Pre vaccination human serum samples were stored at −80°C and heat inactivated at 56°C for 90 min prior to use. The assay was performed as described previously [6] (link), with the following modifications. Briefly, GripTite 293 MSR cells (Invitrogen, catalogue no. R795-07, cultured as per manufacturer’s instructions) seeded the previous day at 3×104 cells per well were infected with a 1∶1 mixture of test sera dilutions and ChAdY25-E–SEAP (secreted alkaline phosphatase) recombinant vector. Recombinant adenoviruses were incubated with five serial dilutions of serum in FBS–DMEM (phenol red free; GibcoBRL catalog no. 31053-028) before infection. The final serum dilutions were 1∶18, 1∶72, 1∶288, 1∶1152, 1∶4608; each serum sample was tested in duplicate. Supernatants were collected and assayed for SEAP concentration using CSPD (Tropix PhosphaLite Chemiluminescent Assay Kit, Applied Biosystems UK) according to the manufacturer’s instructions. Luminescence intensity was measured using a Varioskan flash luminometer (Thermo Scientific). Neutralization titers were defined as the serum dilution required to reduce SEAP concentration by 50% compared to wells infected with virus alone. Neutralization titer was calculated by linear interpolation of adjacent values.
Free full text: Click here
