Alkaline phosphatase conjugated anti human igm

After purification, NS1-DENV1–4 proteins were electrophoresed (SDS-PAGE 12%, stained with Coomassie Blue) and transferred to a nitrocellulose membrane. After transference, the immunolabeling was done. In order to do this, each membrane was blocked with PBS (10 mM phosphate, 137 mM NaCl, and 2.7 mM KCl) supplemented with 3% gelatin, and a pool of 3 polyclonal serum samples from patients infected with dengue virus was used as a primary antibody. Membranes were incubated for 24 h, and after this time were washed 5 times with PBS-Tween-20. Then, a secondary antibody with alkaline-phosphatase-conjugated anti-human IgM (Sigma, St. Louis, MI, USA) was added to the membranes for 2 h. After incubation, the membranes were revealed with BCIP/NBT (Sigma). For the labeling of glycoproteins, a reaction was performed using periodic acid-Schiff (PAS). NS1DENV1–4 proteins were electrophoresed (SDS-PAGE 12%) and transferred to nitrocellulose membrane. After the transfer, membranes were washed with 12% trichloroacetic acid for 5 min. The next steps were carried out at 4 °C in a dark room. Membranes were incubated with 0.5% periodic acid for 15 min, and washed 3× with 15% acetic acid for 5 min. Then, 50 mL of Schiff’s reagent was added and incubated for 30 min. Finally, 6 washes were performed with 7.5% acetic acid for 1 h, and the membranes were dried at room temperature.

Antibody responses were determined in SP-ELISA. Ninety-six well activated polystyrene ELISA plates (NUNC Maxisorp Sigma) were coated with 1 μg per 100 μL of glycopeptide in carbonate buffer 0.05 M (pH 9.6) and incubated at +4°C overnight. After five washes with saline containing 0.05% Tween 20, nonspecific binding sites were blocked by fetal bovine serum (FBS), 10% in saline Tween 20 (100 μL per well) at room temperature for 60 min. Sera diluted from 1 : 100 to 1 : 10,000 were applied at +4°C overnight in saline/Tween 20/10% FBS. After five washes, we added to each well 100 μL of alkaline phosphatase conjugated antihuman IgM (diluted 1 : 200 in saline/Tween 20/FBS; Sigma) or IgG (diluted 1 : 8000 in saline/Tween 20/FBS; Sigma). After 3 h at room temperature incubation and five washes, 100 μL of substrate solution consisting of 1 mg mL⁻¹ p-nitrophenyl phosphate (Sigma) in 10% diethanolamine buffer was applied. After 30 min, the reaction was stopped with 1 M NaOH (50 μL), and the absorbance was read in a multichannel ELISA reader (Tecan Sunrise) at 405 nm. ELISA plates, coating conditions, reagent dilutions, buffers, and incubation times were tested in preliminary experiments. Antibody levels are expressed as absorbance in arbitrary units at 405 nm (sample dilution 1 : 100).