Alkaline phosphatase conjugated anti mouse igg

Whole-cell antigens from selected recombinant E. coli and each His-tagged protein were electrophoresed on 12% (w/v) polyacrylamide gels. The apparent molecular mass of recombinant proteins was determined using markers (kaleidoscope pre-stained standards, Bio-Rad, Hercules, CA, USA). Electrophoresed proteins were transferred to nitrocellulose membranes in a Trans-Blot-semidry-apparatus (Bio-Rad), as described by the manufacturer. Blots were incubated for 1 h at 37 °C with anti-His serum diluted 1:500, sheep sera diluted 1:100 in PBS-2% skim milk and anti-AtlA serum diluted 1:500. After several washings with phosphate-buffered saline with 2% skim milk, blots were incubated for 1 h at 37 °C with alkaline phosphatase-conjugated anti-mouse IgG (Sigma-Aldrich) diluted 1:30.000, or alkaline phosphatase-conjugated anti-sheep antibody (Sigma-Aldrich) diluted 1:10.000. After three more washes, blots were developed with bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT, Promega, Madison, WI) in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris, pH 9.5).

The Ph.D.-C7C Phage Display Peptide Library kit was purchased from New England Biolabs [NEB, Ipswich, Massachusetts, US]. The screening of phage displayed library was performed using the bait 5T33MM Igs, as previously reported [22 (link)]. Briefly, the streptavidin-conjugated beads [Thermo Fisher, Waltham, Massachusetts, US] were coated with 5T33MM Igs and incubated with 1 × 10¹¹ phages overnight at 4 °C. Beads were extensively washed with PBS supplemented with 0.05% Tween-20 to remove unbound phage. 5T33MM Igs-interacting phages were eluted with 0.2 M glycine-HCl (pH 2.2, 1 mg/mL BSA) followed by the addition of neutralizing solution (1 M Tris-HCl pH 9.1). Ultimately, 3 cycles of panning were performed. Plaques of lysis from isolated phages were transferred to nitrocellulose filters, and membranes were blocked with PBS 1X, 0.1% NP-40; 5% milk; 0.02% NaN3 and then incubated for 2 h at RT with 100 μg of purified 5T33MM Igs. After washing, membranes were hybridized with an alkaline phosphatase-conjugated anti-mouse IgG [Sigma Aldrich, Saint Louis, Missouri, US] (dilution 1:5000) and then washed 6 times. Immunoreactive phage clones were detected by BCIP/NBT premixed substrate [Thermo Fisher].

Antibodies levels were determined by ELISA assays. Briefly, 96-well flat bottom plates (Costar, Corning, NY, USA) were coated with 2 μg/mL of recombinant proteins AAEL000793, AAEL007420, or AAEL006347 diluted in carbonate bicarbonate buffer, pH 9.6 (Sigma-Aldrich, Burlington, MA, USA). Plates were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) (25 mM Tris, 150 mM NaCl, pH 7.4). After three washes with TBS with 0.05% (v/v) Tween (TTBS), pooled mouse sera immunized with the AAEL000793, AAEL007420 andAAEL006347 recombinant protein were diluted 1:1000 in TTBS then were added to the plates. Following 1 h of incubation and washing, alkaline phosphatase-conjugated anti-mouse IgG (Sigma-Aldrich, Burlington, MA, USA) at dilution 1:10,000 in TTBS was added. After three more washes, plates were developed with stabilized p-nitrophenyl phosphate (Sigma-Aldrich, Burlington, MA, USA) and absorbance was measured at 405 nm in a VersaMax microplate reader (Molecular Devices, San Jose, CA, USA).

Protein fractions separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to a nitrocellulose membrane according to the method described by Blake [38 (link)]. The membrane was blocked with Tris Buffered Saline with Tween 20 (TBST) 0.5% milk at room temperature for 2 hours, washed 5 times with TBST for 5 minutes, and cut into strips that were incubated for 1 hour with anti-His antibody (Sigma-Aldrich) at 1:10,000 or with a positive control, which consisted of a pool of hyperimmune sera from P. vivax-immune primates at 1:50. Strips were then incubated for 1 h with either alkaline phosphatase-conjugated anti-mouse IgG or anti-human IgG antibodies respectively (Sigma-Aldrich) at a dilution of 1:10,000 and were then washed as before. Finally, formation of immune complexes in the membrane was assessed visually by adding TMB or NBT-BCIP substrates (Sigma Aldrich) accordingly.

The glycoconjugate DHG-SagA and rSagA were analyzed by SDS-PAGE using 4 to 12% Bis-Tris protein gels in morpholineethanesulfonic acid SDS running buffer (NuPAGE Novex; Invitrogen). Electrophoresis was run for 45 min at 170 V in an XCell SureLock minicell (Invitrogen), and immunoblotting was performed at RT for 1 h at 30 V in an XCell II blot module (Invitrogen) using polyvinylidene difluoride membranes (0.2-μm pore size; NuPAGE Novex; Invitrogen). Membranes were blocked with BB overnight at 4°C. The next day, they were washed three times with WB and incubated for 1 h with 2 μg/ml of MAb in BB. The membranes then were washed again as described above and incubated for 1 h with alkaline phosphatase-conjugated anti-mouse IgG (Sigma-Aldrich) diluted 1:1,000 in BB. Finally, three washes with WB were performed and binding was detected by the colorimetric AP substrate reagent kit (Bio-Rad).

AhpC-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated with rAhpC protein (1 μg/well). Sera collected from vaccinated mice were used as the primary antibody. Two-fold serial dilutions of primary antibodies (from 1:50 to 1:6400, v/v) were applied in plates in duplicates. Alkaline phosphatase-conjugated anti-mouse IgG (Sigma, USA) was used as a secondary antibody at a dilution factor of 1:1000 (v/v). The AP Conjugate Substrate Kit was used as a colorimetric reagent (Bio-Rad, USA). Optical density was measured at 405 nm.