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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.

Objective To establish the specificity and sensitivity of alkaline phosphatase activity in testicular tumours for the early detection of testicular germ cell tumours (GCTs). Materials and methods Samples from patients with GCTs of the testis were sectioned and stained using the nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) Sigma Fast substrate technique (referred to as MEDC) and peroxidase anti-peroxidase (PAP) assay. Additionally, several cell lines from GCTs and other tumours were cultured using standard techniques and stained using both methods, and lysates from all samples were stained after gel electrophoresis. Results MEDC showed strong and specific staining of testicular tumours only. The activity on gel electrophoresis appeared as two bands, one at 75 and one at 95 kDa, which was a unique combination for testicular tumours and testis cell lines. Conclusion The MEDC method has extreme sensitivity and intensityfor testicular tumour tissue and has potential use as a powerful tool for the early detection of testicular GCTs by analysing semen samples, particularly in patients with equivocal findings on testicular ultrasonography.