Groopman J D, Trudel L J, Donahue P R, Marshak-Rothstein A, Wogan G N
Proc Natl Acad Sci U S A. 1984 Dec;81(24):7728-31. doi: 10.1073/pnas.81.24.7728.
Monoclonal antibodies specific for aflatoxin B1, aflatoxin B2, aflatoxin M1, and the major aflatoxin-DNA adducts were obtained following fusion of mouse SP-2 myeloma cells with spleen cells of mice immunized with aflatoxin B1 covalently bound to bovine gamma globulin. The aflatoxin-modified protein used to immunize mice was produced chemically by activating aflatoxin B1 to a 2,3-epoxide derivative, which then covalently bound to the protein. One of the monoclonal antibodies isolated (2B11) was found to be a high-affinity IgM antibody with an affinity constant for aflatoxin B1, aflatoxin B2, and aflatoxin M1 of about 1 X 10(9) liters per mol. In a competitive radioimmunoassay using [3H]aflatoxin B1, 3 pmol (1 ng) of aflatoxin B1, aflatoxin B2, or aflatoxin M1 caused 50% inhibition with this antibody. The antibody also had significant cross-reactivity for the major aflatoxin-DNA adducts: 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and 2,3-dihydro-2-(N5-formyl-2',5', 6'-triamino-4'oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1. The antibody was also covalently bound to Sepharose-4B and used in a column-based solid-phase immunosorbent assay system. Aflatoxins added in vitro to phosphate buffer, human urine, human serum, or human milk at levels expected to be obtained in human samples acquired from environmentally exposed individuals were quantitatively recovered by applying the mixture to this antibody affinity column purification system. Preliminary studies using urine samples from rats injected with radiolabeled aflatoxin B1 have also indicated that aflatoxin metabolites can be isolated by these methods. Furthermore, we have found that the monoclonal antibody affinity columns can be regenerated for multiple use. Therefore, the monoclonal antibodies and their application to affinity chromatography represents a useful and rapid technique to purify environmentally occurring levels of this carcinogen and some of its metabolites for quantitative measurements.
将小鼠SP - 2骨髓瘤细胞与用共价结合牛γ球蛋白的黄曲霉毒素B1免疫的小鼠脾细胞融合后，获得了对黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素M1以及主要黄曲霉毒素 - DNA加合物具有特异性的单克隆抗体。用于免疫小鼠的黄曲霉毒素修饰蛋白是通过将黄曲霉毒素B1化学活化成2,3 - 环氧化物衍生物，然后使其与该蛋白共价结合而产生的。分离得到的一种单克隆抗体（2B11）被发现是一种高亲和力的IgM抗体，对黄曲霉毒素B1、黄曲霉毒素B2和黄曲霉毒素M1的亲和力常数约为1×10⁹升/摩尔。在使用[³H]黄曲霉毒素B1的竞争性放射免疫分析中，3皮摩尔（1纳克）的黄曲霉毒素B1、黄曲霉毒素B2或黄曲霉毒素M1会导致该抗体产生50%的抑制。该抗体对主要黄曲霉毒素 - DNA加合物也有显著的交叉反应性：2,3 - 二氢 - 2 - （N7 - 鸟嘌呤基） - 3 - 羟基黄曲霉毒素B1和2,3 - 二氢 - 2 - （N5 - 甲酰基 - 2',5',6' - 三氨基 - 4' - 氧代 - N5 - 嘧啶基） - 3 - 羟基黄曲霉毒素B1。该抗体还与琼脂糖 - 4B共价结合，并用于基于柱的固相免疫吸附分析系统。将体外添加到磷酸盐缓冲液、人尿、人血清或人乳中的黄曲霉毒素以预期从环境暴露个体采集的人类样本中获得的水平进行添加，通过将混合物应用于该抗体亲和柱纯化系统可对其进行定量回收。使用注射了放射性标记黄曲霉毒素B1的大鼠尿液样本进行的初步研究也表明，黄曲霉毒素代谢物可通过这些方法分离。此外，我们发现单克隆抗体亲和柱可以再生并多次使用。因此，单克隆抗体及其在亲和色谱中的应用代表了一种有用且快速的技术，可用于纯化环境中该致癌物及其一些代谢物的含量以进行定量测量。
