Softmax pro v 7

Recombinant human FcγR ectodomains (Sinobiological) (5 μg ml⁻¹) were immobilized into high-binding 96-well microtitre plates (Nunc), and after overnight incubation at 4 °C, plates were blocked with PBS plus 2% (w/v) BSA and 0.05% (v/v) Tween20 for 2 h. IgG immune complexes were prepared by incubating for 1 h at 4 °C Fc variants of the anti-NP (4-hydroxy-3-nitrophenylacetyl) monoclonal antibody Ab 3B62 with NP-BSA (27 conjugation ratio, Biosearch Technologies) at a molar ratio of 1:10 (antigen:antibody). IgG immune complexes or monomeric IgG (for FcγRI) were serially diluted (1:3 consecutive dilutions in PBS starting at 10 μg ml⁻¹ (IgG concentration) for immune complexes or 1 μg ml⁻¹ for monomeric IgG) and applied to FcγR-coated plates. After 1 h incubation at room temperature, bound IgG was detected using horseradish peroxidase (HRP)-conjugated goat F(ab’)₂ anti-human IgG (1 h; 1:5,000; Jackson Immunoresearch). Plates were developed using the TMB (3,3′,5,5′-tetramethylbenzidine) two-component peroxidase substrate kit (KPL) and reactions were stopped with the addition of 1 M phosphoric acid. Absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices) and background absorbance from negative control samples was subtracted. Data were collected and analysed using SoftMax Pro v.7.0.2 software (Molecular Devices).

Recombinant HA (Influenza A H1N1 (A/California/04/2009 or A/Puerto Rico/8/34) or H3N2 (A/x31)), or NA (A/California/04/2009) or NP (H1N1 (A/California/04/2009) or H3N2 (A/x31)) (Sinobiological) (3 μg ml⁻¹) were immobilized into high-binding 96-well microtitre plates (Nunc) and after overnight incubation at 4 °C, plates were blocked with PBS plus 2% (w/v) BSA and 0.05% (v/v) Tween20 for 2 h. After blocking, plates were incubated for 1 h with serially diluted IgG antibodies or serum samples (1:3 consecutive dilutions in PBS starting at 1 μg ml⁻¹ for monoclonal antibodies or 1:10 for serum samples), followed by HRP-conjugated goat anti-human IgG (minimal cross-reactivity to mouse IgG) for human monoclonal antibodies or goat anti-mouse IgG (minimal cross-reactivity to human IgG) for serum samples (1 h; 1:5,000; Jackson Immunoresearch). Plates were developed using the TMB two-component peroxidase substrate kit (KPL) and reactions were stopped with the addition of 1 M phosphoric acid. Absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices) and background absorbance from negative control samples was subtracted. Data were collected and analysed using SoftMax Pro v.7.0.2 software (Molecular Devices).