Abts solution

HBV subviral particles bearing wild-type or alanine mutant L-, M-, and S-HBsAg proteins were produced by transfection of Huh-7 cells with pT7HB2.7 plasmid or mutant derivatives using FuGENE 6 reagent (Roche) as previously described (Salisse and Sureau, 2009 (link)). High-binding 96-well ELISA plates (Costar, Corning) were coated overnight with purified human anti-HBs antibodies (0.5 µg/well in PBS). After PBST washings and a 2 h-blocking step, plates were incubated 2 h with culture supernatants containing wild-type and mutant HBV envelope proteins. After washings, plates were incubated for 1 h with purified His-tagged anti-preS2 1F6 Fab fragments (125 ng/well). Wells were washed before addition of rabbit HRP-conjugated anti-6xHis-tag antibodies (1:4,000-diluted in blocking solution, ab1187, Abcam) and HRP chromogenic substrate (ABTS solution, Euromedex) as described above. Percentage of binding was calculated following the formula ([OD]^mutant / [OD]^wild-type) × 100.

Polyreactivity ELISA was performed as followed. Briefly, high‐binding 96‐well ELISA plates (Costar) were coated overnight with 0.5 μg/well of purified double stranded (ds)‐DNA, KLH, LPS, and 0.25 μg/well of purified insulin (Sigma) in PBS. After washings with 0.001% Tween‐PBS, plates were blocked 2 h with 2% BSA, 1 mM EDTA, PBST (Blocking buffer). After washings, coated plates were incubated 2 h with IgG or IgA antibodies diluted at 26.67 nM and three consecutive 1:4 dilutions in PBS. After washings, the plates were revealed by incubation for 1 h with goat HRP‐conjugated anti‐human IgG (Jackson ImmunoReseach, 0.8 μg/mL final), or anti‐human IgG/IgM/IgA antibodies (Immunology Jackson ImmunoReseach, 0.8 μg/mL final), and by adding 100 μL of HRP chromogenic substrate (ABTS solution, Euromedex) after washing steps. After 1‐h incubation, optical densities were measured at 405 nm (OD_405nm), and background values given by incubation of PBS alone in coated wells were subtracted. High positive (ED38) 38 and negative (mGO53) 8 antibody controls were included in each experiment, and threshold values for reactivity were determined as previously described 39. All experiments were performed using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan).