Furthermore, they have been shown to have a high sensitivity in detecting antibodies directed against the stalk of influenza envelope glycoprotein, the haemmaglutinin. Influenza pseudotype neutralization assays are a safe alternative when potentially pandemic viruses need to be studied. For example, the use of pseudotyped lentiviral vectors as surrogate antigens in neutralization assays is extremely useful in influenza research. Numerous lentiviral vectors harboring glycoproteins of different viruses have been described in the literature, and some of them have been used in neutralization assays. When enveloped viruses are studied, gammaretroviral and lentiviral vectors pseudotyped with the viral glycoproteins of interest are a feasible substitute for viruses.

However, replication-deficient viruses can often be used as a safe alternative. These assays routinely require the use of wild-type viruses, and this limits their application when highly pathogenic human viruses are studied. Neutralization assays are powerful tools to study human and animal antibody responses against viruses elicited by vaccination or natural exposure. Additionally, it will provide a starting point for the development of pseudotype neutralization assays using pseudotypes bearing other viral envelope proteins. This protocol will provide support for the validation and the standardization of the pseudotype neutralization assay for influenza virus serology. We also present the steps necessary to analyse the data and calculate the half maximal inhibitory concentration of the sera analysed.

Here, we describe, in detail, an experimental protocol to perform pseudotype neutralization assays using lentiviral pseudotypes bearing influenza haemagglutinin and expressing firefly luciferase. This could result in discrepancies between the results of different laboratories even when the same pseudotypes and the same samples are analysed.

However, protocols described in the literature differ widely with respect to material, reagents, and methods used to perform these assays and to analyse the raw data generated. Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories.

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