Daduang Sakda, Sattayasai Nison, Sattayasai Jintana, Tophrom Pattara, Thammathaworn Achra, Chaveerach Arunrat, Konkchaiyaphum Monruedee
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
Anal Biochem. 2005 Jun 15;341(2):316-25. doi: 10.1016/j.ab.2005.03.037.
Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.
酶联免疫吸附测定（ELISA）已被改进，用于筛选对眼镜蛇科泰国眼镜蛇毒液具有拮抗活性的植物。研究了植物水提取物对固定在96孔微量滴定板上的抗眼镜蛇毒抗体与抗原（眼镜蛇毒）结合的抑制作用。在随后加入抗蛇毒抗体之前，让提取物中的成分与固定化毒液反应。预计受提取物影响而无法与特异性抗体相互作用的毒液成分将无法与其天然靶点或天然受体结合。莪术，一种古老的泰国药用植物，在ELISA试验中表现出明显的抑制活性。在蛋白质免疫印迹分析中，毒液中的主要成分神经毒素和蛋白质降解酶被确定为该提取物的作用靶点。在免疫沉淀竞争试验中，提取物中的成分对毒素表现出高亲和力。该提取物通过延长中毒后膈肌的收缩时间来减弱毒素活性，并具有保护细胞蛋白免受毒液降解酶作用的能力。小花莪术在ELISA中的活性较低，在两个实验中表现出可接受的结果，但在另外两个实验中结果为阴性，而姜黄在ELISA试验中活性较低，从未显示出任何良好的结果。对36个样品的筛选可将植物的抑制范围分为0至86%。这种改进的ELISA推荐作为一种对大量样品进行抑制剂初步筛选的方法。
