throughput enzyme-linked immunosorbent assay (ELISA) to detect H7N9 subtype avian influenza virus antibodies in chickens. [Methods] Three wild-type hemagglutinin (HA) proteins belonging to W1, W2-A and W2-B clades were expressed by an insect cell-baculovirus expression system, and one recombinant HA (H7-53TM) containing a replaced H3 HA transmembrane domain (TM) was expressed as well. Four HA proteins were purified by ion-exchange chromatography and used as ELISA antigens to detect H7N9 avian influenza virus antibodies. [Results] The results of specificity, sensitivity and repeatability assays showed TM replacement mainly affected the repeatability of the HA antigen. The intra- and inter-coefficient of variables of H7-53TM were less than 10%, showing better repeatability; whereas those of 3 wild-type HA proteins were more than 10%, showing worse repeatability. Therefore, H7-53TM was chosen as ELISA antigen. The results of the Receiver operating characteristic curve (ROC curve) analysis show that the established ELISA could accurately discriminate between H7N9 subtype avian influenza virus-positive and -negative serum specimens. Based on correlation, the established ELISA had significantly strong correlation with HI assay to test 134 chicken serum specimens (r=0.854 6, P<0.000 1), and the established ELISA had significantly correlation with HI assay to test sera collected from chickens vaccinated with vaccine strains belonging to three different clades (r>0.5, P<0.05). [Conclusion] TM replacement can increase the repeatability of the HA protein to detect H7N9 avian influenza virus antibodies, and establishes an indirect ELISA for detecting specific antibodies against H7N9 subtype avian influenza viruses belonging to different clades applied with HA containing a replaced transmembrane domain.