ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF VERSICOLORIN-A AND RELATED AFLATOXIN BIOSYNTHETIC PRECURSORS
ENZYME-LINKED-IMMUNOSORBENT-ASSAY OF VERSICOLORIN-A AND RELATED AFLATOXIN BIOSYNTHETIC PRECURSORS
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DOI：
10.4315/0362-028x-54.2.105
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发表时间：
1991-02-01
影响因子：
2
通讯作者：
PESTKA, JJ
中科院分区：
文献类型：
--
作者：
REYNOLDS, G;PESTKA, JJ
介绍了一种免疫化学方法，用于检测寄生曲霉培养物中杂色蛋白(VA)和其他黄曲霉毒素前体。采用半制备高效液相色谱法从寄生曲霉ATCC 36537培养物中分离纯化出Va，并用质谱仪和紫外吸收光谱进行了确证。为了获得免疫原性，VA被转化为半缩醛，并通过还原烷基化连接到牛血清白蛋白(BSA)。以VA半缩醛-辣根过氧化物酶结合物为标记配体，制备兔抗VA半缩醛-BSA结合物多克隆抗血清，用于竞争ELISA。根据竞争性酶联免疫吸附试验中抑制结合所需的VA类似物的量，与VA的交叉反应性分别为：VA半缩醛、阿维菌素、阿魏酸苷、去甲龙酸、阿维菌素、杂色曲霉毒素和黄曲霉毒素B1分别为106、85、7、6、2和1%。用该酶联免疫吸附试验检测了寄生曲霉ATCC 36537在一种改进的培养程序中提高了VA等价物的产量。VA抗体在黄曲霉毒素生物合成的生化和遗传学研究中是非常有用的。
An immunochemical approach is described for the detection of versicolorin (VA) and other aflatoxin precursors in Aspergillus parasiticus cultures. VA was purified from A. parasiticus ATCC 36537 cultures by semipreparative high performance liquid chromatography and confirmed by mass spectrometry and ultraviolet (UV) absorption. To be rendered immunogenic, VA was converted to a hemiacetal and conjugated to bovine serum albumin (BSA) by reductive alkylation. Rabbit polyclonal antiserum prepared against the VA hemiacetal-BSA conjugate was employed in a competitive ELISA using VA hemiacetal-horseradish peroxidase conjugate as the marker ligand. Based on the amount of VA analogue required to inhibit binding by 50% in competitive ELISA, cross-reactivity relative to VA for VA hemiacetal, averufanin, averantin, norsolorinic acid, averufin, sterigmatocystin, and aflatoxin B1 were 106, 85, 7, 6, 2, < 1, and < 1%, respectively. The ELISA was used to monitor enhanced production of VA equivalents by A. parasiticus ATCC 36537 in a modified culture procedure. The VA antibody should be extremely useful in the biochemical and genetic investigation of aflatoxin biosynthesis.