Nanoparticle-based Antibody Avidity Assay

The protocol was adapted from Langowski et al.¹⁰³ (link) First, recombinant I53_dn5 nanoparticle or H1 MI15-foldon protein was incubated for 1 h on 96-well Nunc MaxiSorp plates (Thermo Scientific) (2.0 μg/mL, 50 μL per well). Then, 200 μL of Tris-Buffered Saline Tween (TBST: 25 mM Tris pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween 20) with 2% (w/v) BSA was added to each well and incubated for 1 hr. Plates were washed 3× in TBST using a robotic plate washer (BioTek). Then, 50 μL of a 1:2,500 serum dilution in TBST with 2% (w/v) BSA was added to each well and incubated for 1 h. To test for avidity, 50 μL of 2 M sodium thiocyanate (NaSCN) or PBS (control) was added to wells for 15 min. After washing plates 3× with TBST, 50 μL of anti-mouse HRP-conjugated goat secondary antibody (CellSignaling Technology) diluted 1:2,000 in TBST with 2% (w/v) BSA was incubated in each well for 1 hr. Following a final 3× TBST plate wash, 100 μL of TMB (SeraCare) was added to each well and rested for 2 min before quenching with 100 μL of 1 N HCl. Absorbance at 450 nm was immediately collected for each well on a SpectraMax M5 plate reader (Molecular Devices). All steps were performed at ambient temperature. Percentage OD450 in the corresponding NaSCN/PBS wells were used to determine the avidity index.