Establishment And Clinical Application Of A Highly Sensitive Method To Detect HER2 Protein In Human Serum

Background and ObjectiveAs the member of human epidermal growth factor receptor family.HER2/P185/ErbB2 ‘s overexpression is closely related to the clinical development of a variety of tumors,including breast cancer,stomach cancer,lung cancer and ovarian cancer.Serum HER2 protein content can directly affect the chemotherapy effect of patients with cancer and survival time.The level of expression of HER2 protein in sera of cancer patients is currently used as a standard for the diagnosis and treatment of various tumors,such as gastric cancer,breast cancer,etc.,which can be considered as an independent risk factor for predicting the prognosis of cancer.The trend of serum HER2 levels is often used as a measure of the therapeutic effect of the tumor and as an evaluation index for the prognosis of cancer patients.However,due to the lack of a technique for the detection of serum HER2 in the present country,highly sensitive detection technology of Serum HER2 protein content and provide a reference for the clinical specimens of the treatment and prognosis of cancer.Materials and MethodsThe laboratory to establish human serum HER2 detection method from the following aspects :(1)Preparation of anti-HER2 monoclonal antibody HuA21 and HerA and high purity antigen standard HER2-ECD: inoculated with the appropriate concentration of cultured in the laboratory culture of CHO cells,to be cultured to 90%density of cells were transfected with the expression vector of HuA21,HerA and the HER2-ECD gene,after cultured for 24 hours and then digested with trypsin.The resistant clones were screened by adding a selective medium containing 0.3 mg /mlZeocin.The purified supernatant was amplified and the culture supernatant was collected.Collecte the culture supernatant and further purified,concentrate.The antibody HuA21 and HerA were purified by SDS-PAGE,and the HER2-ECD was determined by BCA method Standard antigen content,and the results compared with the United States approved clinical use of serum HER-2 / neu ELISA kit.(2)Using double antibody sandwich enzyme-linked immunosorbent assay(ELISA)technology,the anti-HER2 monoclonal antibody HuA21 and biotin-labeled HerA antibody were used as the capture antibody and detection antibody,respectively.HER2 extracellular domain(ECD)were selected as the antigen protein in order to establish a technique for quantitatively detecting HER2 in human serum.And optimization of antibody affinity,specificity,stability and pairing concentration,as well as optimization of reaction reagents and reaction conditions.(3)Serum samples from breast cancer patients in Hefei area were collected and tested with similar kits from Siemens Medical Diagnostic Products Co.,Ltd..The results were analyzed and analyzed by using student's t test and other statistical methods.ResultsThe high purity anti-HER2 monoclonal antibody HuA21 and HerA were successfully prepared by this experiment.Optimize and determine the highly sensitivie serum HER2 detection kit,and use HuA21 as capture antibody and biotin-labeled HerA as detection antibody.The sensitivity of the method was 7.8 pg / mL,and the detection limit was from 0 to 500 pg / ml.The intra-assay coefficient of variation was 0.2%-10.9%,and the coefficient of variation was 1.4% ~ 12.4%.The recovery rate of the antigen in the range of 86.84% to 116.40%.There was not significantly difference on HER2 protein detection of human serum between our kit and Siemens chemiluminescence assay kit(P >0.05),and the correlation coefficient was high(R2 >0.70).The selected clinical samples were determined using our method,and showed that the serum levels of HER2 in patients with HER2-positive metastatic breast cancerwas significantly higher than those in patients with early-stage breast cancer and healthy subjects(P<0.05).The patients receiving trastuzumab plus chemotherapy or chemotherapy alone were followed up and the results showed that the therapy efficacy was positively correlated with the serum HER2 levels decreasing.ConclusionIn this study,we successfully developed a high-sensitivity kit capable of detecting HER2 content in human serum.Serum HER2 levels in patients with HER2-positive metastatic breast cancer were significantly higher than those in early breast cancer patients and healthy subjects(P <0.05).The positive rate of HER2 in metastatic breast cancer patients was significantly higher than that in patients with metastatic breast cancer To 37.5%,consistent with the results reported abroad.