Pnpp substrate

rPfs16 was expressed on a small scale, and the medium and lysate were separated as described above. Samples were taken at 72 h and 96 h postinfection with the recombinant baculovirus. A flat-bottom 96-well plate (Brand) was used to coat equal concentrations of 3 mg/ml protein from either lysate or medium in triplicates at room temperature (RT) for 1 h. The wells were blocked with 2% BSA in 1× PBS and 0.1% Tween-20 (PBST) for 1.5 h at RT. The wells were then incubated with anti-His mAb at 1:1000 dilution in 2% BSA for 1 h at RT. Following three washes with 1× PBST, the wells were incubated with alkaline phosphatase–conjugated anti-mouse secondary antibody at 1:10,000 dilution in 2% BSA for 30 min at RT. After washing with 1× PBST three times, ready-made pNPP substrate (Thermo Fisher Scientific) was added and incubated for 1 h at 37 °C protected from light. Post incubation, the plate was directly read at A₄₀₅ with the Synergy H1 microplate reader (BioTek).

The LCMV Armstrong (LCMV^ARM) strain was kindly provided by R. Ahmed (Emory University, Atlanta, GA). Virus was grown in BHK-21 cells and titres (PFU) in the supernatant were determined as described before58 (link). For acute viral infections, mice were injected intraperitoneally with 2 × 10⁵ PFU of LCMV^ARM and analysed 10 days after infection18 (link)58 (link). LCMV-specific antibodies in the sera of mice were measured as described before18 (link)58 (link). Briefly, lysates of LCMV-infected BHK-21 cells were used as substrate and LCMV-specific IgG and IgM binding was detected in serial dilutions of serum using AP-conjugated goat-anti-mouse IgG or IgM antibodies (both Southern Biotech). Enzyme-linked immunosorbent assays (ELISAs) were developed with pNPP substrate (Thermo Scientific; cat. no. 37621) and absorbance was analysed at 405 nm using a SpectraMax M5 microplate reader (Molecular Devices).

Ninety-six-well plates were coated with 2.0 μg/ml of lipopolysaccharide (LPS, Salmonella enterica serovar Typhimurium, Sigma), salmochelin receptor (IroN), aerobactin receptor (IutA), or 0.25 μg/ml unlabeled chicken IgA (i.e., total IgA; H+L, Thermo Fisher Scientific) overnight at 4°C. LPS is common in gram-negative bacteria, and IroN and IutA are virulence factors involved in iron acquisition. Recombinant IroN and IutA proteins were purified from culture of E. coli BL21 containing the pET-101/D-TOPO vectors (Invitrogen) carrying iroN or iutA genes as previously described (Mellata et al., 2016 (link)). SISs were diluted 1:1 in SEA blocking buffer (Thermo Fisher Scientific), serially diluted 1:2, and incubated for 1 h at room temperature. Goat-anti-chicken-IgA-AP (H+L, Thermo Fisher Scientific) was added, followed by PNPP substrate (Thermo Fisher Scientific), and absorbance was measured at 405 nm. To measure antibody titer, the reciprocal of the highest dilution values doubling the control value (i.e., CON birds) were considered positive. ELISAs were done in duplicate per individual bird and independently replicated twice.