Protein free blocking buffer

3HP-β-LG diluted in PBS was coated onto all wells of a 96-well polystyrene plate (Corning, USA) at 4°C overnight. After blocking with protein-free blocking buffer (Thermo Fisher Scientific, USA), HPV L1 (type16 or 18) protein at 1 μg/ml was added into the wells for 50 μl/well, and the plate was incubated at 37°C for 1 h. After washing with PBST three times, mouse anti-HPV L1 (type16 or 18) antibody (Abcam, UK) was added at a 1: 1,000 dilution. After incubation at 37°C for 1 h and washing again, HRP-conjugated rabbit anti-mouse IgG antibody (Dako, Denmark) was added for 1 h. After washing and adding tetramethylbenzidine (Sigma, USA), the absorbance was measured at 450 nm. Similar protocols were used for detection of 3HP-β-LG in the serum of rhesus monkeys and 3HP-β-LG-specific IgA and IgE in the vaginal swab eluates, which were leftover samples from the clinical trials for evaluating the in vivo safety and efficacy of the intravaginally applied 3HP-β-LG-containing vaginal gels (Registry No.: ChiCTR-TRC-12002016) (Guo et al., 2016a (link),b (link)).

HFF-1 or SK-N-MC cells were grown to semi-confluency overnight in black opaque 96-well plates. The following day, AD169 was preincubated with different dilutions of SPGG for 1 h with agitation and cells were infected with 0.3 MOI of treated or untreated virus for 2 h at RT. The infection media was replaced with SFM and incubated for an additional 5 h under standard conditions. The samples were fixed for 20 min with methanol, washed three times in Tris-buffered saline (TBS), and blocked for 2 h with protein-free blocking buffer (Thermo Fisher, Waltham, MA, USA). Samples were incubated overnight at 4 °C with a mouse monoclonal antibody against immediate early genes 1 and 2 (Virusys, Taneytown, MD, USA, cat. no. P1215) diluted 1:5000 in blocking buffer. Next, samples were washed three times with wash buffer (0.05% tween 20 in TBS) and then incubated for 1 h at RT with goat anti-mouse IgG (H+L) peroxidase-conjugated secondary antibody (Thermo Fisher, Waltham, MA, USA) diluted 1:20,000 in wash buffer. Samples were washed 3 times in wash buffer, Super Signal ELISA Femto Substrate (Thermo Fisher, Waltham, MA, USA) was added, and total luminescence was analyzed using a microplate luminometer (Beckman DTX 880).

Recombinant human single-chain soluble FcRn (sFcRn), containing both β and α chains in a 1:1 molar ratio, was expressed in mammalian cells and purified as described previously (18 (link)). ELISA wells were coated with sFcRn at 50 ng per well in PBS (pH 7.4) overnight at 4°C, and blocked with protein-free blocking buffer (Thermo Scientific) at 37°C for 2 h. Twofold serially diluted protein, prepared with PBS containing 0.2% BSA, pH 6.0 or PBS containing 0.2% BSA, pH 7.4, was added and incubated at 37°C for 1.5 h. The plates were washed with PBST (PBS plus 0.05% Tween-20), pH 6.0 or pH 7.4, and horseradish peroxidase (HRP)-conjugated anti-FLAG tag antibody (Sigma-Aldrich) in PBS (pH 6.0 or 7.4) was incubated with wells for 45 min at 37°C. After extensive washes with PBST (pH 6.0 or 7.4), the binding was detected by the addition of ABTS substrate (Roche, Indianapolis, IN, USA), and monitored at 405 nm.

Recombinant SAV3 E2 protein was used for coating ELISA plates. The preparation of the E2 protein is described elsewhere (35 (link)). Briefly, 96 well Maxisorp plates (Thermo Fisher Scientific) were coated overnight at 4°C with recombinant E2 protein (200 ng/well) and subsequently blocked with a protein free blocking buffer (Thermo Fisher Scientific) before incubation with 1:80 diluted individual sera samples in duplicate. Following overnight incubation at 4°C, wells were washed four times (PBS containing 0.05% Tween-20). Mouse anti-trout IgM mAb (IgF1-18 (6-1-18), 1:500) and HRP conjugated goat anti-mouse (Bio-Rad, 1:1500) were added sequentially and incubated at RT each for 1 h. Plates were developed for 30 min in the dark with OPD substrate (Sigma). Optical densities were measured immediately at 450 nm on a VersaMax microplate reader (Molecular devices).

Peripheral blood mononuclear cell pellets from 10 individuals were used: five with highest and five with lowest methylation at cg19859270. Cells were treated in lysis buffer (Cell Signaling Technology, Beverly, MA, USA) for 15 min and sonicated briefly. Lysates were separated by polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were incubated in protein-free blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature, followed by incubation with primary antibody (1:1000 dilution). Immunoreactive proteins were detected with Amersham ECL (GE Healthcare, Pittsburgh, PA, USA) using a Kodak Imager. Images were analyzed by ImageJ software (NIH).

S100A8 recombinant protein was purified as previously described [8 (link)]. These proteins were immobilized on Maxisorp plates (Thermo Fisher Scientific, Waltham, MA, USA), followed by blocking with protein-free blocking buffer (Thermo Fisher Scientific). TLR4/MD-2 recombinant proteins [25 (link)] with various concentrations of candidate peptides were then added to the indicated S100A8-immobilized ELISA plates for 2 h. The plates were washed, incubated with anti-TLR4 antibody, and HRP-conjugated secondary antibody, followed by the addition of substrate. The reaction was stopped by addition a 2N H₂SO₄ solution. Absorbance OD₄₅₀/OD₆₅₀ was measured using a microplate reader (Thermo Fisher Scientific). Maxisorp plates were also used for peptide coating. After blocking with protein-free blocking buffer, the coated plates were washed, and TLR4/MD-2 recombinant protein (2 µg/ml) was added to each well. The plates were incubated with a TLR4 antibody followed by horseradish peroxidase (HRP)-labeled anti-rabbit IgG. For detection of IL8 and VEGF in culture supernatants, SW480 cells were pretreated with the indicated peptides, and followed by stimulation with S100A8 for 24 h. The amount of interleukin-8 (IL-8) or VEGF in the supernatants were analyzed by ELISA kits (R&D Systems, Mineapolis, MN) according to the manufacturer’s instructions.