Igg2a

Sera collected from SAMR1 and SAMP8 mice were analyzed as described previously.^{49 (link)} Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house),^{50 (link)} IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA). The Ig concentrations were calculated by using the Graph Pad Prism 5.0 software. After in vivo peptide-OVA immunization, anti-peptide-specific IgG titers in sera were determined by indirect ELISA in Nunc 96-microplate wells (Maxi Sorp, 442404, Nunc-Thermo Fisher Scientific). Plates were coated (1 h, 37 °C) with 50 μl of peptide (10 μg/ml PBS); plates were then washed, blocked, incubated with sera (twofold serially diluted in PBS-Tween20/BSA, starting from 1:10), and revealed with goat anti-mouse IgGs (SouthernBiotech, Birmingham, AL, USA), as described.^{49 (link)} The 18-amino-acid peptide is part of the sequence of the H toxin of S. aureus. Coupling agent: Sulfo-SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate).

Microtiter plates (MaxiSorb®, Costar, USA) were coated with 50 μl of 5 μg/ml OVA solution in PBS (pH = 7.2) and incubated overnight at + 4 °C. Following extensive washing with PBS containing 0.05% Tween-20 (PBS-T) and subsequent blocking with 5% BSA in PBS, plates were incubated with serially diluted serum samples at + 4 °C overnight. Next day plates were subsequently exposed to primary biotin-labeled anti-mouse antibodies (BioLegend, San Diego, CA, USA) to either IgEa (clone UH297) or IgG1 (clone RMG1 1) or IgG2a (clone RMG2a 62), and streptavidin-conjugated HRP. Plates were further processed with substrate solution (3,3^′,5,5^′-tetramethylbenzidine) Optical densities (OD) were measured with automatic 96-well plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm with subtraction of optical density at 620 nm that does not correspond to the reaction colored product. Antibody quantities were estimated as OVA-specific antibody titers defined as the serum dilution, in accordance according to 4-PL regression analysis.
For estimation of total IgE levels, plates were coated with unlabeled anti-mouse IgE (clone RME1, BioLegend). Serum samples were added in 1:100 dilution in duplicates with different dilutions of mouse IgE κ (MEA-36, BioLegend). In following steps biotin labeled anti-mouse IgE (UH297) and streptavidin-HRP were added subsequently as described above.

Male C57BL/6N mice (6–8 weeks) were purchased from Janvier (Le Genest, France) and treated according to the International Association for the Study of Pain guidelines. For all experiments the ethics guidelines for investigations in conscious animals were obeyed and the procedures were approved by the local ethics committee (Regierungspräsidium Darmstadt). The animals had free access to food and water and were maintained in climate‐ (23 ± 0.5°C) and light‐controlled rooms (light from 6.00 a.m. to 6.00 p.m.).
Inflammation was induced by injection of 10 μl zymosan (3 mg/ml in PBS; Merck, Darmstadt, Germany) subcutaneously into the plantar side of one hind paw. Eosinophil depletion was achieved by intraperitoneal (i.p.) injection of anti‐Siglec F antibody (0.883 mg/kg; clone 238,047; R&D Systems, Minneapolis, MN) 24 h prior zymosan injection. As control purified rat IgG2a (Biolegend, San Diego, USA) was used. IL‐4c was prepared using IL‐4 (Peprotech, Hamburg, Germany) and anti‐IL4 antibody (Biolegend, San Diego, USA) (Finkelman et al, 1993 (link); Milner et al, 2010 (link); Jenkins et al, 2011 (link)) and administered i.p. (0.166 mg/kg IL‐4 and 0.883 mg/kg anti‐IL4 antibody) 24 h prior zymosan injection. (Finkelman et al, 1993 (link); Milner et al, 2010 (link); Jenkins et al, 2011 (link)).