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Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis. (Mol Cancer Res 2007;5(2):153 -63)

o-Phenylenediamine, 2,2'-azino-di(3-ethylbenzthiazoline sulphonic acid-6) (ABTS), odianisidine and 4-aminoantipyrine were compared as chromogens for the determination of horseradish peroxidase. Highest sensitivity in the determination of horseradish peroxidase-IgG conjugates in dissolved form was obtained with o-phenylenediamine. When these conjugates were used in a two-site binding enzyme immunoassay for hepatitis B surface antigen (HBsAg), the steepest calibration curve and the lowest detection limit were obtained when ABTS was used to determine the immune complexes bound to the solid phase. Non-ionic detergents, such as polyoxyethylene^sorbitol ester, retarded horseradish peroxidase inactivation, resulting in a chromogen-dependent activity rise of horseradish peroxidase. An optimised determination of horseradish peroxidase is reported, in which the sensitivity of the solid phase enzyme immunoassay is doubled by the use of 0-dianisidine. Vergleich von zur Aktivitätsbestimmung von Meerrettich-Peroxidase als Marker im Enzymimmunoassay verwendeter ChromogeneZusammenfassung: Die Empfindlichkeit der Aktivitätsbestimmung von Meerrettich-Peroxidase wurde mit den Chromogenen 0-Phenylendiamin, 2,2'-Azino,di(3-ethylbenzthiazolin-sulfonat-6) (ABTS), o-Dianisidin und 4-Aminoantipyrin verglichen. An IgG gekoppelte Meerrettich-Peroxidase wird in gelöster Form am empfindlichsten mit ö-Phenylendiarnin bestimmt. Nach Einsatz der Konjugate in einem zwei-Seiten Enzymimmunoassay zur Bestimmung des Hepatitis B-Oberflächenantigens (HBsAg) und quantitativer Bestimmung der an die feste Phase gebundenen Immunkomplexe werden die steilste Ständardkurve und geringste untere Nächweisgrenze mit ABTS erhalten. Nichtionische Detergenzien wie Polyoxyethylen*orbitester verzögern die Inaktivierung von Meerrettich-Peroxidase während der Reaktion und fuhren zu einer chromogenabhängigen Aktivitätssteigerung der Meerettich-Peroxidase. Es wird eine .optimierte Bestimmung von Meerrettich-Peroxidase mit <7,Dianisidin vorgestellt, wodurch die Empfindlichkeit des Festphase-OEnzymimmunoassays verdoppelt werden kann.