The non-reducing and non-denaturing environment of native PAGE allows the detection of biological activity. Periplasmic extracts containing the recombinant fusion protein was separated using 10% native PAGE gel. Then Western blot was performed VE-821 mouse to reveal the AP activity using directly

BCIP/NBT AP substrate buffer (100 mM Tris–HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2, containing 0.3 g/l NBT and 0.15 g/l BCIP) onto nitrocellulose membrane. Alkaline phosphatase activity was determined using a biochemical colorimetric test. Briefly, increasing concentrations of SAG1–AP and free AP contained in induced and non-induced periplasmic extracts respectively (20–1500 ng/ml) were diluted in assay buffer (1 M diethanolamine, 0.5 mM MgCl2 at pH 9.8) and incubated with 1 mg/ml pNPP AP substrate (p-nitro-phenyl-phosphate; Sigma-Aldrich, Inc.), in 96-well ELISA plates. The enzymatic AP activity was

assayed by measuring the p-nitrophenol formed from the enzymatic hydrolysis of p-nitro-phenyl-phosphate at 405 nm using a microplate reader (Labsystems Multiskan EX, Finland). All this website assays were conducted in duplicate. The specific activity was calculated as A405 U/μg protein. Immunoreactivity and bi-functionality of the recombinant SAG1–AP fusion protein were tested in anti-T. gondii SAG1 Mab based ELISA. Briefly, 96-well ELISA plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 100 μl/well of 100 mM carbonate–bicarbonate buffer (pH 9.6) containing 5 μg/ml anti-SAG1 Mab and incubated overnight

at 4 °C. Blocking for non-specific binding was performed for 1 h at 37 °C with PBS-T and 5% skim milk powder. The activated plates were incubated for 1 h at 37 °C with 100 μl of twofold dilutions of Ergoloid periplasmic extract containing the SAG1–AP conjugate starting from 1.5 μg/ml. Wells were then incubated with 100 μl of AP substrate (1 mg/ml pNPP diluted in 1 M diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl2) for 30 min at 37 °C. The wells were washed three times with PBS-T between each intermediate step. The absorbance at 405 nm (A405 nm) was measured using a microplate reader. Background was determined by incubating the wells directly with the non-induced periplasmic extract. All assays were conducted in duplicate. Sera samples were provided by the “Laboratoire de Parasitologie Médicale, Institut Pasteur de Tunis” and were collected from pregnant women for systematic toxoplasmosis screening during their first prenatal consultation. The patient’s immune status towards toxoplasmosis and specific IgG titers for positive ones were established using the standard ELISA Platelia™ Toxo IgG kit (Product No. 72840, Bio-Rad, France). Sera samples from Toxoplasma sero-positive and sero-negative patients were diluted 1/20 in 100 mM carbonate–bicarbonate buffer (pH 9.6) and then, volumes of 100 μl/well were used to coat ELISA plates at 4 °C overnight.