Department of Animal Science, University of Connecticut, Storrs, CT 06268, USA.
Toxins (Basel). 2013 Mar 19;5(3):568-89. doi: 10.3390/toxins5030568.
Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.
无烟气烟草制品与口咽癌风险增加有关，部分原因是存在烟草特异性亚硝胺（TSNAs），如 4-（甲基亚硝氨基）-1-（3-吡啶基）-1-丁酮（NNK）。这些强效致癌物是在烟草烘烤过程中形成的，也是口腔中直接亚硝化反应的结果。在目前的工作中，我们描述了一种分泌高亲和力、NNK 特异性单克隆抗体的杂交瘤的分离和鉴定。合成了一种结构相关的苯甲酰衍生物，以促进与 NNK 载体蛋白的偶联，使用格里斯反应对这些载体蛋白进行了存在 N-亚硝基基团的特征分析，并用于免疫 BALB/c 小鼠。携带 NNK 特异性抗体的小鼠的脾细胞用于创建杂交瘤。在其中四个中，选择了一个进行亚克隆和鉴定。来自该克隆的大约 99%的单克隆抗体在存在游离 NNK 的情况下可从板结合的 NNKB 缀合物中竞争性置换。单克隆抗体对 NNKB 缀合物的亲和力通过表面等离子体共振确定为 Kd = 2.93 nM。游离尼古丁是 NNKB 结合位点的不良竞争物。重链和轻链抗体 F(ab)片段被克隆、测序并串联插入表达载体中，在它们之间有一个 FMDV Furin 2A 切割位点。在 HEK 293 细胞中的表达揭示了具有与亲本杂交瘤相似结合特征的功能性 F(ab)。这项研究为合成表达致癌物质隔离特性的转基因烟草奠定了基础，从而使其对消费者的危害降低。
