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Objective To develop specific monoclonal antibodies (mAbs) against human germ cell tumours. Materials and methods A single-cell suspension obtained from tumour tissue fragments (consisting of both tumour and normal compartments) from a patient with seminoma was used as an immunogen. Spleen cells from immunized mice were used to develop mAbs. Tissue specificity, biochemical characteristics and competitive studies were analysed using immunocytochemical staining, dot blots and a Western blot analysis, to identify target antigen(s). Results The immunization protocol led to the development of 107 hybridomas, 90 of which were negative against the original tissue biopsies. The remaining 17 showed positivity against various tissue compartments. One selected mAb (ATC2) showed specific staining on germ cell tumours but not on normal tissues, and positive staining with some human tumour cell lines. The target antigen for ATC2 was confirmed to be placental alkaline phosphatase (PLAP) based on: Western blot analysis compared with commercially available PLAP; comparison of the data with another well-known anti-PLAP mAb (H17E2, although the two mAbs recognized different antigenic epitopes); heat resistance characteristics; high-performance liquid chromatography of the ATC2 target antigen and purified PLAP. Conclusion The selected mAb ATC2 has high specificity for human germ cell tumours, the target antigen for ATC2 being PLAP, although the antigenic epitope(s) differ from those recognized by H17E2. Thus ATC2 may be useful for monitoring serum levels of PLAP in patients with testis cancer and may be relevant for detecting cancer cells in the semen of individuals with suspected testis cancer, particularly in those with equivocal findings on ultrasonography.