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ELISA的方法检测多肽与大鼠血小板生成素(TPO)测定试剂盒的结合情况。准备将合成多肽(其长度大约为8~10个氨基酸)包被于ELISA板,再加入大鼠血小板生成素(TPO)测定试剂盒,一抗、酶标二抗。请问在包被的过程中应采取哪种包被缓冲液?固相载体是否要特殊处理?封闭液用BSA可以吗?
1、多肽抗原的包被一般需先使其与无关蛋白质如牛血清白蛋白质(BSA)等偶联,借助于偶联物与固相载体的吸附,间接地结合到固相载体表面。
2、包被用抗原或抗体的浓度,包被的温度和时间,包被液的pH等应根据试验的特点和材料的性质而选定。抗体和蛋白质抗原一般采用pH9.6的碳酸盐缓冲液作为稀释液,也有用pH7.2的磷酸盐缓冲液及pH7~8的Tris-HCL缓冲液作为稀释液的。通常在ELISA板孔中加入包被液后,在4-8℃冰箱中放置过夜,37℃中保温2小时被认为具有同等的包被效果。
3、zui常用的封闭剂是0.05%-0.5%的牛血清白蛋白,也有用10%的小牛血清或1%明胶作为封闭剂的。脱脂奶粉也是一种良好的封闭剂,其zui大的特点是价廉,可以高浓度使用(5%)。
显色终止后出现黑色絮状沉淀可能为包被板上的蛋白,酶标记物,检测物,封闭液中蛋白变性以及显色反应TMB过量,不溶,沉淀出来。
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