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.

中文翻译：

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两种新型抗HPV16 E7癌蛋白单克隆抗体的制备，表征和诊断评价。

目前，人乳头瘤病毒（HPV）的临床检测方法主要基于PCR方法。但是，该方法只能用于检测HPV DNA和HPV类型，而不能用于准确预测宫颈癌。HPV16 E7是在宫颈癌中选择性表达的癌蛋白。在这项研究中，我们通过原核表达制备了HPV16 E7-组氨酸（HIS）融合癌蛋白，并使用杂交瘤技术获得了几种小鼠抗HPV16 E7-HIS融合癌蛋白单克隆抗体（mAb）。鉴定出两个单克隆抗体69E2（IgG2a）和79A11（IgM）。免疫细胞化学，免疫荧光，免疫组织化学和蛋白质印迹被用来表征这些单克隆抗体的特异性。69E2和79A11抗体的核苷酸碱基和预测氨基酸序列表明它们是新颖的抗体。带有重叠肽段的间接酶联免疫吸附测定（ELISA），间接竞争ELISA和3D结构建模显示，单克隆抗体69E2和79A11特异性结合HPV16 E7的三个暴露肽段（HPV16 E749-66，HPV16 E773-85和HPV16 E791-97）。我们使用这两种抗体（79A11作为捕获抗体，69E2作为检测抗体），基于辣根过氧化物酶（HRP）标记的mAb和四甲基联苯胺（TMB）检测系统建立了双抗体夹心ELISA，用于定量检测HPV16然而，E7-HIS融合癌蛋白并不理想。然后，我们基于标记的抗生蛋白链菌素-生物素（LSAB）-ELISA方法和鲁米诺检测系统建立了化学发光免疫分析法，该方法足以定量检测ng水平的HPV16 E7-HIS融合致癌蛋白，并且适合于检测HPV16阳性宫颈癌组织。总体上，我们获得了两种新型的小鼠抗HPV16 E7癌蛋白单克隆抗体，并建立了用于检测HPV16 E7-HIS融合致癌蛋白的LSAB-lumino-双抗体夹心ELISA方法，这可能是诊断HPV16的有前途的方法型宫颈癌处于早期阶段。