HRP标记的二抗检测heme结合蛋白造成假阳性问题的探讨

为了验证对辣根过氧化物酶(horseradish peroxidase, HRP)作为二抗标记物在检测heme结合蛋白时产生假阳性问题的推测,以菠菜细胞色素b_6f复合体(Cyt b_6f)和铁氧还蛋白-NADP~+氧化还原酶(FNR)为检测对象(前者含有heme结合蛋白Cyt f,后者不含heme基团),采用菠菜FNR一抗、HRP和碱性磷酸酶(alkaline phosphatase,ALP)标记的二抗,设计对比实验。结果表明,在使用HRP标记的二抗体系中,Cyt f不经一抗和二抗孵育即可显色,这是明显的假阳性;而在ALP标记的二抗体系中,Cyt f条带不显色,只有FNR条带显色。在western blot实验中,如果被检测蛋白为heme结合蛋白或者混有少量heme结合蛋白,应避免采用HRP标记的二抗,建议使用ALP标记的二抗或者其他二抗产品;heme结合蛋白具有类似HRP的氧化还原酶活性,其本身可以和显色液反应,从而产生假阳性,干扰对实验结果的判断。

Study on the false positive problem caused by HRP-labeled secondary antibody in detecting heme binding protein

In order to study the false positive problem during detecting heme binding protein with horseradish peroxidase (HRP) as secondary antibody, spinach cytochrome WTBX]b₆fWTBZ] complex (Cyt WTBX]b₆fWTBZ]) and ferredoxin-NADP⁺ oxidoreductase (FNR) are used as the test material (the former contains the heme binding protein Cyt f, and the latter does not contain heme group). A comparative experiment is designed through the use of the primary antibody of spinach FNR and secondary antibodies labeled with HRP and alkaline phosphatase (ALP). The results indicate that in the HRP-labeled secondary antibody system, Cyt f could be colored even without the incubation by the primary antibody and the secondary antibody, which is a significant false positive. In the ALP-labeled secondary antibody system, the Cyt f doesn''t show color, and only the FNR is colored. In western blot experiments, if the protein to be detected is a heme-binding protein or contains a small amount of heme-binding protein, HRP-labeled secondary antibodies should be avoided and ALP-labeled secondary antibodies or other secondary antibodies should be recommended. It is speculated that the heme-binding protein has an HRP-like oxidoreductase activity, namely it can react with the color developing solution, thereby generating a false positive and interfering with the judgment of the experimental results.