抗HPV L1小鼠单克隆抗体及兔多克隆抗体的制备  

Preparation of anti-HPV L1 mouse monoclonal antibody and rabbit polyclonal antibody

作　　者：孙金娥[1] 肖长义[1] 付冰冰[1] 李志英[2] 叶红[3] 黎家华[1] 王雅琴[1] 

机构地区：[1]三峡大学医学院,宜昌443002 [2]三峡大学第二临床医学院妇产科,宜昌443000 [3]三峡大学第一临床医学院妇产科,宜昌443003

出　　处：《免疫学杂志》2013年第12期1079-1083,1092,共6页Immunological Journal

基　　金：国家自然科学基金(30770104);湖北省自然科学基金(2011CDC002)

摘　　要：目的用人乳头瘤病毒主要衣壳蛋白(HPV L1)C-末端保守序列多肽制备抗HPV L1单克隆及多克隆抗体,并对所制备的抗体的检测能力进行鉴定,同时比较其与购买的单克隆抗体的检测能力。方法将HPV L1 C-末端一段30个氨基酸长度的保守序列多肽与载体蛋白KLH偶联,免疫小鼠及兔,制备抗多肽单克隆及多克隆抗体。分别采用ELISA、Western blot、免疫组织化学等方法检测所制备的多肽抗体多种型别HPV L1的反应性,并与市售的多价抗HPV L1单克隆抗体进行比较,以确定多肽抗体的检测能力。结果免疫小鼠及兔后得到的鼠单克隆抗体及兔多克隆抗体,其效价分别在1∶320 000以上及1∶64 000以上;用ELISA方法检测3种抗体对HPV阳性细胞提取蛋白及L1重组蛋白的反应性,均有阳性反应;用Western blot方法检测抗体对HPV阳性细胞提取蛋白及L1重组蛋白的反应性,均出现明显条带,但我们制备的鼠单克隆抗体与细胞提取蛋白未见明显条带;用免疫组织化学方法检测HPV阳性细胞,3种抗体均呈现阳性反应。结论 HPV L1 C-末端保守序列可以诱导产生抗HPV L1单克隆或多克隆抗体,可以检测出多型别的HPV L1,其检测能力与市售的多价抗HPV L1单克隆抗体基本相当。HPV infection plays a decisive role in cervical lesion development. Large-scale screening of HPV infection is an important method for preventing cervical cancer. In this study, we aimed to use the conservative peptide located in the carbon-terminal of human papillomavirus late gene sequence 1 （HPV L1） to prepare anti- HPV L1 monoclonal and polyclonal antibodies, then identify their immune response characteristics and detection capability, and compare the detection capability with the commercial available monoclonal antibody. We obtained the anti-HPV L1 monoclonal antibody （5A4, the titer is above 1：320 000） and polyclonal antibody （rabbit pAb, the titer is above 1：64 000） by immunizing animals with a conjugate which was merged EC31, a 30 amino acid long conservative peptide located in the carbon-terminal of HPV L1 linked a cysteine with carrier protein KLH. ELISA, Western blot and cellular immunohistochemisty were used to test the reaction of multi-type HPV L1 to the antibodies, and to identify the detection capability of the antibodies. Results of ELISA showed that antibodies could react with protein from HPV-positive cells and L1 recombinant protein; results of Western blot showed that antibodies could react with L1 recombinant protein, but 5A4 could not react with protein from HPV-positive cells; results of cellular immunohistochemisty demonstrated positive reaction with HPV-positive cells. These results suggest that the antibodies, which were prepared by the conservative peptide located in the carbon-terminal of HPV L1could be used to test multi--type HPV L1, and they have the same detection capability with commercial available muhivalent monoclonal antibody （muhivalent mAb）.

关 键 词：人乳头瘤病毒 主要衣壳蛋白 单克隆抗体 检测能力 

分 类 号：R392[医药卫生—免疫学]