Development And Application Of Monoclonal Antibodies Against Hemagglutinin Of H4 Subtype Avian Influenza Virus

H4 subtype avian influenza virus (AIV) is mildly pathogenic to poultry and mammals, causing the respiratory symptoms and even death of its host. In the present study, monoclonal antidodies (mAb) against hemagglutinin (HA) of H4 AIV were developed and the flow cytometry method to detect virus-infected MDCK cells was detemined.8-week-old female BALB/c mice were immunized with H4-AIV strain A/Duck/Yangzhou/185/2003 before the spleen cells were collected and fused with Sp2/0-Ag-14 myeloma cells. Hybridoma cells test was screened by hemagglutinin inhibition (HI) test and the positive clones were subjected to limited dilution for subcloning. Four mAbs against HA of H4-AIV were produced after triple subcloning and named as 2C11, 6E7, 4C8 and 5C5 respectively. All the 4 monoclonal antibodies were tested to be IgG2a subtype and the HI titers were between 2⁽120 and 2¹⁵. Cross HI test and indirect immunofluorescence showed that the four mAbs were specific to H4-AIV as they reacted only with H4 AIV but not with NDV, IBV, EDSv-76 and other AIV subtypes. Virus neutralization test indicated that all the four mAbs were able to inhibit the pathogenicity of H4 AIV in cell culture. The HI results revealed that mAbs 2C11, 4C8, 6E7 and 5C5 could inhibit 9, 8, 7 and 7 of 10 H4-AIV isolates respectively, while none of them could inhibit the A/Duck/Suzhou/1/2002 isolate,showing the antigenic difference among the HA proteins of the H4-AIV isolates.A/Duck/Yangzhou/185/2003 isolate showed typical morphology of AIV particles under the transmission electron microscope. The virus was able to grow in Madin-Darby Canine Kiney (MDCK) cell, but better when trpsin-TPCK added.The mAbs and the flow cytometry based on mAb developed in this study provided approaches to the detection of H4 AIV and would be very useful in epidemiological investigation of AIV.