Identification and validation of B cell epitopes from cancer/testis antigen XAGE-1b. (A). ELISA was used to screen candidate peptides from XAGE-1b for recognition by patients' sera. Three patients (#1-3) were positive for either XAGE-1b:1-25 or 57-81. Sera were diluted at 1:25, 1:125, and 1:625 with BSA serving as a control target. The mean OD of 8 HD and the OD of one seronegative patient (#4) are also shown. The use of sera from NSCLC patients for screening is due to higher frequency of Ab against these shared antigens in NSCLC patients. Previous work has shown that peptide epitopes identified using one type of sera are equally recognized by sera from other cancer patients. (B). Western blots confirmed recognition of the full-length XAGE-1b protein. Lane 1, 2, and 3 contained, respectively, lysate from 293 cells transfected with a control plasmid, a plasmid encoding XAGE-1b (denoted with an arrow), and lysate from LNCaP-CL1 cells (expressing XAGE-1b but at a much lower level based on real-time PCR, data not shown).