Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies.

Renjian Hu,Kui Zhang,Zhen Dong,Chongyang Li,Guangzhao Pan,Hongjuan Cui

doi:10.3390/v12030333

Abstract

At present, the clinical detection method of human papillomavirus (HPV) is mainly based on the PCR method. However, this method can only be used to detect HPV DNA and HPV types, and cannot be used to accurately predict cervical cancer. HPV16 E7 is an oncoprotein selectively expressed in cervical cancers. In this study, we prepared an HPV16 E7-histidine (HIS) fusion oncoprotein by using a prokaryotic expression and gained several mouse anti-HPV16 E7-HIS fusion oncoprotein monoclonal antibodies (mAbs) by using hybridoma technology. Two mAbs, 69E2 (IgG2a) and 79A11 (IgM), were identified. Immunocytochemistry, immunofluorescence, immunohistochemistry, and Western blot were used to characterize the specificity of these mAbs. The sequences of the nucleotide bases and predicted amino acids of the 69E2 and 79A11 antibodies showed that they were novel antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) with overlapping peptides, indirect competitive ELISA, and 3D structural modeling showed that mAbs 69E2 and 79A11 specifically bound to the three exposed peptides of the HPV16 E7 (HPV16 E749–66, HPV16 E773–85, and HPV16 E791–97). We used these two antibodies (79A11 as a capture antibody and 69E2 as a detection antibody) to establish a double-antibody sandwich ELISA based on a horseradish peroxidase (HRP)-labeled mAb and tetramethylbenzidine (TMB) detection system for quantitative detection of the HPV16 E7-HIS fusion oncoprotein, however, it was not ideal. Then we established a chemiluminescence immunoassay based on a labeled streptavidin-biotin (LSAB)-ELISA method and luminol detection system—this was sufficient for quantitative detection of the HPV16 E7-HIS fusion oncogenic protein in ng levels and was suitable for the detection of HPV16-positive cervical carcinoma tissues. Collectively, we obtained two novel mouse anti-HPV16 E7 oncoprotein mAbs and established an LSAB-lumino-dual-antibody sandwich ELISA method for the detection of the HPV16 E7-HIS fusion oncogenic protein, which might be a promising method for the diagnosis of HPV16-type cervical cancers in the early stage.

Highlights

The morbidity rate of cervical cancer makes it the fourth most frequent cancer in women worldwide
The predicted amino acid sequence of the HPV16 E7-HIS fusion oncoprotein expressed by the recombinant HPV16 E7 gene was CCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP, which contains a total of 105 aa, including the first 7 amino acids and 98 aa that constitute the HPV16 E7-HIS fusion oncoprotein
HPV16 E7-HIS fusion oncoprotein was about 12 kDa; we found a 15 kDa band that has a molecular weight close to the natural HPV16 E7 protein, which was consistent with previous reports that the MW of HPV16 E7 migrated from 14 to 21 kDa in SDS-PAGE due to the protein negative charge [14] (Figure 1C), indicating the success of the expression of the HPV16 E7-HIS fusion oncoprotein

Summary

Introduction

The morbidity rate of cervical cancer makes it the fourth most frequent cancer in women worldwide. The high-risk human papillomavirus (HPV) integrating the cervix is a direct cause of cervical precancerous lesions and cervical cancer [1]. HPV 16 and 18 account for > 70% of cervical cancers and HPV 31, 33, 35, 45, 52, and 58 account for an additional 20% [2,3]. The prevalence of HPV infection in the cervix among women is 42.7% in the USA [4] and 17.7% in China [5]. HPV detection combined with cytology can improve the detection rate of cervical intraepithelial neoplasia (CIN) and cervical cancer [6]. The clinical detection method of HPV is mainly based on the PCR method [7]

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