Expression Of P185 Protein On T6-17 Cell Surface And Establishment Of Ba- Antibody Microarray Method To Detect P185 In Ovarian Cancer Patients' Serum

Objective: To determine tumor marker P185 protein expression and distribution state on the T6-17 cell surface, and then purified P185 protein as a positive control from cell supernatant. Establish P185 protein detection method by combining the biotin - avidin system with protein chip technology.Methods: Put polylysine-treated cover slips into six cell culture plate, add T6-17 cells and then cultivate 24-48h, 4% formaldehyde fix, 0.1% TrtionX-100 liquid increase permeability, PBS wash. Use immunohistochemistry streptavidin-perosidase (SP) method and DAB color, Compare with the results of paraffin section immunohistochemical which with high expression of P185 protein. The positive control P185 protein was purified by using an affinity chromatography column. In this experiment, Cyanogen bromide activated Sepharose 4B as solid phase medium. We use A18 monoclonal antibody as primary antibody, biotinylated monoclonal antibody Bio-A21 as anti-her-2/neu antibody, gold substrate chip as carriers, and Cy3 fluorescent material as label. GenePix4100A chip scanner read after antigen-antibody reaction.According to fluorescence to explore the best reaction conditions, to determine the P185 protein positive rate in ovarian cancer, and compare with the BA-Elisa results.Results: The immunohistochemical results reveal that P185 protein mainly distributed in the cell membrane. Obtain tumor antigen P185 protein from the culture supernatant of transfected cells called T6-17 successfully. The microarray's efficiency which combine BAS system increased about 5 times. The best reaction conditions are coating concentration of 6.25μg / ml, 37°C incubated for 4h, the sample buffer choose 1% BSA-PBS. P185 positive rate was 17% (11/65) in serum of ovarian cancer patients.Conclusion: T6-17 cell surface express a large number of P185 proteins, the T6-17cell can be considered as a positive control in the clinical application of immunohistochemistry. Confirm T6-17 cell surface expressing P185 protein, establish protein chip detection methods with BAS system. Confirm positive rate of P185 protein has association with ovarian cancer development and has a wide clinical application possibility.