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

Abstract

Human 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 × 106 1/MS, kd = 4.4 × 10−5 1/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 the Tm value 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.