The Influenza Virus Typing Set allows typing of influenza virus A and B subtypes. The Set contains several biotinylated antibodies (two kinds of monoclonal antibodies that distinguish between influenza A subtypes, one monoclonal antibody that binds all types of influenza A viruses, one polyclonal antibody that binds all types of A and B viruses) and Avidin-POD. The sensitivity of this system is equivalent to that of the PAP method.
The protocol involves preparing a virus solution from samples, which is then used to infect Madin-Darby Canine Kidney Epithelial Cells (MDCK) cells in 96-well plates. The culture supernatant is removed, cells are ethanol-fixed, and an avidin solution is added to each well. Following blocking of non-specific binding regions, a biotinylated antibody is added to the well, followed by addition of the streptavidin POD conjugate. Finally, the well is washed and the coloring substrate Diaminobenzidine (DAB, not supplied) is added. Positive subtype determination is made based upon color development within the well.
Influenza viruses are classified into three groups (A, B and C) based upon differences for their nuclear proteins (NP), and are further classified into subtypes according to their surface glycoproteins, neuraminidase (NA) and haemagglutinin (HA). The most prevalent types of influenza virus are influenza A H1, influenza A H3, and influenza B. The influenza A HA protein consists of a head region which contains a target cell receptor domain, and a stem region that contains a special amino acid sequence required for fusion of the virus and the target cell. The HA head region, in particular, has especially strong antigenicity and, thus, most produced antibodies recognize this region. However, mutation readily occurs in this region, and typing represents a significant method for judging the effect of a vaccination. Furthermore, rapid typing is important due to the short infectious period of this virus.
