Anti ttr

The altered expression of TTR in the plasma of all the three case groups, that is, Group A, C, and D was evaluated using Western blot analysis. Plasma samples of all four groups were separated on 12–5% SDS–PAGE and transferred onto nitrocellulose membrane (Millipore, Burlington, MA, USA). To reduce the chance of non-specific binding, the membrane was blocked using 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) in 1× PBS (phosphate buffered saline, Merck Chemicals, Germany) for 1 h at RT with shaking. Anti-TTR (Abcam, Cambridge, MA, USA) (1:3,000) and GAPDH (1:1,000) were used as a primary antibody for 2 h at RT. The membrane was then incubated with horseradish peroxidase (HRP) conjugated anti-mouse antibody for 1 h with mild shaking. After each step, the membrane was washed with PBST to remove unbound proteins and antibody. Enhanced chemiluminescence assay was used to develop the membrane as per manufacturer’s protocol (G Biosciences, St. Louis, MO, USA) (Mishra et al., 2018 (link)). Variation in expression of TTR was analyzed by comparing intensities of the signals observed in the absolute case group (Group A) with that of the absolute control (Group B), only MeS (Group C), and only history of miscarriage (Group D).

Mouse-4G2 (monoclonal antibody against anti-flavivirus group antigen, clone D1-4G2-4-15, ATCC), Hu-4G2 (recombinant monoclonal antibody of clone D1-4G2-4-15 with human IgG Fc produced in Nicotiana tabacum, a gift from Dr. Nobuyuki Matoba), anti-TTR (Abcam), anti-CD31 (Invitrogen), anti- PDGFβR (Abcam), anti-alpha smooth actin (Abcam), anti-Axl (R&D systems), FITC-isolectin B4 (Sigma Aldrich), Alexa Fluor 647-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), Alexa Fluor 549-conjugated goat anti-human IgG (Jackson ImmunoResearch), and Alexa Fluor 488-conjugated goat anti-rat IgG (H+L) (ThermoFisher) were used for the immunofluorescence assay. All antibodies were validated for their specificity using mock or isotype antibody controls.
Mouse IFNAR-1 neutralizing antibody (clone MAR 5A3) and isotype control (clone MOPC-21) were purchased from BioXcell. anti-Axl (AF154), anti-GAS6 (AB885), and isotype control IgG (AB-108-C) were purchased from R&D systems. The human type 1 IFN neutralizing antibody mixture was purchased from PBL Assay Science. Anti-Zika mouse IgM antibody (Clone ZKA185) and an isotype control mouse IgM antibody (anti-fluorescein, clone 4-4-20) were obtained from Absolute Antibody.