H****s
发帖数: 301 | 1 There is good possibility that the epitope is a conformational epitope, not
a linear one. Swapping polypeptides might disrupt the conformational epitope. |
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t*******1
发帖数: 55 | 2 偶不是搞生物的，最近被分配一个任务协助查找具有high affinity 和non-
overlapping epitopes的抗体，这要哪里下手呀。急 |
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c*******y
发帖数: 1657 | 4 是啊,太多抗体了,只有一些特殊的才知道其epitope |
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h**********r
发帖数: 671 | 5 必须是pESC系列。
当时我还问大家要不要加kozak序列，也没说出个所以然来。后来我终于等到了客服的
答复。下面就是：
The GAL1 and GAL10 yeast promoters from the pESC-Leu vector have the FLAG or
the MYC epitopes in the two MCS of the vector. Both include ATG codons at
the 5’ end of the FLAG and the MYC epitopes. If you clone into the Bgl II
or the Xho I site in frame with the ATG of the epitope tag, you will have
the start codon, kozak sequence and should get good expression of your N-
tagged protein. If you need the epitope tag at the C-terminal, ... 阅读全帖 |
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V******1
发帖数: 8 | 6 作为一个没做过膜蛋白的伪structure biologist，赞一下专业回答。
再补充一点生物大分子的。结构可能对于大分子比如抗体的指导作用没有receptor/
ligand或者enzyme/substrate那么看起来那么直接，但也有很大帮助。比如知道target
的结构，就可以有针对性地display更感兴趣的epitope，尤其是那些non-linear的
epitope单单根据序列是没法预测的。反过来说也可以突变掉unwanted epitope来避免
screen到不想要的抗体。拿到antibody/antigen complex的结构，可以根据结构做有针
对性的affinity maturation来进一步优化抗体。design bi-specific甚至tri-
specific antibody，需要考虑几个target epitope在空间上的位置。做vaccine的，可
以根据结构来design engineer的antigen，使之更有效地产生neutralizing antibody
。还有找到一个好的抗体，patent的时候也得知道epitope的不是，虽... 阅读全帖 |
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a*******s
发帖数: 117 | 7 以下为部分论文。 若有错误，欢迎指正。
夏宁邵所在领域还是偏应用，非基础研究。 亮点是2010年的一篇柳叶刀。
2012年度：
Quantitative hepatitis B core antibody level may help predict treatment
response in chronic hepatitis B patients.
Yuan Q, Song LW, Liu CJ, Li Z, Liu PG, Huang CH, Yan Y, Ge SX, Wang YB, Peng
CY, Zhang J, Kao JH, Chen DS, Chen PJ, Xia NS.
Gut. 2012 Jun 14. [Epub ahead of print] No abstract available.
影响因子： 10.614
Hepatitis E virus: neutralizing sites, diagnosis, and protective immunity.
Zhang J, Li SW, Wu T, Zhao Q, Ng MH, Xia NS.
... 阅读全帖 |
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A***l
发帖数: 302 | 8 3. Linear or branched covalently attached T- and B-cell epitopes (Gamete
antigens).
As we have mentioned, short synthetic peptides representing B-cell
epitopes are incapable of inducing B-cells to differentiate and produce
antibody unless associated with epitopes that are recognized by helper
T-cells. The early study of the immune response to synthetic antigens has
shown that uncoupled peptides can realize their potential as vaccines if
they contain domains that react with helper T-cell receptor |
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A***l
发帖数: 302 | 9 . Discussion and conclusion
These four approaches are among the most popular approaches in the design of
synthetic peptide vaccine. Of course, they are not incompatible with each
other. For example, synthetic peptide combinatorial libraries offer a
convenient way to find epitopes, which are basic components of Gamete
antigens and MAPs. The two-epitope MAP could be looked as highly branched
covalently attached T- and B-cell epitopes. Adjuvants have been connected to
MAP to make more powerful mala |
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A***l
发帖数: 302 | 10 1. Identification of antigenic determinant peptides and synthetic peptide
combinatorial libraries
Since peptide vaccines must contain B-cell/T-cell recognized epitopes, it is
crucial to identify them. So far several methods have been used to identify
such epitopes. If the amino acid sequence of a protein antigen is known,
overlapping peptides can be synthesized and screened as targets for B cells,
MHC class I molecules/CTL cells (typically 8-10 amino acids) and MHC II
molecules/helper T cells (t |
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g***m
发帖数: 465 | 11 the principle is quite the same as DNA microarray except it
employs protein-protein interation in stead of annealing of
oligo DNA. Normally a chip coated with epitopes is used to
detect a cell flow. The opitic character will change if any
cell surfact would interact with any epitope on the chip. It
is very cool to study the surface protein interaction. Johns
Hopkins has a website and service for this tech.
It is a nascent technique as I know.
Another powerful tech for protein identification is m |
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p*****m
发帖数: 7030 | 12 特定抗原每个个体对应产生的抗体是不一样的：抗原被处理成epitope的方法不会一样；
就算epitope一样 每个人的BCR repertoire也不一样。
至于反应的差别，这个影响因素很多 未必就一定是BCR（质/量）不一样导致的 从anti
gen presentation开始之后每一步都有被调节的可能
产生 |
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k****4
发帖数: 43 | 13 即使对同一个epitope产生的抗体也不同吗？
我说的突变体不是指免疫系统整个失灵。而是说对于某个特定epitope是否
存在某些个体不产生抗体。HIV是获得性免疫缺陷，应该不是DNA产生了突变吧。
同？ |
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c********d
发帖数: 75 | 14 有个蛋白在细胞表达过程中降解很厉害，不同的条件下表达也不同。我能用ELISA来检
测该蛋白在不同条件下的相对表达总量吗？用的是anti-peptide antibody，标准曲线
只用一抗二抗。
我觉得是不太合适的。
1. 抗体不能检测到失去epitope的蛋白
2. 保留epitope的蛋白在不同大小蛋白上面向有利抗体结合的构象几率不一样
3. 不同蛋白降解产物的结合孔板的亲和性不一样。
我的理解正确吗？请大家指点！谢谢！ |
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l**********1
发帖数: 5204 | 15 Y-2-H 用多重标记 做对照和比较
比如以下的 split-luciferase+GST+HA-epitope tagged
三重标记
they found new proteins interaction paper:
//www.ncbi.nlm.nih.gov/pubmed/21530591
Protocol details:
>the wheat germ in vitro translation system for use in the splitlu
ciferase
assay and co-purifications with glutathione S transferase
(GST)- and HA-epitope tagged proteins (Fig. 2A).
>
In the split-luciferase assay, proteins of interest are fused to
N- and C-terminal fragments of luciferase. If the proteins interact,
the N... 阅读全帖 |
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C**S
发帖数: 522 | 16 wiki:
http://en.wikipedia.org/wiki/FLAG-tag
Unlike some other tags (e.g. myc, HA), where a monoclonal antibody was first
isolated against an existing protein, then the epitope was characterized
and used as a tag, the FLAG epitope was designed first, and then monoclonals
were raised to recognize it. |
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m******5
发帖数: 1383 | 17 in such case, I guess it would be sufficient to just include a 'no epitop'
group for Chip-Seq.Do peak calling against both input and non epitop.
a |
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b*****n
发帖数: 34 | 18 这两个文章结果不错，但是病人样本太小，病人可能都是精挑细选，更倾向对免疫疗法
有反应的（这个是根据以前一些一期试验的经验，没有依据）。个人对cancer vaccine
持怀疑态度，主要是因为：
1. 这个mutational load theory本身就很有瑕疵，如果看看几篇N Eng J Med的文章，
有疗效和没有疗效的患者mutational burden本身重叠部分就很大，加上现在很多被认
为是low mutational burden的肿瘤类型，对checkpoint反应率也没差太多。所以
antigen response 到了一定程度后，就是看microenvironment的抑制情况了。如果只
是作为maintenance therapy，那就回到20年前了，很难和新一代的制剂抗衡。
2. 即便是早期的cancer testis antigen vaccine, long peptide vaccine 也可以产
生类似于这个文章里的T 细胞反应，不过疗效在established tumour里微乎其微。这两
个文章也是看的复发率，大量的suppressive fact... 阅读全帖 |
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h*******r
发帖数: 182 | 19 After a full course of immunization with a new vaccine consisting a
recombinant polypeptide, 10% of adults fail to make antibody to the
polypeptide. The nonresponsers have an increased frequeny of one HLA type.
What is the most likely explannation for the failure fo theis group to
respond to immunization.
B
A. Failure of B lymphocyte to recognize the polypepetide
B. Failure of T lymphocyte to recognize the polypepetide
C. Lack of class I MHC presentation of the polypeptide
D. Lack of class II MH... 阅读全帖 |
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d****o
发帖数: 32610 | 20 那是当然
对现在的流感疫苗俺最担心的是年年这么reinforce learning最后会over train
影响俺自己免疫系统今后对抗类似结构epitope的潜力 |
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l*******g
发帖数: 28502 | 21 ☆─────────────────────────────────────☆
Jessicat (smiley cat) 于 (Fri Jul 24 23:54:56 2009, 美东) 提到:
昨天看了三藩版一篇关于后院养鸡的帖子，正好刚买房子，后院里有个现成的笼子，一
时兴起，就决定养鸡了。以下是我所作的一些研究，供有兴趣的童鞋们参考。
1. 查一下当地的zoning code，有些地区可能不让住宅区养鸡。我地区允许养，但数量
有限制，对公鸡（rooster）可能发出的噪音也有规定。
2. 到哪里去买鸡？一个方法是到当地的craigslist.com上去找，一般5到10块钱一只母
鸡，公鸡便宜，但如果你养鸡的目的是下蛋，不用公鸡也可以。二是到网上order，比
如www.mypetchicken.com上面一只小鸡才两三块钱，可以要求打疫苗。最少数量要求是
3-5只，但是邮寄需要35块钱。
3. 养什么种类的鸡？我做了一下研究，如果是下蛋为目的，以下几种可以考虑：Rhode
Island（首选，蛋又多又巨大），Star（Black Star or Red Star），A... 阅读全帖 |
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l*h
发帖数: 4124 | 22 it depends on what antigens are included in the vaccines. even for the same
bacteria, different vaccines may use different antigens or epitopes.
you will not be able to get this information from pediatricians. you will
have to talk to vaccine scientists to get the specific technical info. |
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s*********g
发帖数: 68 | 23 if you are interested and with strong background in Structure and Immunology
, send you official address and real name to my mitbbs mail-box. |
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s**********g
发帖数: 551 | 24 3分左右杂志，病毒免疫。 article "Analysis of murine B-cell Epitopes on
Eastern Equine Encephalitis Virus Glycoprotein E2"
请这方面的expert，站内联系，对自己的专业背景简单介绍。而且希望能尽早review完
，最好是一个星期。 |
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C********7
发帖数: 1035 | 25 关键词：Gold nanorod, vaccine, respiratory virus, In vitro studies using
ELISA, circular dichroism, epitopes, Human dendritic cells, T cells, immune
responses
Impact factor ~ 4
Send me your name, email, affiliation, list of publications,a couple
sentences describing your expertise. Candidate with matching background(
preference in Nano-Bio area) and earliest correspondence will be recommended
. |
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s*******n
发帖数: 10426 | 26 同正常人一样，CD4+ T cell 恢复到正常水平，功能也不受影响。
如果你非要根治，这个现在也做得到，好像5-6年前，德国的医生就可以把患者的免疫
细胞取出来，在体外清除病毒，然后回输替代，彻底根治。不过好像对患者的情况有特
殊要求，具体的记不住了，太长时间了。
不过，同样的方法也被用来治疗癌症，从患者身上取自体的CD8+ T，体外用装载
epitope的DC刺激之后回输，这些CD8+ T cell就会专门杀死癌细胞，治愈癌症。现在这
样的病例报道越来越多了。
这个如果以后能常规化，就可以实现中医理想中的“千人千方”个体化治疗，这个也是
西医发展的一个方向，不过现在看，技术上、经济上还得再等几十年.. |
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A***l
发帖数: 302 | 27 CURRENT PROBLEMS AND THE APPROACHES TO RESOLVE IT
More than 37 years ago, protein fragments of tobacco mosaic virus were
proven to elicit antibodies that neutralized the whole virus (7). Today
there are still few, if any, synthetic peptide-based vaccines commercially
available. There are a number of contributing reasons for this, among
which are the facts that: (1) the peptide sequences used to represent
epitopes are generally short and as a consequence contain insufficient
information to fold i |
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A***l
发帖数: 302 | 28 4. Multiple antigen peptide (MAP) approach
In order to avoid possible undesirable side effects of adjuvants/carriers,
another approach, MAP has been applied to the development of synthetic
vaccines. The goal of it is to design chemically unambiguous, multivalent
vaccines containing the relevant B- and T-cell epitopes but requiring no
adjuvants/carriers. This approach uses a small peptidyl core matrix bearing
radially branching synthetic peptides as dendritic arms. The simplest
design is to util |
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n*******e
发帖数: 27 | 30 hehe,//humble. that is a good question,做SEMINAR时,会答不上别人的文题时,通常
先用这招缓一缓:).我看了一下书,又得到一些INFO.首先,这些抗原都是糖蛋白,开始我
以为是由蛋白质和共价修饰它的多糖共同决定了抗原的表面决定簇EPITOPE.但是现在看
来仅仅是由一个由6个糖苷组成的多糖决定了抗原的种类.再说的明白些,O抗原是最基本
的多糖抗原,在绝大多数人体中都有(BOMBAY型没有),然后由一个糖甘转移酶(glycosyl-
transferase)转移不同的糖基至O抗原上.A型和B型的差别就在于这一个转移酶,它的基因
位于第9染色体的长臂上.这些多糖结构,在植物和微生物中也有,可以想见,如果人体把
这些自己没有的东西当成抗原来识别并产生相应的抗体,那这中抗体也能识别同样结构
的人体中的抗原.这似乎与免疫系统在长期进化中的适应性和它自身的发育有关.免异是
个生物学中最复杂,也是当前最热门的话题.象AIDS,AUTOIMMUNE都是很难解决的.为什麽
机体会产生一种抗体去识别它从来没见过的东东,为什麽不产生抗体识别自身的抗原(如
果有,人 |
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no
发帖数: 560 | 31 单抗的话，Roche的非常好，据说AbCam的单抗和多抗都不错，但价钱都很贵，多抗里面cl
ontech自己的多抗就挺不错，不过得是对GFP fulllength的那个，另一个peptide Ab效价
太低，不好用。以上的抗体在WB和IP都很好使。
但是，单抗的问题在于protein folding和epitope exposure，我们有几中GFP的融合蛋白
，蛋白折叠的构象使其不能被GFP单抗IP，甚至不能在WB中被识别，只能用多抗。 |
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w******e
发帖数: 1187 | 32 要做比如一个diagnostic kit，限制因素是做出compatible 的Ab pair吗？
现在做target 不同epitope site的Ab，有什么好方法？
多谢指点！ |
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c*********t
发帖数: 340 | 33 如果想证明A和B相互作用，在CELL LINE里表达FLAG epitope-tagged A
然后证明B也能一起被PULL DOWN
这个方法有什么缺点吗（我觉得这种大量表达某种蛋白可能会造成某些ARTEFACT？）
为什么要用FLAG TAGGED A呢
直接拿cell lysate做CoIP为什么不行呀
请达人指点下，多谢！：） |
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V***b
发帖数: 3419 | 34 鼠肝脏切片，癌症和正常，有些蛋白染的好，有些背景很大，有的染不上，有的
pattern比较奇怪，
以前没做过，所以没经验，那位给指点一下？
epitope unmask？antibody quality？什么是关键因素？ |
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V***b
发帖数: 3419 | 35 多谢各位回复。
抗体重要是肯定的。我觉得epitope unmask/retrieval也很重要，不够不行，过了估计
也不行。你们都是怎么做的？我是用买的retrieval buffer，autoclave，20 min。还
是用citrate buffer，100C，boiling？ |
|
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r********7
发帖数: 229 | 37 愿闻其详limitation：）
他们网站上介绍的优点：
http://www.ablynx.com/research/research.htm
recognising uncommon or hidden epitopes
binding into cavities or active sites of protein targets
drug format flexibility
gastro-intestinal stability
tailoring of half-life
ease and speed of drug discovery
ease of manufacture
与一般抗体相比可以识别更多的抗原位点，avidity/efficacy相似，一期临床
验证了安全性, 并且可以做到抗酸性，还可以调节half-life. 如果再来个定向
运输，再加上控制制造成本简直就无敌了。感觉就是完胜一般的单克隆抗体
呀。 |
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c*******y
发帖数: 1657 | 38 有好几个抗体想知道它们的结合位点
查了santa cruz，给出了大致的结合位点（是c还是n端）
还有些抗体santa cruz没有
要怎样才能知道呢？
难道要查很多文献吗？ |
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s******y
发帖数: 28562 | 39 打电话直接问他们的技术服务人员。就说你用了他们的抗体，需要知道这个信息
来理解一些实验结果。
不要浪费时间查文献。你在公司网站上如果查不到，文献上一般来讲也就查不到。
因为写文献的人都是你和我这样的人，并不会额外知道什么。 |
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S*****s
发帖数: 287 | 42 I think it is project dependent, and probably investigator dependent, so it
is my personal opinion here. In mammalian cells, GFP shows up as soon as it
is translated, so it can be hard to tell membrane expression from
cytoplasmic expression. In my study, the wild type protein had efficient
membrane trafficking, and mutant protein decreased trafficking efficiency.
GFP fusion protein was not able to show the weak membrane expression of the
mutant protein. When I used HA-tagged protein, I detected ... 阅读全帖 |
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E*C
发帖数: 1629 | 43 我自己画了个图，不知道能不能看清楚。
抗体XX是针对蛋白片断AD得到的。ELISA检测XX和AD结合值很高。
把这段蛋白重组成2个片段AC，BD （中间有重复片段BC）。 ELISA检测XX和这2个片段
都有结合。
把这段蛋白重组成3个片断AC，BC，CD，ELISA检测，XX和他们都不结合。
现在问题是：
1， 怎么找到最可能的抗体表位？
2， 怎么解释XX和3个片段都不结合？
各位大人请赐教 |
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W****C
发帖数: 1937 | 44 没看懂。。。是不是３个片段的那里打错了？怎么跟两个片段的一样 |
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E*C
发帖数: 1629 | 45 yes,corrected right now |
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H****s
发帖数: 301 | 46 一个比较简单的法子，把这段蛋白的基因随机片段化（最好是50-100bp大小）。然后把
片段文库克隆到噬菌体展示载体（phage display vector）上去，用你的抗体来筛。把
筛到的克隆测序，除去假阳性，然后做序列比较，（一般来说）你就可以找到抗原表位
了。 |
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E*C
发帖数: 1629 | 47 是啊,噬菌体展示可能是最好的办法了.
谁能解释一下第二个问题吗 |
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S*****s
发帖数: 287 | 48 能不能在 C 端或者 N 端带上一个 epitope tag？如果可以的话，不破细胞直接染细胞
表面蛋白，另一组样品破了细胞再染。两组一对照就知道这个蛋白是怎么表达的了。 |
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s****9
发帖数: 932 | 49 I understand it can be the problem of the antibody and will never be solved,
but want to try as hard as possible.
Could you suggest a few antigen retrieval method? Do you think 5min acetone
fixation is enough to expose the antigen epitope?
Very few background on the staining. Do you think it is worthy to try
different blocking reagents?
Thanks so much. Really appreciate your suggestions.
or |
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s****9
发帖数: 932 | 50 Thanks. I will have a try of PFA fixation with protein retrieval.
The purpose of the IHC is to in situ visualize the cell type which has the
enzyme expression(located on the ER membrane). My laser capture data
suggests that the enzyme selectively expressed on certain regions but that
regions contains many cell types. IP is not useful for me.
My concern is that the epitope is intramembrane and thus cannot expose out
after fixation.
t
if |
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