Detection of influenza A and B neutralizing antibodies in vaccinated ferrets and macaques using specific biotin-streptavidin conjugated antibodies

Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.