Streptavidin alkaline phosphatase

96-well MaxiSorp polystyrene plates (NUNC, Thermo Fisher Scientific, Rochester, NY) were coated with 2 μg/mL recombinant Ag85B (BEI Resources) in 100 μL PBS and incubated at 4°C overnight. Plates were washed 4 times with PBS containing 0.05% Tween 20 (Sigma-Aldrich, wash buffer) and blocked with 200 μL 5% NFDM at 37°C for 2 hours, washed 4 times with wash buffer and serum samples serially-diluted in PBS/1% FCS (ELISA diluent), were added to wells and incubated at 37°C for 1 hour. Wells incubated with ELISA diluent or naïve mouse serum were used as negative controls. Plates were then washed 6 times and biotinylated goat anti-mouse IgG antibody (Southern Biotech, Birmingham, AL) diluted in ELISA diluent was added, and plates were incubated at room temperature and left for 45 minutes. Plates were washed again 6 times and 100 μL streptavidin-alkaline phosphatase (GE Healthcare UK Limited) diluted in ELISA diluent was added at room temperature for 30 minutes. Plates were subsequently washed 8 times and developed with 100 μL pNPP AP substrate (BioFx Laboratories, Owing Mills, MD) at room temperature for 15–25 minutes. Color development was stopped with 50 μL Stopping Solution (BioFx Laboratories) and plates were read at 405 nm on a Synergy HT Multi-Mode Microplate Reader (BioTek). Data are expressed as mean endpoint antibody titers ± SEM.

Ninety-six-well plates (Costar 3590) were coated with mouse anti-human IgG (against C_H3 domain) (MCA878G, 1 μg/ml, AbD serotec, Oxford, UK) or NIP-BSA. Plates were blocked with 0.1% BSA in PBS, and supernatants from HEK293E cells were transiently transfected with 1 μg of vaccine plasmids, or purified vaccine proteins were added to wells in triplicates. Plates were now incubated with biotinylated mAb against IgG (Fc fragment) (HP-6017, 1 μg/ml, Sigma-Aldrich, Germany) or a C179 mAb specific for stem HA (1 μg/ml, kind gift from Yoshinobu Okuno, Osaka University, Japan) followed by biotinylated anti mouse IgG2a [1 μg/ml IgG2a(a) 553502, BD Pharmingen]. Next, plates were incubated with streptavidin alkaline phosphatase (1:3000, GE Healthcare, USA), developed with phosphatase substrate (P4744-10G, Sigma-Aldrich, Germany), and read at 405 nm with a Tecan reader using the Magellan v5.03 program.

96-well MultiScreen IP plates were coated with 2 μg/mL recombinant Ag85B protein (BEI Resources: NR-14870) in 100 μL PBS overnight at 4°C. Wells coated with 100 μL CM10 served as background controls. Plates were then washed 5 times with PBS and blocked with 200 μL 5% “Blotting Grade Blocker” nonfat dry milk (NFDM, Bio-Rad, Hercules, CA) for 1 hour at room temperature. A total of 2 x 10⁵ cells were seeded per well and incubated at 37°C in 5% CO₂ for 4–6 hours. Cells were then discarded, and plates were washed 5 times with PBS and incubated with biotinylated goat anti-mouse IgA antibody (IgA-BIOT; Southern Biotech, Birmingham, AL) diluted in PBS containing 1% BSA for 3 hours at room temperature. Plates were subsequently developed by incubation with 100 μL streptavidin-alkaline phosphatase (GE Healthcare, UK Limited, Little Chalfont Buckinghamshire, United Kingdom) diluted in PBS for 1 hour at room temperature and then washed 5 times with PBS,100 μL of BCIP/NBT substrate (Moss Inc, Pasadena, MD) was added and spots were allowed to develop for 20–30 minutes at room temperature. Numbers of antibody-secreting cells (ASCs) were counted using an AID-ELISPOT counter (AID, Strasburg, Germany). Data are presented as spot-forming cells (SFCs) per million cells ± SEM.