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目的制备人乳头瘤状病毒(human papillomavirus,HPV)16型E6蛋白单克隆抗体并对其进行检测。方法 15只Balb-c小鼠分为3组,各5只。设计3条HPV16E6抗原多肽分别对3组小鼠进行免疫,筛选得到对B细胞表位有较高活性的线性多肽HPE601,并用该组的小鼠脾脏制备杂交瘤细胞。采用间接ELISA法对小鼠血清进行阳性克隆筛选和检测抗体效价,采用考马斯亮蓝染色鉴定抗体纯度,采用标准曲线计算抗体浓度,采用Western blot法检测并采用间接免疫荧光法验证纯化E6抗体对Caski、Hela和正常宫颈上皮细胞核蛋白特异性结合情况。行宫颈组织活检患者20例,其中HPV16型宫颈组织7例,HPV18型宫颈组织6例和正常宫颈组织7例,采用免疫荧光法检测单克隆抗体对3种组织的检出率。结果制备得到单克隆抗体Whu001,经鉴定抗体纯度较好,该克隆能特异性识别HPV16型E6蛋白;Whu001对HPV16型组织的检出率(80%)明显高于HPV18型宫颈组织(15%)和正常宫颈组织(0),差异有统计学意义(P<0.05)。结论成功筛选出了针对HPV16E6抗原的单克隆抗体,其对HPV16的诊断具有特异性,为在蛋白水平上检测HPV E6蛋白提供了新途径。
Objective To prepare monoclonal antibody against human papillomavirus (HPV) 16 E6 and to test its expression. Methods Fifteen Balb-c mice were divided into 3 groups with 5 mice each. Three HPV16E6 antigen peptides were designed to immunize three groups of mice, respectively, and the linear peptide HPE601, which has high activity on B cell epitopes, was screened and the hybridoma cells were prepared from the mouse spleen. The positive clones were screened by ELISA and the antibody titers were detected by indirect ELISA. The purity of the antibody was identified by Coomassie brilliant blue staining. The antibody concentration was calculated by standard curve. Western blot and indirect immunofluorescence were used to confirm the purified antibody against E6 Caski, Hela and normal cervical epithelial cell nucleoprotein specific binding. 20 cases of cervical biopsy patients, including HPV16 type of cervical tissue in 7 cases, HPV18 type of cervical tissue in 6 cases and normal cervical tissue in 7 cases. The detection rate of monoclonal antibodies to three kinds of tissues by immunofluorescence method. Results The monoclonal antibody Whu001 was obtained. The purity of the identified antibody was good. The clone could specifically recognize HPV16 E6 protein. The detection rate of Whu001 to HPV16 was significantly higher than that of HPV18 (15%) And normal cervical tissue (0), the difference was statistically significant (P <0.05). Conclusion The monoclonal antibody against HPV16 E6 antigen was successfully screened, which is specific to the diagnosis of HPV16. It provides a new way to detect HPV E6 protein at the protein level.