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longxy金蟲(chóng) (小有名氣)
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[求助]
投稿后大修，關(guān)于SPR的，求高人幫忙看看如何回答審稿人意見(jiàn)！
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實(shí)驗(yàn)中用到Biacore測(cè)抗體和抗原的親和力，文章寫(xiě)道抗體A比抗體B親和力高（A是我挑選得到的）審稿人對(duì)Biacore結(jié)果提出質(zhì)疑，以下是一些問(wèn)題，我本人對(duì)這個(gè)領(lǐng)域不熟悉，求高人能否幫忙回答一下。 Reviewer #1: The Biacore Data presented in the publication is everything but convincing for several reasons: 1.) The authors state that association and dissociation constants were calculated by using the Biacore evaluation version 4.1 software. What is missing, is the information which models have been used for the fits that are necessary to derive the kinetic data. 2.) Looking at the sensogramms CWPa in Figure 5 A. It seems that the curves for 0.36 µM and 0.71 µM as well as 1.79 µM and 1.43 µM overlap in the dissociation phase, even though different capture levels were reached (at least before correction of RI, which is not shown). How did the software deal with this? 3.) In Figure 5C two sensograms are shown at 0.36 µM here we clearly see a slower Offrate Koff for the CWP2 than for CWPa, in the Table 3 the authors claim that Koff for CWPa is 6 times slower (1,3E-3) that the Koff for CWP2 (7,34E-3). Looking at this, together with the missing fits, the large RI in all the curves the numbers presented in the table are not very convincing. 4.) The high signals of >2000 RU indicate that the target epitope is very abundant in the CWP preparation, for a monovalent scenario, even if the size of the protein is 5 kDa only >20% of the CWP preparation should be the target protein. This seems a lot. It would be nice if the authors would comment on this in the context of what is known about the target protein. Therefore, the authors should provide some more insight into the generation of the kinetic data presented in Table 3 since these Kinetic measurements are the basis for the claim that a >10 fold increase of affinity was achieved. [ Last edited by wg423 on 2011-12-25 at 19:38 ] |
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魏氏一家
新蟲(chóng) (初入文壇)
- 應(yīng)助: 0 (幼兒園)
- 金幣: 4
- 帖子: 1
- 在線: 30分鐘
- 蟲(chóng)號(hào): 1483317
- 注冊(cè): 2011-11-09
【答案】應(yīng)助回帖
longxy(金幣+5): 謝謝您的幫助！我早已經(jīng)回復(fù)過(guò)審稿人了！您的回復(fù)大多指出我試驗(yàn)的錯(cuò)誤并沒(méi)有指出如何回復(fù) 2011-11-11 14:33:05
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Answer for reviwer #1 1.) you need is: the global fit 1:1 (one to one) binding model 2.) In figure A, show different binding level (5 lines) but the dissociation see some overlap (only 3 lines). The reviwer can see they are different from unshown data. So, there is some mistake in the fitting. 3.) The CWPa Koff is quicker than CWP2, see from figure C. The large RI, means you have not have control or in line control Fc in the test. or not fit the data by deducted the refernce cell. 4.) Normally for Kd analysis, the max RU should <2000 RU |
2樓2011-11-09 10:06:09
yitianmao
新蟲(chóng) (初入文壇)
- 應(yīng)助: 0 (幼兒園)
- 帖子: 6
- 在線: 1.5小時(shí)
- 蟲(chóng)號(hào): 1364206
- 注冊(cè): 2011-08-09
| Reviewer很內(nèi)行，重做吧，我們公司也有，海歸博士專門(mén)值守，Biacore 3000，經(jīng)典機(jī)型，儀器很新，價(jià)錢(qián)也很公道。聯(lián)系Email：guanyanbin@crownbio.com |
3樓2012-04-12 14:44:35
廿四
鐵蟲(chóng) (小有名氣)
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- 專業(yè): 生物技術(shù)藥物
4樓2014-01-09 00:08:00
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