Rabbit anti human igg

Maxisorp^TM plates (Nunc, Waltham, MA, United States) were coated with rabbit anti-human IgG (Sigma, Darmstadt, Germany) or mouse anti-human IgG2 (Sigma) overnight at +4°C. Plates were washed in wash buffer (PBS pH 7.4, 0.05% Tween20), blocked with blocking buffer (PBS, 1% skimmed milk, 0.05% Tween20) for 1 h at room temperature (RT) followed by an additional wash. Patient and calibration sera for IgG (Dako, Glostrup, Denmark) and pure IgG2 (The Binding site, San Diego, CA, United States) were diluted in a blocking buffer, titrated on plates and incubated for 1 h at RT. After one final wash, all wells were incubated for 20 min with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (Dako P0214). The optical density was measured at 405 nm, and absorbance between calibration sera and patient sera was compared.

A total of 5 × 10⁶ C2C12 cells were lysed in the RIPA buffer, pre-cleared, and incubated with the anti-PKCe antibody (Abcam, Cambridge, UK, 5 μg/mL) or anti-Nrf2 antibody (Cell Signaling, Danvers, MA, USA, 5 μg/mL) overnight at 4 °C. Rabbit anti-human IgG (Sigma Aldrich, St. Louis, MO, USA, 1 μg/mL) was used as a negative control. Samples were incubated with Protein A/G PLUS-Agarose beads (Santa Cruz, Dallas, TE, USA) for 1 h at 4 °C and resolved with SDS-PAGE.

Sera were tested for B. burgdorferi antibodies by a home-made indirect immunofluorescence assay (IFA). The Borrelia employed as the antigen was B. burgdorferi sensu stricto (strain B31 ATCC 35210). These bacteria were propagated in Barbour Kelly medium and fixed on spot slides. The fluorescein-labelled conjugates used were rabbit anti-human IgG and IgM (Sigma, St Louis, MO, USA), diluted 1/128 in PBS. Briefly, two-fold dilutions of each serum sample were added to the antigen spots and incubated in a humidity chamber for 30 min at 37 °C. After washing, the conjugate was added to each sample. The slides were then incubated for 30 min, washed, and examined using a BH2 fluorescence microscope (10 × 40, Olympus, Tokyo, Japan). Positive and negative control sera were also examined. Sera showing a typical pattern of fluorescence at IgG titres of ≥1:256 and IgM titres of ≥1:32 were deemed positive (Figure 1). All positives sera were tested for infection by Treponema pallidum by the haemagglutination test (TPHA-BioMérieux, Marcy l´Etoile, France) to rule out syphilis.