Polyclonal rabbit anti human igg

All antibodies were used FITC, PE, Cy3, PE-Cy5, PE-Cy7, APC, eFluor660 or biotin conjugated. The biotinylated mAb were revealed with Streptavidin-PE-Cy5. mAb were used at 0.1–1 µg/1×10⁶ cells in 100 µl PBS/2% FCS/0.02% NaN₃ (FACS buffer) or HBSS/0.3% BSA incubated on ice for 20–30 min. Antibodies used were: anti-CD16/CD32 (Fcγ III/II Receptor) (2.4G2), anti-CD90.1 (Thy1.1) (HIS51), anti-CD8α(Ly-2) (53–6.7), anti-CD3ε (145-2C11), anti-Vα2 T cell receptor (B20.1), anti-α4β7 (DATK32), anti-IA^b (AF6-120.1) and anti-CD11c (HL3) all from BD Biosciences (Heidelberg, Germany), anti-CCR9 (B cell hybridoma supernatant, kindly provided by Prof. R. Förster), anti-rat IgG (secondary for CCR9) (from Jackson ImmunoResearch Europe Ltd.), anti-CCR10 (248918), E-selectin/human IgG-Fc-Chimera (both from R&D) and polyclonal rabbit anti-human IgG (secondary for E-lig staining) (DakoCytomation, Hamburg, Germany).
All stainings were performed following a standard protocol except of E-lig staining. This staining was performed as described [16] (link). Control staining for E-lig and CCR9 was done with secondary Ab only, while corresponding isotype control mAb or fluorescence minus one (FMOs) were used as a control for all other stainings.
Data were acquired and analysed on a FACScan using CellQuestPro software (BD Biosciences) or on a FACS Canto II (Becton Dickinson) and analysed using FlowJo.

All ELISAs were performed in principle as described by Schweiger et al., (2012) [20 (link)] and the antigens were coated according to previous titration studies [20 (link)]. In this study, an anti-human-immunoglobulin G (IgG; polyclonal rabbit anti-Human-IgG, specific for the Fc part of the heavy chain; DAKO, Glostrup, Denmark) conjugated to alkaline phosphatase (Roche Applied Science, Rotkreuz, Switzerland) was used with a dilution of 1:500. Positive standard sera of human patients with confirmed AE or CE and negative standard sera of blood donors were included in all test runs and ELISA plates (5 sera per plate).

IgG levels in culture supernatants were detected by ELISA. Briefly, microtiter plates (Greiner, Milan, Italy) were coated with polyclonal rabbit anti-human IgG (Dako, St. Clara, CA, USA) and incubated 3 h at 37 °C and then overnight at 4 °C. After washes, culture supernatants were incubated for 2 h at 37 °C and horse-radish peroxidase (HRP)-conjugated rabbit anti human IgG (Dako) was added. After 2 h of incubation, substrate solution (o-Phenylenediamine dihydrochloride) (Sigma Aldrich) was added. Colorimetric reaction was read at 492 nm (Sunrise, Tecan, Switzerland). Data were calculated by Magellan Software (Tecan). IgG levels were expressed as µg/mL.
IL-6 levels were detected by ELISA using Duo set antibody pairs (R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. Results were expressed as pg/mL.