AFLATOXIN DNA ADDUCTS DETECTED BY MONOCLONAL ANTIBODIES

黄曲霉毒素B1(AFB1)是已知的最强的致癌物质之一 实验动物，以及一些流行病学研究发现 AFB1暴露与人类肝癌之间存在强烈的正相关关系。 这些数据激发了我们开发监测技术的兴趣 个体暴露于黄曲霉毒素B_1及其代谢。为了实现这一目标， 高度特异的单抗用于补体 分析测定黄曲霉毒素代谢物的分析化学方法 在生物样本中。在下面的研究中，我们建议：(1)生产 并鉴定了检测主要黄曲霉毒素DNA的单抗 加合物：AFB1-N7-GUA和AF-FAPYR。抗原产生的两种策略 将会被使用。(A)一种新的DNA加合物-肟抗原和(B)一种半抗原 由黄曲霉毒素环氧化物合成黄曲霉毒素鸟苷加合物 将尝试诱导对DNA的抗体特异性和亲和力 以前的半衰期不能得到的加合物；(2)发展 分析免疫分析和制备亲和层析方法 足以在细胞、组织和排泄物中检测黄曲霉毒素B 1及其衍生物 长期服用黄曲霉毒素B1的动物 肿瘤发生学。由于主要的AFB-N7-Gua不能提纯 排除使用32P-后标记方法，高亲和力DNA加合物 必须产生特定的抗体来检测低水平的 非放射性标记的细胞AFB-DNA损伤；以及(3)评估 (3)中开发的分析程序对相关剂量的适用性 对细胞内AFB1-DNA加合物和代谢物浓度的反应， 单次和长期给予黄曲霉毒素B_1大鼠的组织和排泄物。这些 研究将改进探索饮食与饮食之间的对应关系的方法 黄曲霉毒素摄入量与生物标志物，如排泄的黄曲霉毒素DNA 加合物，以确定这些标记是否具有必要的属性 用于非侵入性地评估人们的暴露状态。

Aflatoxin B1 (AFB1) is one of the most potent liver carcinogens known for experimental animals, and a number of epidemiological studies have found strong positive associations between AFB1 exposure and human liver cancer. These data have spurred our interest to develop technologies to monitor individual exposure to and metabolism of AFB1. To accomplish this goal, highly specific monoclonal antibodies are used with complementary analytical chemistry methods to analyze and quantify aflatoxin metabolites in biological samples. In the following study, we propose to: (1) Produce and characterize monoclonal antibodies to detect the major aflatoxin-DNA adducts: AFB1-N7-Gua and AF-FaPyr. Two strategies for antigen production will be used. (A) A novel DNA adduct-oxime antigen and (B) a hapten made from aflatoxin guanosine adduct produced by synthetic aflatoxin epoxide will be tried to induce an antibody specificity and affinity for the DNA adducts not attained by previous haptenization procedures; (2) Develop analytical immunoassays and preparative affinity chromatographic methods adequate to detect AFB1 and its derivatives in cells, tissues and excreta of animals chronically dosed with aflatoxin B1 at levels which induce tumorigenesis. Since the lability to depurination of the major AFB-N7-Gua precludes the use of 32P-post-labeling methods, high affinity DNA adduct specific antibodies must be produced for the detection of low levels of non-radiolabeled cellular AFB-DNA lesions; and (3) Evaluate the applicability of assay procedures developed in (3) for relating dose response to concentrations of AFB1-DNA adducts and metabolites in cells, tissues and excreta of rats dosed singly and chronically with AFB1. These studies will refine methods to probe the correspondence between dietary intake of aflatoxins with biological markers, such as excreted AFB-DNA adducts, to determine whether these markers have the requisite properties for non-invasive assessment of the exposure status of people.