The identification of tumor antigens remains a major objective in tumor immunology, especially in pediatric malignancies where solid tumors often do not express a single dominant antigen. Methods such as the Serological Screening of Recombinant cDNA Expression Libraries (SEREX) have been used in the discovery of tumor-expressed proteins by virtue of their ability to induce an antibody response. To focus and accelerate this approach, we first identified candidate antigens by gene expression profiling data from clinical neuroblastoma specimens and then used an animal model to generate an antibody response to an engineered cell-based vaccine. Candidate tumor antigens were expressed as recombinant proteins in a mammalian system and screened for antibody recognition using serum from mice vaccinated with a neuroblastoma cell-based vaccine engineered to express CD80 and CD86, with or without Treg depletion. Through this procedure, the never in mitosis A (NIMA)-related kinase NEK2 was identified as a tumor-associated antigen. Direct testing of serum from patients newly diagnosed with neuroblastoma showed specific serological responses in two of 20 patients. Although NEK2 was not universally recognized, it may serve as a tumor antigen for some patients.