Anti human igκ

Immunoblotting was carried out as previously described²⁴ with some modifications. The cells were harvested, lysed by sample buffer containing 2-mercaptoethanol and separated by SDS-PAGE using a 4–20% gradient gel. Blotting was carried out using an iBlot 2 Gel Transfer Device and iBind Western Device (Thermo Fisher Scientific). Human immunoglobulin HC, Igλ, Igκ, and chicken IgM were detected by HRP-conjugated goat anti-hIgG-Fc (Bethyl), anti-human Igλ (Southern Biotech), anti-human Igκ (Southern Biotech) and anti-chicken IgM (Bethyl), respectively. ECL western blotting detection reagents (GE Healthcare) and LAS-4000 Mini (Fujifilm) were used for signal detection.

ELISA plates (Thermo Scientific, Schwerte, Germany) were coated overnight with whole normal human brain lysate (Novus Biologicals; 10 μg/ml) or anti-human Igκ (SouthernBiotech; 2.5 μg/ml), respectively, both diluted in PBS or with PBS alone. As for the ELISPOT assay, whole normal human brain lysate and anti-human Igκ were titrated to their optimal concentration for use in the antibody ELISA. Plates were blocked with 10% FBS in PBS containing 0.05% Tween 20 for 2 h at room temperature. The plates were incubated overnight with serum at 4°C. All serum samples were diluted 1:400 in 10% FBS solution containing 0.05% Tween 20 detergent. Biotinylated anti-human IgG (Hybridoma Reagent Laboratory) diluted in 0.5% FBS/0.05% Tween 20 solution was used as a detection antibody at 0.05 μg/ml. All plates were developed with tetramethylbenzidine substrate (eBioscience, Frankurt, Germany) after incubation with streptavidin-horseradish peroxidase (eBioscience) at 1:1000 dilution. The reaction was stopped with 0.16 M sulphuric acid and the optical density (OD) in the wells was read at 450 nm using a Perkin Elmer Victor 3 1420 Multilabel Counter and Wallac 1420 software version 3.00 revision 5.