Liu Yong-sheng, Ding Yao-zhong, Zhang Jie, Chen Hao-tai, Zhu Xiao-ling, Cai Xue-peng, Liu Xiang-tao, Xie Qing-ge
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
Hybridoma (Larchmt). 2010 Feb;29(1):37-43. doi: 10.1089/hyb.2009.0058.
Monoclonal antibodies (MAbs) against prion protein (PrP) are powerful tools for diagnosis and research in transmissible spongiform encephalopathies. Ten MAbs to recombinant/native cellular PrP (PrPc) in mammals were prepared with a simple method and identified in detail. Normal BALB/c mice were immunized with the recombinant bovine mature PrP (rbomPrP) and PrP27-30 (rboPrP27-30) expressed in Escherichia coli. The immunized splenocytes were fused with SP2/0 mouse myeloma cells, and positive hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA). The characterizations of these MAbs, such as Ig, Ig subclass, titer, affinity index, specificity, epitopes recognized, and binding to recombinant/native PrPc of cattle, sheep, or human beings, were evaluated by Western blotting and indirect or sandwich ELISA. Ten MAbs could be divided into five groups depending on the results of indirect ELISA additivity test and their reaction to E. coli-expressed truncated-PrPs. Isotyping of the MAbs revealed that they belong to IgG1, IgG2a, and IgG2b subclass. Their indirect ELISA titers were between 10(3) and 10(6). Affinity constants were between 10(9) and 10(12) M(-1). Ten MAbs specifically reacted with the rbomPrP, without binding to prion-like protein Doppel and the lysates of E. coli. These MAbs could also respond to the recombinant mature PrP (rmPrP) of sheep and human beings. Also of interest was the ability of the MAbs to bind with dimer of rmPrP and PrP extracted from the brain tissue of cattle or sheep. We conclude that anti-PrP MAbs successfully prepared with a simple method could potentially be useful in mammalian prion research.
抗朊病毒蛋白（PrP）的单克隆抗体（MAbs）是传染性海绵状脑病诊断和研究的有力工具。采用一种简单方法制备了针对哺乳动物重组/天然细胞PrP（PrPc）的10种单克隆抗体，并进行了详细鉴定。用在大肠杆菌中表达的重组牛成熟PrP（rbomPrP）和PrP27-30（rboPrP27-30）免疫正常BALB/c小鼠。将免疫后的脾细胞与SP2/0小鼠骨髓瘤细胞融合，通过间接酶联免疫吸附测定（ELISA）筛选出阳性杂交瘤。通过蛋白质印迹法以及间接或夹心ELISA对这些单克隆抗体的特性进行评估，包括免疫球蛋白（Ig）、Ig亚类、效价、亲和指数、特异性、识别的表位以及与牛、羊或人重组/天然PrPc的结合情况。根据间接ELISA加和性试验结果及其对大肠杆菌表达的截短型PrP的反应，10种单克隆抗体可分为五组。单克隆抗体的亚型鉴定显示它们属于IgG1、IgG2a和IgG2b亚类。它们的间接ELISA效价在10³至10⁶之间。亲和常数在10⁹至10¹² M⁻¹之间。10种单克隆抗体与rbomPrP特异性反应，不与朊病毒样蛋白多普蛋白（Doppel）和大肠杆菌裂解物结合。这些单克隆抗体也能与绵羊和人的重组成熟PrP（rmPrP）反应。同样有趣的是，这些单克隆抗体能够与rmPrP二聚体以及从牛或羊脑组织中提取的PrP结合。我们得出结论，用简单方法成功制备的抗PrP单克隆抗体可能对哺乳动物朊病毒研究有用。
