Purification Of The Oncoprotein P185 And Establishment Of BA-ELISA Method To Detect P185 In Breast Cancer Patients' Serum

Objective Use p185 by affinity chromatography from the culture supernatant of T6-17 which transfected the Her-2/neu gene as the positive control, to detected p185 in breast cancer patients' serum by BA-ELSIA, and discuss the significance of serum P185 in breast cancer diagnosis. Methods The p185 oncoprotein was purified by using an affinity chromatography column. In this experiment, the anti-p185 oncoprotein monoclonal antibody was obtained from A18 hybridoma ascite with octoic acid-saturated ammonium sulfate precipitation. The antibody activity was coupled with CNBr-activated Sepharose 4B as solid phase medium to pack the affinity chromatography column. The p185 oncoprotein antigen was binded with the anti-p185 antibody when the Culture supernatant run through the column. The gradient salt solution(0.2M HAC PH2.5)as elute buffer to dissociate the p185 antigen from the solid phase medium and elution fraction was collected. After be concentrated, the p185 antigen protein was identified by using ELISA,SDS-PAGE and Western blot. Use the biotinylation Her-2/neu mcAb made by ourselves, and P185 by affinity chromatography from the culture supernatant of T6-17 as the positive control, to detect the p185 in breast cancer patients'serum and normal control serum by BA-ELSIA, meanwhile perform IHC in these patients tissues to detect P185,then analyze these experimental results by statistics software. Results The oncoprotein p185 was purified successfully from the Culture supernatant of transgene T6-17 cells; detect 122 cases of breast cancer patients' serum by BA-ELISA method, 39 cases are serum p185 positive; detect 110 cases of normal human serum by BA-ELISA method, 0 case is serum p185 positive; detect 122 cases of breast cancer patients' tissues by IHC, 43 cases are p185 positive, in which 38 cases serum p185 increased. Conclusion The BA-ELISA method to detect p185 in breast cancer patients'serum has taken shape initially, the serum p185 is significant to breast cancer in diagnosis, and the serum p185 level is highly relevant to tissue expression level of p185.