C t l immunospot plate reader and counting

Freshly sorted cells or day-2-post culture cells were tested for antibody secretion by ELISPOT. Sorted cells were plated on goat anti-human IgM or goat anti-human IgG coated plates in medium alone, and incubated in 5% CO2 for 14-18 hours at 37°C. Spot-forming cells were detected by incubation with Alkaline Phosphatase-conjugated goat anti-human IgM or anti-human IgG (Southern Biotech), and developed with VECTOR Blue, Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Spots in each well were counted using a C.T.L ImmunoSpot plate reader and counting software (Cellular Technology Limited).

On days 6, 7, or 8 after each dose of H7N7 PLAIV in the first cohort (15 subjects), circulating IgG Antibody-secreting cells (ASCs) specific for the vaccine and the H7 HA were enumerated by ELISpot assay. The ELISpot assay was performed largely as described previously [21 (link)]. Immobilon P membrane-based 96-well plates (Millipore, Billerica, MA) were coated wi th β-propiolactone-inactivated vaccine virus diluted in PBS to 5000 HAU/ml or with recombinant H7 from A/Netherlands/219/2003 (H7N7) (BEI Resources) at 1 μg/ml. PBS only was added to negative control wells. Enriched B cells were resuspended in complete medium containing alkaline phosphatase-conjugated goat anti-human IgG (H + L) (KPL, Gaithersburg, MD) at .2 μg/ml. Serial 2-fold dilutions of the cell suspensions were prepared in coated and blocked plates and incubated overnight. After washing and spot development, spots were counted using a CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH). Spot counts are expressed as a frequency of input CDI 9+ cells. The enriched B cells were also characterized by flow cytometry as described previously [22 ].