Streptavidin horseradish peroxidase hrp

Purified vaccine proteins were either kept native or mixed with 0.1 M dithiothreitol (DTT) and heated to 95°C for 5 min. Vaccine proteins (0.1 μg per lane) were loaded on 4 to 12% NuPAGE bis-tris gels (cat. no. NP0326BOX, Thermo Fisher, Massachusetts, USA) and separated with Bolt MOPS (morpholinepropanesulfonic acid) SDS running buffer (cat. no. B0001, Thermo Fisher). Proteins were then transferred to iBlot 2 polyvinylidene difluoride (PVDF) transfer stacks (cat. no. IB24002, Thermo Fisher), blocked in 2% skim milk in PBS with Tween 20 (PBST) at room temperature for 60 min, and incubated with biotinylated anti-HA MAb (1:3,000; clone: H36-4-52) at 4°C for 18 h. Next, the blot was incubated with streptavidin-horseradish peroxidase (HRP; 1:10,000; cat. no. 7105-05, SouthernBiotech) for 30 min at room temperature. Detection was done with WestPico chemiluminescence substrate (cat. no. 34578, Thermo Fisher). Separately, recombinant PR8 protein was deglycosylated with peptide-N-glycosidase F (PNGase F) according to the manufacturer’s instructions (cat no. P0704S, New England Biolabs, Massachusetts, USA) to determine the size contribution of glycan moieties on HA.

Tissue and cells were lysed in radioimmunoassay precipitation (RIPA) buffer and western blotting performed as previously described [24 ] using the primary antibodies indicated. Band density was quantified using Image J software (NIH, Bethesda, MA, USA). ACC1 protein was identified by avidin–agarose bead pull-down, SDS-PAGE, transfer to polyvinylidene difluoride (PVDF), incubation with streptavidin–horseradish peroxidase (HRP) (ThermoFisher) and chemiluminescent detection: this method utilises the affinity of the endogenous ACC1-bound biotin for avidin/streptavidin and has been described previously [20 (link)].

ELISA high binding plates (Thermo Fisher Scientific) were coated with 1 μg/mL of mouse anti‐VEGF (R&D Biosystems) in phosphate‐buffered saline (PBS) overnight at room temperature. Plates were then blocked with 300 μL of blocking buffer (1% BSA in PBS) for 1 hour at room temperature. After washing with wash buffer (PBS with 0.2% Tween‐20), 100 μL samples of conditioned medium from siRNA‐transfected 60 mm plates were added to appropriate wells and incubated at room temperature for 2 hours. A standard curve of recombinant VEGF from 0 to 2000 pg/mL was run alongside experimental samples. Plates were washed again and incubated with 1.2 μg/mL goat anti‐VEGF (R&D) for 2 hours at room temperature. After washing, streptavidin‐horseradish peroxidase (HRP, Thermo Fisher Scientific) was added for 1 hour followed by detection with 3,3′,5,5′‐tetramethylbenzidine (TMB, eBioscience). Data were analysed using one‐way ANOVA with Tukey's post hoc testing.

Levels of myeloperoxidase were determined using the human myeloperoxidase DuoSet ELISA kit (R&D Systems, Abingdon, UK). Ninety-six-well enzyme-linked immunosorbent assay (ELISA) plates were coated with 4 μg/ml capture antibody in phosphate-buffered saline (PBS) at room temperature (RT) overnight. Plates were washed 3 times with PBS (Sigma-Aldrich Co. Ltd, Irvine, UK) containing 0.05% Tween 20 (Sigma-Aldrich Co. Ltd, Irvine, UK) between each step. Wells were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at RT. Samples and standards were diluted in 1% BSA-PBS in the precoated plates and incubated at RT for 2 h. Detection was performed by incubating plates with detection antibody at 50 ng/ml for 2 h at RT, followed by 20 min incubation with streptavidin-horseradish peroxidase (HRP) (1:200) (Fisher Scientific, Loughborough, UK) at RT. Signal was developed using TMB-Turbo substrate (Fisher Scientific, Loughborough, UK) for 20 min and stopped by adding 2 N H₂SO4 in a 1:1 ratio. Optical density reading was performed at 450 nm and corrected for optical imperfection (540 nm). All samples were run in duplicate. Results are expressed as micrograms per milliliter and calculated using an MPO standard curve.

iPSC-MGLCs were seeded at 4 × 10⁴ cells/well in 96-well plates (Corning, USA) in macrophage end-differentiation medium. The following day, the medium was changed to fresh medium, and supernatants were collected after 24 hr. Quantification of sTREM2 from cell culture supernatants was performed using an in-house-generated ELISA using MaxiSORP 96-well plates (Nunc, Thermo Fisher Scientific, USA) coated with 1 μg/mL of a rat anti-mouse/human TREM2 monoclonal antibody (clone 237920, R&D Systems, USA) overnight at 4°C. After a blocking step, cell culture supernatant samples and standards (recombinant human TREM2-His; Life Technologies) were incubated for 2 hr at room temperature (RT) with biotinylated polyclonal goat anti-human TREM2 capture antibody (0.1 μg/mL; AF1828, R&D Systems). After incubation with streptavidin-horseradish peroxidase (HRP) (0.1 μg/mL; Invitrogen), followed by the addition of a chromogenic substrate solution (TMB, Life Technologies), the reaction was terminated with the addition of stop solution (0.16 M H₂SO₄) and absorbance was read at 450 nm (GENios, Tecan, USA). In addition, a second method using a mesoscale assay validated the results (see Supplemental Experimental Procedures).