Streptavidin peroxidase polymer

Plasma FD was measured using an in-house ELISA assay, optimized for use in plasma. ELISA plates (MaxiSorp^®, Nunc) were coated with 100 ng/well of mouse monoclonal anti-human FD (GAU 01-04-02, Invitrogen, Carlsbad, CA, USA) diluted in carbonate-bicarbonate buffer pH 9.3 (overnight, 4 °C). Plates were blocked with PBS-BSA 1% and washed with PBS-BSA 0.1% (both incubations 1 h at 37 °C). A standard curve ranging from 0 to 80 ng/mL of purified FD (Complement Technology, Tyler, TX, USA) was included on each plate, alongside one internal control plasma sample used to assess interassay variability. The interassay coefficient of variation was below 10%. Plasma samples were diluted in PBS-BSA 0.1% at 1/50, 1/100, and 1/200 dilutions for analysis. FD detection was made using a mouse monoclonal anti-human FD conjugated with biotin (GAU 008-01B-02, Invitrogen) (1/2500 dilution, 1 h at 37 °C) and then with streptavidin-peroxidase polymer (S2438, Sigma Aldrich, St Louis, MO, USA) (1/400, both incubations 45 min at 37 °C). The reactions were developed using ABTS as substrate and read at 405/620 nm using an Epoch™ Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). All samples were measured in duplicate.

RSV long strain was propagated in HEp-2 cells (ATCC, Manassas, VA) at 37°C and purified by sucrose gradient as described (14 (link)–16 (link)). Viral titer was determined by immunostaining in HEp-2 cells using biotin-conjugated goat anti-RSV primary antibody (Cat #: 7950-0104, Bio-rad, Hercules, CA), followed by the incubation with streptavidin peroxidase polymer (Cat#: S2438, Sigma-Aldrich, St Louis, MO), as previously described (14 (link), 16 (link)).

Proteins were C-terminally biotinylated using an EZ-Link® Micro Sulfo-NHS-Biotinylation Kit (Pierce) according to the manufacturer's instructions. Successful biotinylation was detected via Western blotting using an ultrasensitive Streptavidin-Peroxidase Polymer (Sigma).

After incubation with diluted patient sera, the biotinylated anti-human IgM (Thermo Scientific, IL) or anti-human IgG (Santa Cruz Biotechnology, CA) at 1 : 5000 dilution in phosphate buffered saline (PBS) with 5% bovine serum albumin (BSA) was added. After 1 h of incubation at room temperature, the plates were washed as previously described then incubated with streptavidin-peroxidase polymer (Sigma, MS) at 1 : 8000 dilution in PBS with 5% BSA for another hour. At the end of incubation, the plates were washed again before the addition of the ABTS substrate. Optical densities at 405 nm (OD₄₀₅) were measured after 30 minutes as previously described.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blue-native PAGE (BN-PAGE), and western blot analyses were performed as described elsewhere (55 (link)). For western blot analysis, samples were separated through 3%–12% NuPAGE Gels and transferred onto polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked in a solution of Tris-buffered saline containing 5% non-fat dry milk and 0.05% Tween 20 and subsequently probed with anti-Env antibodies (2G12 and VRC01) or biotinylated-goat anti-mouse C3d polyclonal antibody. Proteins were visualized with HRP-conjugated anti-human IgG antibody or streptavidin-peroxidase polymer according to the manufacturer’s instructions (Sigma).

When subcutaneous tumors reached a diameter of approximately 8 mm, tumors were irradiated with 30 Gy of X-rays at a rate of 3.9 Gy/min with a TITAN-320 X-ray generator (Shimadzu, Kyoto, Japan). Other parts of the mouse body were covered with a brass shield. On post-exposure days 1, 3, and 7, tumors (n = 3 per time point) were sampled and fixed in 10% (v/v) neutral buffered formalin and embedded in paraffin for sectioning. Nontreated tumors were used as controls. Sections (thickness, 1 μm) were immunostained with antibody 3–6 (diluted 1:200) followed by horseradish peroxidase-conjugated anti-rat immunoglobulin from a kit (BD, Franklin Lakes, NJ, USA). Nuclei were counterstained with hematoxylin.
To compare antibodies 3–6 and 12–2–7, six subcutaneous A375 tumors were fixed with 4% paraformaldehyde overnight in 0.1 M sodium phosphate (pH 7.2). After dehydration in ethanol, the tissues were embedded in polyester wax. Adjacent sections were immunostained with anti-TNC antibody 3–6 (6 μg/mL) or 12–2–7 (8 μg/mL) as the primary antibody; the secondary antibody was goat anti-rat IgG biotin conjugate (SC-2041, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:1000). A streptavidin-peroxidase polymer (S2438, Sigma; diluted 1:1000) served as the detection reagent. Coloring was done with diaminobenzidine, and nuclei were stained with methyl green.