Autoantibody Detection in Rabbit Serum

ELISAs were performed using 4 different neuronal antigens to detect autoantibodies in rabbit serum. Ninety-six well plates were coated overnight in 0.015 M carbonate/0.03 M bicarbonate (pH 9.6) buffer with each of the 4 antigens (Lysoganglioside, tubulin, human dopamine D1 receptor [D1R] antigen, and human dopamine D2 receptor [D2R]) as described previously [48 (link)]. ELISAs were then performed similar to what was described [49 (link)]. In short, washes were performed with PBS containing 0.05% Tween (PBS-Tween) five times and then blocked for 1 h. The wells were washed and sera from immunized or placebo rabbits were diluted and incubated overnight. After five washes, the samples were incubated with alkaline-phosphatase-labeled goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA). Then substrate and p-nitrophenyl phosphate were added. The optical density (OD) was measured at 405 nm. The results are expressed as the mean of triplicate wells. All ELISAs were validated with known positive and negative serum controls for each assay to maintain a standardized assay.