| Background and AimsColorectal cancer (CRC) is one of the most common forms of cancer. In Western countries, it is the second most frequent cause of cancer death. And it is the fourth most common cancer in Shanghai, China. Moreover, in the past few years, the morbidity and mortality of CRC became gradually higher in developed and developing countries. CRC, like many other tumors, is curable if detected in an early stage. Survival is directly related to the extent of disease at the time of diagnosis. Those diagnosed at an advanced stage have an estimated 5-year survival rate of 7%, in contrast with a survival rate of 92% for individuals detected at an early stage. Therefore, it is an important issue to find early cancer.Non-invasive screening methods for CRC generally include fecal occult blood test (FOBT), flexible sigmoidoscopy, colon X-ray examination, colonoscopy, MR or CT colonography, exfoliated colonocytes test, etc. Although FOBT is the most widespread test, its sensitivity and specificity are poor. Furthermore, its cost-effectiveness are lower. Colonoscopy is undoubtedly the most effective method, but its potential disadvantages, for example invasiveness, cost, inconvenience and higher risk should be considered. So detection of the tumor markers from stool or exfoliated CRC cells from stool may be the key approaches to screen early CRC.In order to make a sensitive, simple and specific approach for screening CRC, we prepared some correlative antibodies against CRC using differential expression protein of CRC, and constructed a nanometer magnetic cell sorting system (MACS) for exfoliated colorectal cancer cell.Materials and methodsThis study consists of three sections.Section 1 is about construction and application of a magnetic cell sorting system for exfoliated colorectal cancer cells.1. Determination of the differential soluble protein of colorectal cancer compared with that of the normal colon tissues by SDS- polyacrylamide gel electrophoresis (SDS-PAGE).①All the soluble proteins from 42 cases with CRC and 42 normal colon tissues (control tissues) were abstracted. ②Samples of the soluble proteins were isolated by SDS-PAGE. ③The electrophoresis images were analysed by Adobe Photoshop 6.0 software, and the molecular weight (MW) of the up-regulated proteins that occurred frequently in CRC tissue were calculated. ④Enriched the target protein (soluble colorectal cancer associated protein, molecular weight 47KD, SCAP47) by SDS-PAGE, and identified the purity.2. Preparation of the associated antibodies for CRC (1). Preparation of the polyclonal antibodies (PcAb) from rabbit serum ①3 New Zealand White rabbits, male, 6 months, 2.5Kg±. ②After injection of BCG, every rabbit was subsequently given emulsive mixture of the separated protein and Freund's adjuvant(complete or incomplete) and the separated protein solution only for several times. ③Detection of the titer of PcAb by enzyme-linked immunosorbent assay (ELISA) and double diffusion precipitation test. ④Separated and collected the serum from the rabbits with higher titer polyclonal antibodies against SCAP47. (2). Preparation of mice monoclonal antibody (McAb) ①6 BALB/c mice, female,4 weeks. ②The emulsive mixture of the separated protein and Freund's adjuvant(complete or incomplete) were injected into abdominal cavity for several times, and the separated protein solution was injected into spleen. ③The titer was detected by ELISA, and the spleen of the mice with higher titer were dissected. ④The hybridoma cells which SP2/0 cell fused with murine immuno-spleencell cultured in carbon dioxide gas incubator at 37℃. ⑤The positive cell line was cloned with the limited dilution method. ⑥The mice abdominal cavity were inoculated with the clonal cells, and their ascites were collected. ⑦The McAb was purified according to the procedure of saturated ammonium sulfate deposition, Sephadex G-50 column desalination, DEAE-Sephadex A-50 column chromatography. ⑧Identified the purified McAb. |
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