Ltd, Tokyo, Japan). HAI assays were performed in V-bottomed 96-well microtitre plates (Nunc Roskilde, Denmark), as previously described [8, 9]. Sera were subjected to 2-fold serial dilutions (from 1:8 to 1:16 384) in phosphate-buffered saline (PBS) prior to incubation with 4 HA units of the influenza A/California/7/09 (H1N1)
virus [provided by the WHO Influenza Collaborating Centre, National Institute for Medical Research (NIMR), London, UK]. Glutaraldehyde-fixed turkey red blood cells (0.4%) were added at room temperature and after 30 min a reading was taken[10, 11]. To minimize assay variation, sera from one positive and one negative healthy INK 128 cell line subjects were used in each plate for plate validation, paired samples Bortezomib clinical trial were assessed in the same test, samples were repeated at least twice in independent experiments, plates were read twice in flat and tilted positions by two or three trained individuals and the geometric mean of the different readings was calculated. HAI titres were considered valid if two independent readings did not differ by more than one dilution. Results were expressed as the reciprocal of the highest dilution showing a positive HAI. Negative samples were assigned a titre of 1:4 for computational purposes and individual values were log-transformed to calculate the geometric mean antibody titres (GMTs). The MN assay was adapted from a previously described procedure . Briefly,
decomplemented sera were serially diluted 2-fold (starting at 1:10) in flat-bottomed 96-well microtitre plates. Virus [2 × 104 tissue culture infective dose 50 (TCID50)/mL] was added and neutralization PI-1840 allowed to proceed for 1 h at 37°C prior to the addition of Madin Darby Canine Kidney (MDCK) cells (5 × 105 cells/mL). Sixteen hours later, monolayers were scored for confluency, fixed and treated with a monoclonal antibody (MCA400, clone AA5H, AbD Serotec, Duesseldorf, Germany) against influenza A nucleoprotein. Staining was revealed by adding anti-immunoglobulin G (HRP-IgG; Dako, Glostrup, Denmark) followed by tetramethyl benzidine (TMB) substrate
(Invitrogen, Zug, Switzerland), prior to measuring the absorbance at 650 and 450 nm (for background subtraction). The average optical density (OD) values from five replicate wells containing virus and cells (V+C) and cells only (C) were used to calculate the 50% neutralizing endpoint. The endpoint titre was expressed as the reciprocal of the highest dilution of serum with an OD value less than X, where X = [(average of V+C wells) − (average of C wells)]/2 + (average of C wells). Assay variations were limited by several means: positive and negative control samples were included in one plate per run, samples were tested at least twice in independent experiments and plates were validated using stringent criteria. Negative samples were assigned a titre of 1/5 for computational purposes.