2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts)

The IgG titers were measured by ELISA as described using soluble gp350, gB, gp42 and gH/gL as target antigens [51 (link),58 (link)]. First, 96-well microtiter plates (Nunc-Immuno Plate Maxisorp) were coated with 25 ng/well of the target antigen in 1×PBS (pH 6.2) at 4 °C overnight and blocked with 0.1% Tween in PBS + 1% BSA. Equal amounts of sera from each animal for each treatment group and timepoint were pooled and serially diluted in 1×PBS (1:100, 1:300, 1:900, 1:27,000, and 1:81,000), added to the plate in quadruplicates, and incubated for 2 h at RT. The plate was washed 3 times and then incubated with HRP-labeled anti-rabbit secondary antibody at RT for 1h. Plates were washed three times and the substrate ABTS (Sera Care) was added. The reactions were stopped with ABTS stop solution (Sera Care). To determine antibody titer, optical density (OD) was read at 405 nm with a spectrophotometer (Filermax® F3, Molecular Devices). The assay was independently repeated two times with either individual animal serum or pooled sera.

Serum samples belonging to individual mice in the DOX-loaded TBSV-CooP-treated group were analyzed by ELISA to determine the concentrations of anti-TBSV CP antibodies. Briefly, ELISA Maxisorp plates (ThermoFisher Scientific, Waltham, MA, USA) were coated overnight with 100 μL 1× Carbonate Buffer (0.2 M Na₂CO₃/NaHCO₃ at pH 9.4) containing 0.5 μg of purified TBSV-WT. After washing three times with 1× PBS supplemented with 0.1% Tween-20, and twice in PBS, plates were blocked in PBS-5% low-fat milk for 2 h at 37 °C. Then, after washing as before, 100 μL of individual mouse serum dilutions in 1× PBS were added in triplicate to wells and incubated overnight at 4 °C. The presence of bound murine IgG antibodies was detected using an HRP-labelled sheep anti-mouse IgG polyclonal antibody (GE Healthcare Europe GmbH, Opfikon, Switzerland; 1:2500), incubated for 1 h at 37 °C and revealed, after washing, with 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS; SeraCare, Milford, CT, USA) as substrate. The colorimetric reaction was measured at OD_405nm using a microplate absorbance reader (Tecan, Mannedorf, Switzerland). Endpoint titers were defined as the reciprocal of the highest serum dilution giving an absorbance ≥ 0.1 OD above the blank (absorbance of the sera of sham-treated mice). Geometric mean titers were determined for each group.

Biotinylated GAGs were diluted to 40 ng/ml with 10 mM sodium phosphate, pH 7.4, containing 150 mM NaCl and 0.1% w/v Tween 20 (PBS-T), applied to streptavidin-coated 96-well plates (Thermo Fisher Scientific), and rinsed with PBS-T. Purified cochlin solutions expressed by CHO cells at 1.5 µg/ml in PBS-T were added to the wells and incubated at 4°C overnight. After washing with PBS-T, 100 µl horseradish peroxidase (HRP)-conjugated anti-human IgG-Fc antibody was added and incubated for 1 h at room temperature. After washing with PBS-T, 100 µl of ABTS (5120-0032, Sera Care, Milfold, MA, U.S.A.) was added to each well and incubated for 20 min at room temperature. Absorbance was measured at a wavelength of 405 nm. Each experiment was conducted in duplicate. In case of binding inhibitory assay with several GAGs, mCOCH(FL)-Fc was preincubated with the indicated concentration of an inhibitor for 2 h at 20°C and then measured the binding of mutated PNA-Fc (10 µg/ml) to immobilized heparin was performed as described above.