Enzyme-linked Immunosorbent Assay (ELISA) for NY-ESO-1 or XAGE1 antibodies
Serum samples were obtained at baseline, 4 and 8 weeks after the first KW-0761 treatment, and every 4 weeks during the continuous treatment until the point of study discontinuation. Recombinant NY-ESO-1 and the synthetic XAGE1 protein (1 μM) in ELISA Buffer kit (PeproTech, Rocky Hill, NJ) were adsorbed onto a 96-well ELISA plate (Nunc, Roskilde, Denmark) and incubated at 4°C overnight. Plates were washed with 0.05% Tween-20 in PBS and blocked with 1% BSA/PBS (200 μl/well) at room temperature for 1 hour. After washing, 100 μl of serially diluted serum was added to each well and incubated at room temperature for 2 hours. After washing, horseradish peroxidase-conjugated goat anti-human IgG (MBL, Nagoya, Japan) was added to the wells, and the plates were incubated at room temperature for 1 hour. After washing and development, absorbance was read at 490 nm [