這是一篇American journal of gastroenterology的文章 Autoantibodies as Potential Biomarkers for the Early Detection of Esophageal Squamous Cell Carcinoma 主旨是若從病人身上可檢驗出癌症抗原(如下述P53等)對應之抗體 有助於早期診斷食道癌 實驗方式是用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 小弟的問題是 1. 一開始為什麼又要加入檢體血清 (當然是測裡頭有沒有抗P53蛋白抗體) 同時又要加兔子多株抗體(specific for capture proteins)呢? 2. 後來加入的secondary ab 為甚麼可以加anti-human IgG "或" anti-rabbit IgG? 另 文內的意思是說只有羊抗人 IgG有接HRP 還是抗兔IgG也有HRP? 3. 為如果血清有抗P53抗體(就是實驗想看的東西)，就會接上P53蛋白質，接著用 附HRP羊抗人IgG去抓，抓到的地方顯然就是實驗想測的地方，但是THB在HRP作用下會變 成藍色，反而不是實驗裡要看的顏色，實驗裡要看的是450nm應當是沒有HRP作用下的 THB加酸後的顏色，換句話說就是沒有人血清抗體的地方，感覺頗矛盾，是不是哪邊我 誤會了呢? 感謝好心大大幫我解答一下這段 尤其是第三個問題 謝謝 --

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