这是一篇American journal of gastroenterology的文章 Autoantibodies as Potential Biomarkers for the Early Detection of Esophageal Squamous Cell Carcinoma 主旨是若从病人身上可检验出癌症抗原(如下述P53等)对应之抗体 有助於早期诊断食道癌 实验方式是用ELISA 原文如下 Briefly, purified recombinant antigens, p53, NY-ESO-1, MMP-7, Hsp70, Prx VI, and Bmi-1, were diluted in 50 m M bicarbonate buff er (pH 9.6) to a final protein concentration of 0.1, 0.1, 0.6, 0.8, 1.5, and 0.6 μ g /ml, respsectively. Serum samples and quality control samples (a pooled plasma sample collected randomly from 100 patients with ESCC) were diluted 1:110 in blocking buffer, and then incubated at 37 ° C for 1 h, as well as were appropriate control rabbit polyclonal antibodies specific for capture proteins. After washing, horseradish peroxidase-conjugated goat anti-human IgG or anti-rabbit IgG were used as secondary antibodies at the dilution recommended by the manufacturer. After a 60-min incubation, the plates were washed, and ready prepared 3,3 ′ ,5,5 ′ - tetramethylbenzidine and hydrogen peroxide were added. Colors formation was allowed to proceed for 15 min and then stopped with 0.5 M H 2 SO 4 . The absorbance of each well was read at 450 nm within 5 min by a plate microplate reader 小弟的问题是 1. 一开始为什麽又要加入检体血清 (当然是测里头有没有抗P53蛋白抗体) 同时又要加兔子多株抗体(specific for capture proteins)呢? 2. 後来加入的secondary ab 为甚麽可以加anti-human IgG "或" anti-rabbit IgG? 另 文内的意思是说只有羊抗人 IgG有接HRP 还是抗兔IgG也有HRP? 3. 为如果血清有抗P53抗体(就是实验想看的东西)，就会接上P53蛋白质，接着用 附HRP羊抗人IgG去抓，抓到的地方显然就是实验想测的地方，但是THB在HRP作用下会变 成蓝色，反而不是实验里要看的颜色，实验里要看的是450nm应当是没有HRP作用下的 THB加酸後的颜色，换句话说就是没有人血清抗体的地方，感觉颇矛盾，是不是哪边我 误会了呢? 感谢好心大大帮我解答一下这段 尤其是第三个问题 谢谢 --

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