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 90 (ka = 8.1 x 10(6) 1/MS, kd = 4.4 x 10(-5) 1/S, KD = 5.5 x 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 90 had a commendable thermal stability with the T-m value of 73 degrees C. Analyses of western blot, immunohistochemistry and immunofluorescence showed that the 90 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. (C) 2017 Elsevier Inc. All rights reserved.