Amoureux et al. BMC Cancer 2010, 10:91 http://www.biomedcentral.com/1471-2407/10/91 first verified the specificity of the ELISA by digesting PSA-NCAM from an extract of TE 671 cells known to express PSA-NCAM [17]. After verifying that endoN indeed digested PSA-NCAM by Western blot (not shown), a serial dilution of TE671 extract treated or not with endoN was prepared and tested by ELISA. Data shown on Figure 1B indicate that the signal obtained in absence of endoN is specific for PSA-NCAM as it was completely abolished at most dilutions of the extract after enzymatic digestion. In these diluted extracts, PSA- NCAM was more concentrated than in the GBM biop- sies, ensuring that the signal measured in GBM biopsies was entirely PSA-NCAM specific. However, a limitation of our test is that although the anti-PSA antibody allows to reveal PSA, it does not discriminate between different lengths of PSA chains. In order to accurately assess PSA-NCAM level from GBM biopsies, its expression in normal brain tissue had to be determined. Adult human brain cortex expresses PSA-NCAM only on rare cells [22]. In non tumoral adult human brain tissue, a signal equivalent to 8 pg PSA-NCAM/μg of protein was mea- sured, and PSA-NCAM was below detection in adult mouse cortical tissues (n = 4). Moreover, the inter assay variability for these samples was of 10 pg/μg of protein. Page 5 of 12 Therefore, the threshold of significance for specific detection of PSA-NCAM was set to 10 pg PSA-NCAM/ μg of protein. This value was chosen to classify groups of GBM patients that express either less or more than 10 pg PSA-NCAM/μg of protein. Embryonic human brain extract served as positive control since it is known to express high level of PSA- NCAM, as do medulloblastoma (MB) tissues [16], which was confirmed in the current study by immunohisto- chemistry (Figure 2C) and ELISA for MB (150-1030 pg PSA-NCAM/μg of protein, n = 3) and embryonic human brain (1600 pg PSA-NCAM/μg of protein). PSA-NCAM measurement by ELISA is correlated to immunohistochemistry analysis Immunohistochemistry has been so far the standard method in anatomopathology laboratories, including using the PSA-NCAM antibody [16,23]. Therefore, we verified that the results obtained using the ELISA test correlated with the immunohistochemistry results. Figure 2A shows biopsies with various degrees of PSA- NCAM immunohistochemistry staining with the corre- sponding ELISA measurements. They illustrate weak or medium PSA-NCAM immunostaining in two GBM Figure 2 PSA-NCAM measurements by immunohistochemistry and ELISA, and distribution of PSA-NCAM measurement by ELISA in 56 GBM biopsies. PSA-NCAM immunohistochemistry on two GBM biopsies with below detection (A) or positive (B) staining, and on a MB biopsy highly expressing PSA-NCAM (C); scale bar: 33 μm. (D) Individual measurements of PSA-NCAM in the 56 GBM, by ELISA and Immunohistochemistry. (E) Individual measurements of PSA-NCAM by ELISA and Immunohistochemistry in two groups (PSA-NCAM<10 pg/μg of protein and PSA-NCAM>10 pg/μg of protein). Lines indicate the median in each group (F) Distribution of PSA-NCAM content from 56 biopsies, represented as boxplot with first quartile (9 pg PSA-NCAM/μg of protein), median (25 pg PSA-NCAM/μg of protein) and third quartile (40 pg PSA-NCAM/μg of protein). The dotted line corresponds to 10 pg PSA-NCAM/μg of protein (level of sensitivity of PSA-NCAM detection in GBM tissue).