[求救] paper裡實驗方法看不懂 求解惑
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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03/04 11:35, , 1F
03/04 11:35, 1F
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