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Research paper : Development of basic tools for glycoscience and their application to cancer diagnosis (H. Narimatsu)−210−Synthesiology - English edition Vol.5 No.3 (2012) and specificity and the suitable probe (e.g., lectin A), an antibody-probe sandwich detection kit is established and verified with more than 100 samples.9.If the advantage over current markers is statistically confirmed, verification of the kit is further conducted with more than 1,000 samples.10.Furthermore, the modification of glycan structure is determined by MSn. As the available samples obtained from patients are generally limited, it is difficult to identify the glycan structure of the samples by MSn. In such cases, we first confirm that cultured cancerous cells indicate the same lectin reactivity, and then the target glycoproteins are purified in large amounts from supernatant of the cultured cells, and are determined for the glycan structure by MSn. 11.At this point, we ask for cooperation of domestic clinical doctors. With the objective data presented under the cooperation of many doctors, the kit is evaluated comparatively with the current biomarkers, and applied for production and health insurance approval by the collaborative public company for the final production. We have targeted many diseases. The followings are successful examples in development of the liver fibrosis marker and cholangiocarcinoma marker. 5.2 Development of liver fibrosis markerWe have developed a liver fibrosis marker, which is almost ready to be applied for production approval by the collaborative company.Infection with hepatitis B virus (HBV) and C virus (HCV) progresses to acute hepatitis, chronic hepatitis, cirrhosis, and finally hepatocarcinoma in 20-30 years after infection. The patients infected with HBV and HCV are estimated to be several hundred million worldwide, 7 % of the Japanese population (about 8 million) and 10 % of the Chinese population (about 150 million). In the course of fibrosis, hepatocytes are disrupted after infection and replaced with fibrin molecules, and the stiffness of the liver increases. Currently the definitive diagnosis is made by needle biopsy; however, this is an invasive and burdening method for patients, requiring 2-3 days of hospitalization. The level of fibrosis (liver stiffness) is classified into F0 (fibrosis 0) to F4. The incidence of hepatocarcinoma increases along with the progression of fibrosis. At F3, the fibrosis level is considerably progressed as a result of chronic hepatitis, and F4 is the state of cirrhosis. Medication with interferon and ribavirin is mostly effective at F3, but cannot be expected at F4. Development of the biomarker for simple blood tests to evaluate the fibrosis level is keenly awaited for the evaluation of therapeutic effect and prediction of the risk of hepatocarcinoma.According to the above-described developmental scheme, alpha-1 acid glycoprotein (AGP) was selected as the first candidate for the fibrosis marker. AGP is a glycoprotein abundantly present in blood, and mainly secreted from the liver. We premised that AGP would well reflect the status of liver fibrosis, and its lectin avidity would be strong as it has five N-glycans. Moreover, it has been long known that the glycan structure of AGP is altered along with liver fibrosis. We could use the serum samples provided from clinical doctors, available with the data of fibrosis levels diagnosed by biopsy. The most suitable lectin for distinguishing F4 and F3 was selected via immunoprecipitation of AGP followed by lectin array. Three lectins, AOL, MAL, and DSA were able to evaluate the fibrosis level quite accurately.[41][42] We established the sandwich detection system consisting of the anti-AGP antibody and three lectins in collaboration with Sysmex Corporation, and optimized the system for an automatic clinical chemistry analyzer, HISCL produced by Sysmex. This system can measure one sample in 17 minutes. However, AGP was not suitable enough for clinical diagnosis, as AGP must be immunoprecipitated from serum before analysis, which requires 2 hours of processing. We further searched for an appropriate glycoprotein for direct measurement of serum by HISCL, and found “molecule X” (the name cannot be disclosed as it is before publication). Glycans on molecule X detected by lectin Y well reflect the status of fibrosis. We prepared a monoclonal antibody against molecule X and established a sandwich assay system with this antibody and lectin Y. This system enabled evaluation of fibrosis within 17 minutes by HISCL without preprocessing of serum. In the future, a patient visiting a clinical doctor would receive the serum test before seeing the doctor, and the doctor would be able to have the fibrosis level of the patient on the same day before consultation. 5.3 Development of cholangiocarcinoma markerWhen a hepatic mass is found by an imaging diagnosis method, intrahepatic cholangiocarcinoma derived from bile duct epithelial cells and hepatocarcinoma derived from hepatic cells must be clearly distinguished. Prognosis of intrahepatic cholangiocarcinoma is bad, and its therapeutic strategy is totally different from that of hepatocarcinoma.In accordance with the developmental strategy of the cancer marker mentioned above, we incised small tissue specimens of 1-mm diameter from the cholangiocarcinoma tissue and the normal region by microdissection. As a result of the lectin array of the fluorescence labeled glycoprotein extract, the WFA lectin signal was remarkably different between the cancer tissue and the normal tissue. We identified many candidate molecules (230 glycoproteins) of the cholangiocarcinoma marker that bind to WFA by the IGOT method. These molecules were prioritized based on the estimated blood concentrations by bioinformatics. The antibodies were purchased for the 10 glycoproteins of

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