3 amino 9 ethylcarbazole tablets

Serum HEL-specific IgG1 were detected by ELISA using a modified version of the protocol previously described^{44 (link)}. Briefly, plates were coated with 5 μg/ml of HEL in PBS overnight at 4 °C. Sera were added to the saturated plates and were incubated for 2 h at 37 °C. Pooled sera from hyperimmune mice (immunised with SRBC-HEL twice) were used as a standard. Plates were developed with the BD Opteia kit (BD Biosciences). Serum dsDNA-specific IgG1 was detected by ELISA as previously described^{65 (link)}.
For detection of HEL-specific antibody-forming cells (AFCs) ELISPOT plates (Millipore) were coated with 5 μg/ml of HEL in PBS overnight at 4 °C. Single-cell suspensions of the spleen were added to saturated ELISPOT plates in quadruplicate and incubated overnight at 37 °C in 5% CO₂ in a humidified incubator with culture medium. AFCs were detected with goat anti-mouse IgG1 antibody conjugated to horseradish peroxidase (Southern Biotech). Plates were developed using 3-amino-9-ethylcarbazole tablets (Sigma-Aldrich). Plates were read using an AID ELISpot reader according to the manufacturer’s instructions.