Nunc polysorp 96 well plates

Anti‐NP and anti‐HA antibodies in mouse sera were measured on Nunc PolySorp 96 well plates (Thermo Fisher). For NP‐ELISAs plates were coated overnight at 4 ° with 2 µg mL⁻¹ NP‐9 (high affinity) or NP‐27 protein conjugated to BSA (Biosearch Technologies)in carbonate buffer (Sigma). For anti‐HA ELISA, plates were coated with 2 µg mL⁻¹ of recombinant HA1 (Sino biologics) in carbonate buffer. Plates were then blocked with TBS/Tween 20/0.5% BSA for 2 h at room temperature. Sera was diluted in TBS/Tween 20/0.5% BSA at concentrations noted in the figures and incubated at room temperature for 2 h. Alkaline phosphatase‐conjugated anti‐mouse IgG antibodies (Southern Biotech) were diluted in TBS/Tween 20/0.5% BSA and incubated for 2 h at room temperature. Plates were developed using phosphatase substrate (Sigma) were used for detection. ODs were read at 405 nm on a SpectraMax Microplate Reader (Molecular Devices).

Tosyl-activated superparamagnetic microbeads M-450 (MBs, Dynabeads, Cat. no. 14013, 4.5 μm diameter, 4 × 10⁸ beads/mL, 30 mg/mL) were from Invitrogen. TDM from Mycobacterium bovis (Cat. no. T3034), and fatty acid-free bovine serum albumin (BSA, Cat. no. A7030) were from Sigma. Goat F(ab’)₂ anti-human IgG (H + L) labelled with biotin was from Southern Biotech. Nunc PolySorp 96-well plates, 1-step ultra TMB ELISA, and Pierce streptavidin poly-HRP were obtained from Thermo Scientific. Mycobacterial recombinant 38 kDa and Ag85A proteins were obtained from Mybiosource (CA, USA). Neodymium disc magnets (diameter 5 mm, thickness 2 mm) were from AliExpress Global Retail. Unless otherwise specified, all experiments were performed using PBS buffer without Ca⁺² and Mg⁺² ions.