Streptavidin hrp conjugate

Affinity purified proteins or cell lysates were electrophoresed in SDS-PAGE and transferred into nitrocellulose membrane (Hybond ECL). Immunodetection was performed as described earlier (6 (link)). In brief, the membrane was incubated with specific antibodies against the protein of interest followed by incubation with either fluorescently (Li-Cor and Invitrogen) or biotin labeled (Dako Cytomation) secondary antibodies. The fluorescently labeled secondary antibodies were detected with Odyssey infrared Imaging System (Li-Cor). The biotinylated secondary antibodies were detected on X-ray film using streptavidin-HRP conjugate (GE Healthcare) followed by enhanced chemiluminecence (Amersham ECL Western Blotting Detection Reagent, GE Healthcare). Primary antibodies used, were as followed: monoclonal anti-β-tubulin (Upstate), monoclonal anti-pEIF2α (Abcam), polyclonal anti-eIF2α (Abcam), monoclonal anti-GRP78 (Abcam), monoclonal anti-MBP (New England Biolabs), monoclonal anti-α-smooth muscle actin (Sigma Aldrich) polyclonal anti-GAPDH (Trevigen), polyclonal anti-TIA-1 and monoclonal anti-TIA-1/TIAR (Santa-Cruz) and polyclonal anti-TIA-1 (BioVision).

For serum IgG1 quantification, rat anti–mouse IgG1 (LO-MG1-13; AbD Serotec) antibody was used for coating and rat anti–mouse/HRP (LO-MK-1; AbD Serotec) was used for the secondary detection step. For IgE, rat anti–mouse IgE (LO-ME-3; Serotec) antibody was used for coating. Biotin-conjugated rat anti–mouse IgE mAb (BD) and streptavidin–HRP conjugate (GE Healthcare) were used for detection. Quantification standards were established using mouse IgG1 and IgE mAb (Serotec).

The NPLA assay for scFv-Fc fusions and complete IgG antibodies was performed by a modified version of the procedure described by Jensen.⁷⁷ All assays were performed in 96-well microtiter plates, with Vero cells used as the host for infection. A dilution series (50 µL) of scFv-Fc fusions or mAbs were incubated with an equal volume of WEE virus strain 160/99, with a TCID₅₀/mL of 5 × 10⁴, for 2 h at 37 °C, in 96-well plates. Following this incubation, 100 µL of freshly trypsin-treated Vero cells, at a density of 2 × 10⁵ cells/mL, was mixed with the antibody virus mixture. As a positive control for infection, virus samples, not previously incubated with antibody, and virus samples previously incubated with WEEV-specific antibodies with no neutralizing activity, such as mAb 8742 or mAb SFV12/2, were used. Non-infected Vero cells were used as a negative control. Cell monolayers were fixed 20 to 24 h post infection, by incubation with 3% formalin in PBS for 2 h at 4 °C. The monolayers were washed with PBS-T, overlaid with 100 µL of a 1:5,000 dilution of the WEEV-specific biotinylated mAb SFV 12/2 and incubated for 90 min at room temperature. Bound biotinylated mAbs were detected by incubation with streptavidin-HRP conjugate (GE Healthcare, 1:4,000) as described above. An Olympus CK2 inverted microscope (Olympus) was used for bright field microscopy.