Ap conjugated anti human igg

Ni²⁺-coated ELISA plates (Pierce) were used to capture 2 ng/μl (in TBS) rE proteins (DENV1-4) for 1 hr at 37°C. The plates were blocked using TBS + 3% skim milk + 0.05% Tween-20 for 1 hr at 37°C and subsequently washed 3 times with TBS + 0.05% Tween-20. Next, plates were incubated for 1 hr at 37°C with 2 ng/μl (in blocking buffer) of mouse and human derived mAbs: 4G2 (mouse, cross-reactive), 3H5 (mouse, DENV2 specific), 12C1.5 (mouse, cross-reactive), 8A1 (mouse, DENV3 specific), 1M7 (human, cross-reactive), 1F4 (human, DENV1 specific), 2D22 (human, DENV2 specific), 5J7 (human, DENV3 specific), 5H2 (chimpanzee, DENV4 specific) and DV4 141 (human, DENV4 specific) (Table 1). Following incubation, the plates were washed and accordingly treated with AP-conjugated anti-mouse IgG (Sigma, 1:1000) or AP-conjugated anti-human IgG (Sigma, 1:2500) for 45 mins at 37°C. Finally, the plates were washed, developed with AP-substrate (Sigma) and absorbance was measures at 405 nm.

Maxisorp 96-well microtiter plates were coated overnight with 5 µg/mL streptavidin (50 µL/well) in NaHCO₃ buffer (50 mM, pH 9.6), then washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) for one hour. Peptides (2 µg/mL) were then loaded on the plate for 30 min. Rabbit sera, individual phage clones or purified antibodies were added to the plates for a one-hour incubation. The plates were then washed with PBST and incubated with the detecting antibody: alkaline phosphatase (AP)-conjugated goat anti rabbit for rabbit serum (Sigma-A8025), horseradish peroxidase (HRP)-conjugated anti-M13 antibody (GE healthcare, Little Chalfont, UK) for phage clones and AP-conjugated anti-human IgG (Sigma-A3187) for scFv-Fc antibodies. Detection of HRP conjugates was carried out with 3,3′,5,5′-tetramethybenzidine (TMB/E, Millipore, Billerica, MA, USA). Detection of AP-conjugated antibodies was carried out with SIGMAFAST p-nitrophenyl phosphate tablets (Sigma-N2770).

Chimeric MAb and rabbit sera were tested for specificity by Western blot. Lysates containing equal concentrations of total protein, as measured by the Bradford assay, were loaded to a NuPAGE 10% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA), and the electrophoresed proteins were transferred to an Amersham Protran Premium 0.45 µm nitrocellulose membrane (GE Healthcare 10600096). The membrane was then blocked (1% skim milk in PBS) and probed with chimeric MAb or immunized rabbit sera. Rabbit anti-SNAP-25 polyclonal antibody raised against a synthetic peptide corresponding to the N-terminus of human SNAP-25 (Sigma S9684), was used as a positive control to detect both the cleaved and the intact SNAP-25. AP-conjugated anti-human IgG (Sigma A3187) and AP-conjugated anti-rabbit IgG (Jackson 711-066-152) were used as secondary antibodies for chimeric MAb and rabbit sera, respectively. 5-Bromo-4-Chloro-3-Indolyl-phosphate (BCIP, Sigma B8503) was used as the chromogenic substrate. Densitometry analysis showed that the total SNAP-25 (cleaved and intact) in the BoNT/E treated samples produced similar signals, further confirming that equal amounts of the subject protein were loaded to the gels (Figure S1).