Anti mouse igg horseradish peroxidase conjugates

Pooled human umbilical vascular endothelial cells (HUVECs) and endothelial cell growth medium 2 (EGM2) were purchased from PromoCell (Heidelberg, Germany). Antibodies to ICAM1 and GAPDH were obtained from New England Biolabs UK Ltd. (Hertfordshire, UK). Anti-SOCS3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SuperSignal™ West Pico Chemiluminescent Substrate was from Fisher Scientific (Loughborough, UK). Secondary antibodies, anti-rabbit-IgG horseradish peroxidase, anti-goat-IgG horseradish peroxidase and anti-mouse-IgG horseradish peroxidase conjugates, were from Sigma-Aldrich Company Ltd. (Dorset, England). Forskolin, rolipram and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) were obtained from Calbiochem (Paisley, UK). I942 (N-(2,4-dimethylbenzenesulfonyl)-2-(naphthalen-2-yloxy)acetamide) was purchased from MolPort (Riga, Latvia). Recombinant human interleukin 6 (IL6) protein and recombinant human soluble IL6 receptor α (sIL6Rα) proteins were purchased from R and D Systems (Abingdon, UK).

Immulon 4HBX 384-well ELISA plates (Thermo Scientific) were coated with 40 ng/well of NiV Pre-F or NiV monomeric G protein in BupH buffer (Pierce) at 4°C for 16h. The NiV pre-F protein used to coat ELISA plates is the same antigen used in immunization studies (also contains thrombin-his-strep tag) while the NiV monomeric G protein has the thrombin-his-strep tag and no multimerization domain, differing from the antigen used in immunization studies. After standard washes and blocks, plates were incubated with 4xfold serial dilutions of heat-inactivated sera for 30-45 min at room temperature. Following washes, anti-mouse IgG-horseradish peroxidase conjugates (Sigma) were used as secondary antibody and 3,5,3’,5’-tetramethylbenzidine (TMB) (KPL) was used as the substrate to detect antibody responses. Endpoint titers were calculated as the dilution that emitted an optical density exceeding background (secondary antibody alone) with a predetermined absorbance cut-off of 0.2 (approximately 3-4x average background value).