Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC. 1 DOI:10.21769/BioProtoc.2936 www.bio-protocol.org/e2936 A Lentiviral Pseudotype ELLA for the Measurement of Antibodies Against Influenza Neuraminidase Fabrizio Biuso 1 , George Carnell 2 , Emanuele Montomoli 1, 3 and Nigel Temperton 2, * 1 VisMederi Research s.r.l., Siena, Italy; 2 Vial Pseudotype Unit, Medway School of Pharmacy, University of Kent, Chatham Maritime, Kent, United Kingdom; 3 Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy *For correspondence: n.temperton@kent.ac.uk [Abstract] This protocol describes the rapid and safe production of lentiviral pseudotypes characterized by a lentiviral core containing a reporter, in conjunction with avian influenza haemagglutinin (HA) and human neuraminidase (NA) glycoproteins on the surface. Production is optimized with Endofectin Lenti TM transfection reagent in 6-well plate format. These pseudotyped viruses can be employed for serological assays of surface glycoproteins HA and NA. They can be efficiently used to perform the ELLA (Enzyme-linked lectin assay) to measure NA inhibiting antibodies in lieu of using reassortant virus or Triton X-100 inactivated wild-type virus as source of antigen, which may require higher biosafety levels. Keywords: ELLA, PV, OD, Assay, Neuraminidase, Antibody, Neutralisation [Background] The production of influenza virus pseudotypes has been extensively described previously (Nefkens et al., 2007; Temperton et al., 2007; Carnell et al., 2015). The need for a safe and rapid system to evaluate antibodies targeting the NA via the ELLA assay, avoiding the employment of reassortant mismatched virus or wild-type virus, has been met by producing influenza NA bearing pseudotypes (Prevato et al., 2015). A recent study (Biuso et al., 2017) confirmed that the co-expression of HA with NA improves the release of newly formed pseudotyped lentiviruses. Here we report a simple, widely applicable and optimized protocol for PV production by using the Endofectin Lenti TM transfection reagent in 6-well plate format, and the methodology to perform an ELLA assay with the resulting influenza pseudotypes. While PV production for HA-based assays has made use of the lentiviral genome containing a reporter gene, the ELLA assay utilizes solely the surface NA glycoprotein, rendering a PV-incorporated reporter irrelevant to this protocol. The original ELLA assay from 1990 was recently improved (Couzens et al., 2014). This assay enables the detection of exposed galactose residues resulting from the enzymatic action of NA on sialic acids present on the fetuin substrate. This assay allows the measurement of NA inhibiting antibodies, through detection of a drop in enzymatic NA activity. The ELLA overcomes the limits of the cumbersome thiobarbituric acid assay (TBA) that employs hazardous materials, allowing for large-scale screening of serum samples. The basis of the assay is simple, 96-well plates are coated in the carbohydrate fetuin, which is then exposed to NA through NA bearing PV. The NA enzyme cleaves terminal sialic acid residues from the fetuin, exposing galactose that is then bound by the peanut agglutinin from Arachis hypogeal, conjugated to horseradish