Nagata S, Soutome H, Tsutsumi T, Hasegawa A, Sekijima M, Sugamata M, Harada K, Suganuma M, Ueno Y
Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Japan.
Nat Toxins. 1995;3(2):78-86. doi: 10.1002/nt.2620030204.
Six monoclonal antibodies (MAbs) to microcystin-LR (MCLR), a cyclic heptapeptide hepatotoxin isolated from the cyanobacterium Microcystis aeruginosa, were produced. They showed the protective effects on hepatotoxicity of MCLR in vitro and in vivo, and on the inhibition of protein phosphatase by MCLR. Competitive enzyme-linked immunosorbent assays with various microcystins revealed that the six MAbs recognized a part of the molecule, in particular, a tertial structure around Adda, 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyldeca-4,6-dienoic acid. The specificity of these MAbs varied slightly. In primary rat hepatocyte cultures, all MAbs showed protective effects against the MCLR-induced cell damages, assessed by morphological changes, lactate dehydrogenase release into the medium, and a calorimetric assay to measure the cell viability using a tetrazolium dye. The M8H5 MAb showing the highest affinity for MCLR blocked the lethal effects and hepatocellular damage to mice. In addition, M8H5 MAb recovered protein phosphatase 2A inhibition by MCLR in a dose-dependent manner, while phosphatase inhibition by okadaic acid was not affected. Thus, the MAbs specifically reacted with the microcystins and prevented their biological activities. This is the first report on the protective effects of specific monoclonal antibodies on MCLR-induced toxicity.
制备了六种针对微囊藻毒素-LR（MCLR）的单克隆抗体（MAb），微囊藻毒素-LR是一种从铜绿微囊藻中分离出的环状七肽肝毒素。它们在体外和体内均显示出对MCLR肝毒性的保护作用，以及对MCLR抑制蛋白磷酸酶的保护作用。用各种微囊藻毒素进行的竞争性酶联免疫吸附试验表明，这六种单克隆抗体识别该分子的一部分，特别是Adda（3-氨基-9-甲氧基-2,6,8-三甲基-10-苯基-4,6-癸二烯酸）周围的三级结构。这些单克隆抗体的特异性略有不同。在原代大鼠肝细胞培养物中，所有单克隆抗体均对MCLR诱导的细胞损伤表现出保护作用，通过形态学变化、培养基中乳酸脱氢酶的释放以及使用四氮唑染料测量细胞活力的比色法进行评估。对MCLR亲和力最高的M8H5单克隆抗体可阻断对小鼠的致死作用和肝细胞损伤。此外，M8H5单克隆抗体以剂量依赖性方式恢复了MCLR对蛋白磷酸酶2A的抑制作用，而冈田酸对磷酸酶的抑制作用不受影响。因此，这些单克隆抗体与微囊藻毒素特异性反应并阻止其生物活性。这是关于特异性单克隆抗体对MCLR诱导毒性的保护作用的首次报道。
