ELISA plates (Nunc Immunoplate) were coated with protein overnight (4uC) at a concentration of 1 mg/ml in 16 PBS. The plates were blocked with ELISA dilution buffer (1% BSA and 1% Triton x-100) before the serial diluted serum was added to the plate. Secondary horseradish peroxidase-conjugated (HRP) antibodies added in the following dilutions: anti-rabbit-IgG 1:2000 (P0448, Dako), anti-mouse-IgG 1:2000 (P0260, Dako), and antirat-IgG 1:3000 (A9037, Sigma). The optical density was measured at 490 nm in an ELISA plate reader (VersaMax, Molecular Devices). Antibody titration curves were determined using GraphPad Prisma5.