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首页 » 临床研究 » 抗体特异性检测提议问答-国际抗体验证工作组

抗体特异性检测提议问答-国际抗体验证工作组

来源:互联网 2017-12-28 09:23


The International Working Group on Antibody Validation:
国际抗体验证工作组
Proposal for Antibody Specificity Testing Q&A
关于抗体特异性检测提议的常见问题及回答

1.What is the IWGAV?
1. IWGAV是什么?

The International Working Group on Antibody Validation (IWGAV) is an ad hoc committee of international scientists with diverse research interests, but a shared goal of improving standards for antibody use and validation. It is the first initiative of its size and scope to establish a set of standards for antibody validation for both antibody producers and users. Thermo Fisher Scientific, the world leader in serving science, provided financial support to the IWGAV in 2015 to spearhead the development of industry standards and help combat the common challenges associated with antibody specificity and reproducibility. 
国际抗体验证工作组(IWGAV)是一个专门成立的委员会,由研究兴趣各异的各国科学家组成,共同致力于优化抗体的使用和验证标准。就其规模和影响力而言,IWGAV是首家为抗体生产商和使用者制定抗体验证策略的组织。Thermo Fisher Scientific公司作为科学服务领域的世界领导者在2015年为IWGAV提供了资金支持,以推进行业标准的建立,并帮助解决抗体特异性和可重复性方面面临的挑战。

2.Who is part of the IWGAV?
2.IWGAV的成员都有谁?

Dr. Mathias Uhlen, Chair of IWGAV, Royal Institute of Technology
Mathias Uhlen博士,IWGAV主席,瑞典皇家工学院
Dr. Anita Bandrowski, University of California at San Diego
Anita Bandrowski博士,加州大学圣地亚哥分校
Dr. Steven Carr, Broad Institute?and MIT
Steven Carr博士,布洛德研究所和麻省理工学院
Dr. Aled Edwards, Structural Genomics Consortium
Aled Edwards博士,结构基因组协会
Dr. Jan Ellenberg, European Molecular Biology Laboratory
Jan Ellenberg博士,欧洲分子生物学实验室
Dr. Emma Lundberg, Royal Institute of Technology
Emma Lundberg博士,瑞典皇家工学院
Dr. David Rimm, Yale University
David Rimm博士,耶鲁大学
Dr. Henry Rodriguez, NCI and NIH
Henry Rodriguez博士,美国国家癌症研究所和美国国立卫生研究院
Dr. Michael Snyder, Stanford University
Michael Snyder博士,斯坦福大学
Dr. Tadashi Yamamoto, Niigata University
Tadashi Yamamoto博士,新泻大学

3.Why was this proposal developed?
3. 为什要出台这份提案?

The IWGAV created these recommended validation strategies with a primary focus on specificity testing to address the lack of a comprehensive framework of best practices and standardized methods in determining the validity and binder specificity of antibodies. As a result, there is a wide variation in the quality and consistency of antibodies, which is proven to have significant adverse implications for the rigor and reproducibility of biomedical research.
IWGAV主要针对抗体特异性检测而提出这些建议,以强调在确定抗体有效性和结合特异性时,缺乏一个由最佳方案和标准化方法所组成的整体框架的问题。因为缺少上述框架,导致抗体在质量和性能一致性方面存在巨大的差异,并对生物医学研究的严谨性和可重复性造成了显著的不良影响。(2018第一届中国抗体验证大会)

4.What does the IWGAV hope to achieve by developing the recommended strategies?
4. IWGAV希望通过出台这些策略而达到什么样的目标?

The primary objective of the validation strategies was to create optimal approaches, or “conceptual pillars,” for validating antibody specificity for commonly used applications. The secondary objective was to provide recommendations that ensure antibody reproducibility over time.
这些验证策略的主要目标是为常见应用建立验证抗体特异性的最优方法,或者称之为“支柱策略”。次要目标是就长期而言为确保抗体性能的可重复性提供建议。

5.What are the key “conceptual pillars”?
5. 关键的“支柱策略”是什么?

The five validation pillars in the IWGAV’s proposal include:
IWGAV提案中的五个支柱验证策略包括:
  • Genetic strategies: Measure the relevant signal in control cells or tissues in which the target gene or epitope has been knocked out or knocked down using techniques such as CRISPR/Cas or RNAi.
  • 基因策略:利用CRISPR/Cas 或RNAi等技术,将对照细胞或组织中的目标基因敲除或敲低,并检测这些对照细胞或组织中的相关信号。
  • Orthogonal strategies: Use an antibody-independent method for quantification across multitudes of samples and then examine the correlation between the antibody-based and antibody-independent quantifications. 
  • 正交策略:使用非抗体依赖方法对大量样品进行定量,并检测基于抗体和非抗体定量间的相关性。
  • Independent antibody strategies: that recognize different epitopes on the target protein and confirm specificity via quantitative comparisons.
  • 独立抗体策略:使用2种或多种可识别目标蛋白表面不同抗原决定簇的独立抗体,并通过对比或定量分析确定特异性。
  • Expression of tagged proteins: Modify the endogenous target gene to add sequences for an affinity tag or a fluorescent protein. The signal from the tagged protein can be correlated with detection through antibody-based methods.  
  • 标签蛋白的表达:为内源性目标基因添加亲和标签或荧光蛋白标签。利用抗体检测方法可对来自标签蛋白的信号进行校正。
  • Immunocapture followed by mass spectrometry (MS): Couple immunocapture, the technique of isolating a protein from a solution through binding with a target-specific antibody, with MS analysis to identify proteins that interact directly with the purified antibody (as well as proteins that may form a complex with the target protein). 
  • 免疫捕获及质谱分析(MS):免疫沉淀(IP)技术可通过使用与目标蛋白特异性结合的抗体分离出目标蛋白,其与MS分析结合(IP-MS)可识别与纯化抗体直接发生相互作用的目标蛋白以及可能与目标蛋白形成复合物的蛋白。

6.What is next step for this proposal?
6. 该提案的下一步是什么?

While this recommended proposal will serve as a foundation for developing universal antibody validation standards, the IWGAV encourages all stakeholders to provide feedback through various ongoing efforts to develop consensus guidelines for antibody use.
这份建议将作为建立通用抗体验证标准的基础,同时IWGAV鼓励所有利益相关方通过各种渠道给出反馈以便建立抗体使用的统一指南。

7.Who is this proposal for?
7. 提案的受众是谁?

The IWGAV’s proposal is designed to support the efforts of the research community, including publishers, funding agencies, antibody producers and researchers. Ultimately, the lack of a comprehensive framework of best practices and standardized methods for determining the validity of antibodies has significant adverse and costly implications for biomedical research and the advancement of science. As such, these principles are pertinent for everyone within the research community.
这份IWGAV提案的目的是支持科研相关业界,包括出版机构、资助机构、抗体生产商及研究人员。在确定抗体有效性和结合特异性时缺少一个由最佳方案和标准化方法所组成的整体框架,最终将对生物医学研究和科学进步造成显著的不良影响,且带来巨大的损失。因此,这些提议和业界每个人都有关。


8.Do you think these strategies will be widely adopted?
8. 你认为这些策略会被广泛采用吗?

Widespread adoption of the group’s comprehensive validation strategies will require continued input from the global research community, publishers, funding agencies and antibody producers.  Each of these stakeholders will have important roles to play in the adoption and implementation of this proposal. 
该组织提出的综合验证策略的广泛实施需要全球研究界、出版机构、资助机构和抗体生产商的持续投入。每个利益相关方都将在这些建议的采纳和实施过程中扮演重要角色。

9.What do you recommend antibody users do?
9. 你们对抗体使用者有何建议?

We recommend that users adhere to expanded validation testing and target identification when this data isn’t readily provided by antibody suppliers. This includes publishing as much information as possible in manuscripts, such as methods used to determine antibody specificity, catalog number, and lot number, and perhaps Resource Identifiers (RRIDs)1 to ensure research reagents such as antibodies can be unambiguously identified.
我们建议抗体使用者坚持进行抗体验证检测和抗体目标鉴定,尤其是当抗体生产商没有提供相关数据时。可以在论文中发表尽可能多的信息,例如确定抗体特异性的方法、所使用抗体的货号以及批号,以及可能的资源标识(Resource Identifiers,RRIDs)1以确保研究所用试剂如抗体可以被清晰的识别。

10.What do you recommend antibody producers do?
10. 你们建议抗体生产商如何行动?

The IWGAV recommends producers confirm antibody target binding through any number of techniques available (e.g. CRISPR, immunocapture followed by MS, tagged protein expression, siRNA, independent antibody validation and orthogonal strategies) and repeat validation experiments for each new lot to ensure that data remain current and relevant. RRIDs could also supplement the (I) catalog number and (II) lot number.1 Producers are also encouraged to provide as much additional information regarding the antibodies as possible, including antibody concentration in characterization assays, details of the immunogen used for antibody production, antibody-epitope affinity, antibody isotype, buffer formulation, and the material data sheet. When possible, the standard operating procedures should be provided, as sometimes changing an experimental parameter can alter reagent performance.
IWGAV建议生产商使用任何可用的技术(如CRISPR、免疫捕获后进行MS分析、标记蛋白表达、siRNA、独立抗体验证及正交策略)验证抗体与目标的结合,并对每一新批次的抗体重复验证以确保验证数据仍然有效。资源标识(RRIDs)也可以提供(I)货号和(II)批号1。IWGAV还建议生产商提供尽可能多的有关抗体的信息,包括鉴定实验中的抗体浓度、抗体生产所用的免疫原相关细节、抗体-抗原表位亲和力、抗体亚型、缓冲液配方以及物料数据表。由于实验参数的改变有时候会改变试剂的性能,因此尽可能实行标准操作程序。

11.What are the strategies’ potential impact on biomedical research?
11. 这些策略对于生物医学研究有哪些潜在影响?

Because antibodies can be used in different types of applications, it is important that they are tested and validated for use in each specific setting. Validation is critical to ensure that antibodies specifically recognize their intended target, perform similarly over time, and ultimately provide the same results in a given experiment for all users when used according to the appropriate experimental protocol. These validation strategies by the IWGAV are an integral first step towards validating antibody specificity and reproducibility.
抗体可以在不同类型的应用中使用,因此在特定使用背景下对抗体进行检测和验证非常重要。对抗体的验证可以确保抗体特异性识别其目标抗原、其性能不受时间影响,并且最终所有使用者都可以根据正确的实验方案得到相同的结果。 IWGAV提出的这些策略向着验证抗体特异性和可重复性迈出了坚实的一步。

Antibodies with minimal validation have the potential to deliver unreliable results that waste time, effort, materials, and money, and – most disconcertingly – may lead to incorrect scientific conclusions and critical research delays. The use of poorly characterized antibodies has been estimated to cost $350 million each year in the United States alone and $800 million worldwide as a result of failed or unreliable experiments.2
缺少充分验证的抗体可能会导致不可靠的实验结果,造成时间、精力、材料和金钱的浪费,并且最重要的是可能导致错误的科学结论以致明显的研究停滞。据估算,使用缺乏验证的抗体造成的损失,仅在美国就高达3亿五千万美元,而在世界范围内由于失败的或不可靠的结果而造成的损失甚至高达8亿美元。

12.What about other specificity determination methods (e.g., affinity measurements, DNA sequencing, isotype determination, etc.) – why were they not included in the strategies?
12. 其它特异性验证方法(如亲和测定、DNA测序、亚型确定等)为什么没有被包括在提案中?

Other methods such as affinity measurements, DNA sequencing, isotype determination, epitope mapping, adsorption, and protein arrays can yield highly useful information, but they do not directly gauge antibody specificity or cross-reactivity. Therefore, they are not recommended as primary methods to validate antibody specificity.
其它验证方法如亲和测定、DNA测序、亚型确定、抗原表位定位、吸附实验以及蛋白芯片可以产生非常有用的信息,但它们并不直接测定抗体特异性和交叉活性。因此,不推荐使用上述方法对抗体特异性进行验证。

13.How do these strategies differ from strategies being developed by other groups?
13. 这些策略和其它组织提出的策略有什么不同?

Currently, there are no universally accepted guidelines for antibody validation and specificity assessment. The IWGAV’s recommended strategies are the first of its kind to be published from an independent working group of this size and scope to establish a set of standards for antibody validation for both antibody producers and users.
目前还没有广泛被接受的抗体验证和特异性评估指南。就其规模和影响力而言,IWGAV是首家为抗体生产商和使用者制定抗体验证策略的组织。

14.Do the IWGAV strategies take specific use of the antibody into consideration when validating?
14. IWGAV的策略在进行抗体验证时考虑抗体的特殊应用了吗?

The intended use of antibodies are taken into account when different pillars are used to validate specificity for antibodies in different applications. 
当用不同策略验证不同应用中的抗体特异性时,会充分考虑该抗体的目标应用。

15.How will the IWGAV address application specific techniques in the future?
15. IWGAV未来将如何解决应用特异性技术的问题?

The IWGAV hopes to incorporate feedback from members of the community and revisit the principles to further to develop comprehensive guidelines for antibody use, either independently or through other ongoing efforts.
IWGAV希望整合来自学会成员的反馈,重新审视这些原则并进而提出针对抗体使用的综合指南,这一指南可以独立提出也可以通过其它正在进行的项目而提出。

16.What is Thermo Fisher Scientific’s relationship with the IWGAV?
16. Thermo Fisher Scientific和IWGAV之间是什么关系?

Thermo Fisher Scientific, the world leader in serving science, provided financial support to the IWGAV in 2015 to spearhead the development of industry standards and help combat the common challenges associated with antibody specificity and reproducibility.  The IWGAV is an independent group and Thermo Fisher had no active role on the IWGAV in order to preserve the group’s independent perspective.
作为科学服务领域的世界领导者,Thermo Fisher Scientific公司在2015年为IWGAV提供了资金支持,以推进行业标准的建立并帮助解决抗体特异性和可重复性方面面临的挑战。IWGAV是一个独立组织,Thermo Fisher对IWGAV保持其独立观点没有任何影响。

17.Did Thermo Fisher Scientific help to develop the strategies?
17. Thermo Fisher Scientific在策略制定过程中起了帮助作用吗?

No, the IWGAV is an independent group. The strategies were created by the members of the IWGAV.
没有。IWGAV是一个独立组织。这些策略由IWGAV的成员制定。


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