Preparation and characterization of a high-affinity monoclonal antibody against human epididymis protein-4

摘要

AbstractHuman epididymis protein-4 (HE4) may serve as a putative biomarker for the early diagnosis, therapy and especially prognosis of ovarian, lung and breast cancer. Detection and targeting of HE4 using the anti-HE4 antibody could be one of the effective strategies for the cancer diagnosis and treatment in clinical practice. In this study, a high-efficiency expression system was established to purify recombinant HE4. We obtained high purity HE4 in 400?mg quantity from 1?L culture supernatant of HEK293F cells. CCK-8 and cell cycle assays indicated that the purified recombinant HE4 protein could promote SKOV3 cell cycle and proliferation at the concentration of 0.1?mg/L. Furthermore, an anti-HE4 high-affinity monoclonal antibody 9C3 (ka?=?8.1?×?1061/MS, kd?=?4.4?×?10?51/S, KD?=?5.5?×?10?12?M) was prepared using hybridoma technique and analyzed by surface plasmon resonance technology using this HE4 protein. Differential Scanning calorimeter (DSC) analysis showed that 9C3 had a commendable thermal stability with theTmvalue of 73?°C. Analyses of western blot, immunohistochemistry and immunofluorescence showed that the 9C3 was highly specific to HE4 in human cancer cells and tissues. In conclusion, our study designed a method to prepare human recombinant HE4 with high yield and generated a high-affinity anti-HE4 monoclonal antibody that might have potential for basic research and clinical application.展开▼