Peroxidase conjugated streptavidin

An enzyme-linked immunosorbent assay (ELISA) was carried out using Maxisorp plates (Nunc, Roskilde, Denmark) coated with Ae. albopictus SGE (2 µg/ml in PBS) at 37 °C for 150 min. Plates were blocked using 250 µl of protein-free Blocking-Buffer (Pierce, ThermoFisher, France) for 60 min at room temperature. Individual sera were incubated in duplicate at a 1/100 dilution in PBS-Tween 1%, 4 °C overnight. Monoclonal mouse biotinylated Ab against human IgG (BD Pharmingen, San Diego, CA) was incubated at a 1/1000 dilution for 90 min at 37 °C. Peroxidase-conjugated streptavidin (GE, Orsay, France) was added at 1/1000 for 60 min at 37 °C. Colorimetric development was carried out using ABTS (2,2′-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium) in 50 mM citrate buffer (pH 4) containing 0.003% H₂O₂ and absorbance was measured after 120 min at 405 nm. Each serum was assessed in duplicate wells and in a blank well without antigen (ODn) to measure non-specific ELISA reactions. Individual results were expressed as the ΔOD value calculated using the equation ΔOD = ODx-ODn, where ODx represents the mean of the OD readings in the two antigen wells.

Enzyme-linked immunosorbent assay (ELISA) was performed as previously described [32 (link)]. Briefly, the peptide (20μg/mL in 100 μl of Phosphate Buffer Saline, i;e. PBS) was coated for 150 minutes at 37°C into Maxisorp plates (Nunc, Roskilde, Denmark). Plates were blocked by Protein-Free Blocking-Buffer (Pierce, Thermo Scientific, France). Each eluate was incubated in triplicate at 4°C overnight at 1/20 dilution in PBS-Tween 1%. Mouse biotinylated Ab to human IgG (BD Biosciences, San Diego, CA) was incubated at a 1/1000 dilution in PBS-Tween 1% and peroxidase-conjugated streptavidin (GE Healthcare, Orsay, France) was added (1/1000 dilution in PBS-Tween 1%). Colorimetric development was carried out using 2, 2’-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium (ABTS; Thermo Scientific, France) and absorbance (OD) was measured at 405 nm. Individual results were expressed as the ΔOD value calculated according to the formula ΔOD = ODx − ODn, where ODx represented the mean of individual OD values in the two wells containing antigen and ODn the OD value in well without antigen. A subject was considered as an “immune responder” if ΔOD was higher than the cut-off (Cut-off = mean (ΔOD_unexposed) + 3SD = 0.181) calculated from specific IgG level in negative “not exposed” controls (n = 10).

Inhibition assays were performed using enzyme-linked immunosorbent assay (ELISA). Purified hPDPN-Fc was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc.) at 1 μg/ml for 30 min. After blocking with SuperBlock T20 (PBS) Blocking Buffer, LpMab-7 or NZ-1 was added at 1 μg/ml for 30 min. The plates were incubated with biotinylated CLEC-2-Fc (1 μg/ml) followed by 1:1000-diluted peroxidase-conjugated streptavidin (GE Healthcare, Piscataway, NJ). The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories Inc., Philadelphia, PA).