Maxisorp elisa plate

96-well Maxi-Sorp ELISA plates (Nunc Inc, Roskilde, Denmark) were coated with 100 µl gp120 in 50 mM carbonate buffer pH 9.6 (2 µg/ml) over night at 4 °C and then blocked with PBS-T (phosphate buffered saline, pH 7.4 with 0.05% Tween 20) for 1 h at 37 °C. The experiment was performed with reaction mixtures containing 1 µM Trx1 + 100 nM TrxR1 + 240 µM NADPH + indicated concentrations of PX-12 (0–100 µM), 5 mM dithiothreitol was used as positive control and PBS as a negative control. The reaction mixtures were added to the gp120-coated wells for 30 min at 37 °C and then washed four times with PBS-T to remove any unbound material. Subsequently, the wells were incubated with 10 µM biotin-maleimide for 30 min at room temperature, then washed and incubated with steptavidine-ALP (Mabtech, Nacka, Sweden) diluted 1:1,000 in PBS for 30 min at room temperature. The wells were washed four times with PBS-T and 1 mg/ml p-nitrophenyl phosphate dissolved in 10% diethanolamine pH 9.8 with 0.5 mM MgCl₂ was added. The absorbance at 405 nm was measured using a microplate reader (Tecan).The data is based on three separate experiments. The value for 1 µM Trx1 + 100 nM TrxR1 + 240 µM NADPH was set to 100% in each individual experiment and all other values were normalized against this reference value. The data shown are means of three independent experiments and the error bars represent the SEM.

Maxisorp ELISA plates (NUNC) were either coated directly with skimmed milk or fractionated milk samples (in 1:10 dilution, unless stated otherwise) in 0.2 M NaHCO₃ buffer overnight at 4°C, or in case of human milk protein capturing ELISA, with purified lactoferrin (100 ng/ml) or with antibodies to MUC1 (unbiotinylated, 3 μg/ml), MUC4 (40 μg/ml), κ-casein (20 μg/ml), or α-lactalbumin (30 μg/ml). The plates were blocked in Tris-sodium buffer containing 1% BSA, followed by incubation with skimmed human milk in case of milk protein capture. For C-type lectin binding, plates were washed in Tris-sodium-0.05%Tween and incubated with DC-SIGN-Fc or MGL-Fc for 1 h at room temperature (RT). For MUC1 detection, plates were incubated with biotinylated MUC1 antibody (2 μg/ml). To detect Lewis-antigen expression following MUC1 capture, plates were incubated with antibodies to Lewis x or Lewis y (0.5 μg/ml). After washing, binding of C-type lectins was detected by PO-labeled goat anti-human IgG, binding of MUC1 by PO-labeled streptavidin and binding of Lewis antigens by PO-labeled goat anti-mouse IgM (Jackson Immunoresearch). The reaction was visualized in 100 μg/ml 3,3′-5,5′-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) and optical density was measured by a microplate absorbance spectrophotometer (Biorad) at 450 nm.