Goat anti human igg fc fragment specific

The subset of sera from the original study was analysed with a microarray as described earlier [5] (link)–[8] (link). Briefly, recombinant proteins were produced in human embryonic kidney cells (HEK293) and purified by HIS-tag purification (purity more than 95%), as specified by the manufacturer (Immune Technologies, New York, USA). Oncyte avid nitrocellulose film-slides containing 64 pads per slide were used (Grace bio-labs, Bend, USA), and spot signals were quantified by the use of a Scanarray scanner (Perkin Elmer, Waltham, USA) using an adaptive circle quantification method. Finally, conjugates consisted of goat anti-human IgG (Fc-fragment specific) conjugated with Dylight649-fluorescent dye (Jackson Immuno Research, West Grove, PA, USA).
Table 1 shows the antigens included in the study. Notice that next to the antibody response against the A/2009 (H1N1) pandemic virus, we tested the samples against a range of other antigens, among which A/1918 (H1N1). The hemagglutinin of H1N1 virus of 1918 is genetically and antigenically related to the 2009 virus [11] (link)–[14] (link). Readers of each test (HI and microarray) were blind to results of the other tests, and had no access to ancillary information (age, sex).

As serum is known to induce astrocyte activation, before treatment primary mouse astrocytes were washed once with HBSS and subsequently serum-starved for a variable time (1–24 h) in Sato’s serum-free medium (Insulin 10 μg ml⁻¹, transferrin 100 μg ml⁻¹, bovine serum albumin 300 μg ml⁻¹, putrescine 16 μg ml⁻¹, thyroxine 400 ng ml⁻¹, tri-iodo-thyronine 300 ng ml⁻¹, progesterone 60 ng ml⁻¹, sodium selenite 40 ng ml⁻¹, 1 mM Glutamax in DMEM, low glucose, pyruvate, Life Technologies). After serum starvation, astrocytes were treated with either pre-clustered rat recombinant EphB1-Fc (1, 5, 10 µg ml⁻¹, R&D Systems) or IL-6 (50 ng ml⁻¹, R&D systems) in Sato’s medium. For all experiments clustered EphB1-Fc was used unless stated otherwise and was referred to as ‘EphB1’ in the text. Before treatment hiPSC-derived astrocytes were cultured for 72 h in absence of BMP4 and LIF and then treated with either human recombinant EphB1-Fc (10 µg ml⁻¹, Gentaur) or IL-6 (50 ng ml⁻¹, R&D systems). Clustering of EphB1-Fc was obtained by incubating EphB1 with a clustering antibody (goat anti-human IgG, Fc fragment specific, 1:10, Jackson ImmunoResearch) for 30 min at RT. At the appropriate time point after the stimulation, cells were further processed for immunocytochemistry, WB or RNA extraction.