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

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 E7 49-66 , HPV16 E7 73-85 , and HPV16 E7 91-97 ). 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.Viruses 2020, 12, 333 3 of 23 carcinoma, and 5 cases of cervical cancer tissues derived from stage IIa HPV16-positive cervical cancers. All cancerous specimens were obtained by using the extensive hysterectomy, while normal specimens were obtained by using the panhysterectomy. A Mini Shaker MH-1 microplate oscillator (Kylin-Bell Lab Instruments, Haimen, Jiangsu, China), a 1510 MULTISKAN GO full-wavelength microplate reader (Thermo Fisher Scientific, Waltham, MA, USA), and an Infinite ® M200 PRO microplate reader (TECAN, Männedorf, Switzerland) were used for general experiments. Preparation of HPV16 E7-HIS Fusion OncoproteinAccording to the full-length HPV16 E7 gene, specific primers with 6 histidine (HIS) tags were designed. The sequences of primers F and R were as follows: F: 5 -GCCCATGGACCATCACCATC ACCATATGCATGGAGATACACCTAC-3 ; R: 5 -GCAAGCTTTTATGGTTTCTGYGAACAGATGG GGCACAC-3 . Using the total genome DNA of CaSki cells, which was pre-e...

Immunodiagnosis and Immunotherapeutics Based on Human Papillomavirus for HPV-Induced Cancers

“…For instance, two mouse mAbs induced by a recombinant HPV16 E7 oncoprotein were used to establish a chemiluminescent immunoassay based on a labeled streptavidin-biotin (LSAB)- enzyme-linked immunosorbent assay (ELISA) method and a luminol detection system that was suitable for the detection of HPV16 E7 oncoprotein in HPV16-positive cervical carcinoma tissues. In this method, an IgG2a-type anti-HPV16 E7 mAb was used as the detection antibody, and an IgM-type anti-HPV16 E7 mAb was used as the capture antibody ( 21 ). A Rabbit polyclonal antibody to the HPV58 E7 protein was also developed for detection of cervical cancer; it can specifically recognize the endogenous and the recombinant HPV58 E7 proteins ( 33 ).…”

“…Minor capsid proteins, making up the capsid of the virus and involving in the proliferation of the virus mAbs) against various types of HPV proteins, have been developed for both diagnosis and therapy of HPV-positive carcinomas (21,(29)(30)(31). Even other kinds of biomimetic antibodies, such as affibody, nanobody, intracellular singlechain antibodies (scFvs), as well as T-cell-based therapies, have also been developed for the diagnosis or therapeutics of HPVassociated cancers.…”

“…At present, the clinical detection method of HPV is mainly based on the polymerase chain reaction (PCR) method. This method can only be used to detect HPV DNA and HPV types and cannot be used to accurately predict HPV-positive cancers ( 20 , 21 ). For instance, the serum DNA level of HPV has been shown to have a significant prognostic impact on advanced anal carcinoma before first-line chemotherapy, and HPV-circulating tumor DNA (ctDNA) becomes negative after chemotherapy completion ( 22 ).…”

Monoclonal antibodies in cervical malignancy-related HPV

“…This novel therapeutic strategy for HPV-related cervical cancer is very effective, specific, and non-toxic ( 7 ). In this regard, over several decades, various forms of HPV protein antibodies, either polyclonal or monoclonal antibodies, have been designated against multiple types of HPV proteins or cancer cells’ surface proteins and stimulated pathways during the infection to improve cervical cancer diagnosis and treatment ( 17 – 19 ). For example, monoclonal antibodies have been commonly used to detect HPV16 virus-like particles (VLP) epitopes ( 20 ).…”