Zhang Y J, Chen C J, Haghighi B, Yang G Y, Hsieh L L, Wang L W, Santella R M
Comprehensive Cancer Center, Columbia University, New York, New York 10032.
Cancer Res. 1991 Mar 15;51(6):1720-5.
A quantitative indirect immunofluorescence technique was developed utilizing a monoclonal antibody (6A10) recognizing the imidazole ring-opened form of the major N-7 guanine adduct of aflatoxin B1 (AFB1). This method was used to investigate adduct formation in woodchuck hepatocytes treated in culture and in liver tissue of rats treated i.p. with AFB1. Fluorescein isothiocyanate-labeled secondary antiserum was used for adduct localization in conjunction with 4',6-diamidino-2-phenylindole dihydrochloride staining to localize nuclei. Quantitation of AFB1-DNA adducts was carried out by densitometric analysis of photographic slides. Specific nuclear staining was observed in both woodchuck hepatocytes and rat liver tissue. There was a dose-response relationship between fluorescence intensity and AFB1 dose in treated animals. Turnover of adducts could also be followed in animals over 48 h with this method. DNA was isolated from liver tissue of treated animals and adduct levels were quantitated by competitive enzyme-linked immunosorbent assay with antibody 6A10 and by fluorescence spectroscopy. There was a significant correlation of the quantitative immunofluorescence intensity with levels of AFB1 adducts detected by enzyme-linked immunosorbent assay (r = 0.61, P less than 0.05) and spectrofluorescence (r = 0.78, P less than 0.01). This immunohistochemical method should be applicable to the detection of adducts in liver tissues of humans exposed to high levels of dietary AFB1.
利用一种单克隆抗体（6A10）开发了一种定量间接免疫荧光技术，该抗体可识别黄曲霉毒素B1（AFB1）主要N-7鸟嘌呤加合物的咪唑环开环形式。该方法用于研究培养的土拨鼠肝细胞以及经腹腔注射AFB1处理的大鼠肝脏组织中的加合物形成情况。异硫氰酸荧光素标记的二抗与4',6-二脒基-2-苯基吲哚二盐酸盐染色结合用于加合物定位以定位细胞核。通过对照片幻灯片进行光密度分析来定量AFB1-DNA加合物。在土拨鼠肝细胞和大鼠肝脏组织中均观察到特异性核染色。在处理过的动物中，荧光强度与AFB1剂量之间存在剂量反应关系。使用该方法还可以在48小时内跟踪动物体内加合物的周转情况。从处理过的动物的肝脏组织中分离DNA，并使用抗体6A10通过竞争性酶联免疫吸附测定和荧光光谱法定量加合物水平。定量免疫荧光强度与通过酶联免疫吸附测定（r = 0.61，P <0.05）和荧光光谱法（r = 0.78，P <0.01）检测到的AFB1加合物水平具有显著相关性。这种免疫组织化学方法应适用于检测暴露于高剂量膳食AFB1的人类肝脏组织中的加合物。
