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實習醫生日記之頑固失眠

實習醫生日記之頑固失眠

今日去我院某教授跟門診,有一位中年女性患者因“反復失眠20余年”來就診。在此之前我并不知道真正意義上的熊貓眼,不過今日可真的見識到了,特拍了一張照片:

實習醫生日記之—妊娠劇吐

實習醫生日記之—妊娠劇吐

劉某,女,32歲,第一次懷孕,停經已12周。該患者停經的第九周開始出現惡心嘔吐,開始時嘔吐尚不多,3-5次每天。后來嘔吐逐漸加重,7-8次每天,嘔不能食,嘔出食物及黃膽水。

實習醫生日記之豬蹄腳

實習醫生日記之豬蹄腳

組成 黃芪10克,黨參(或太子參)10克,丹參10克,炒白術10克,薏苡仁15克,仙鶴草15克,白花蛇舌草15克,甘草5克。功能 益氣活血,健運脾胃。主治 適用于治療慢性萎縮性胃炎,或伴有腸上皮化生等

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抗體特異性檢測提議問答-國際抗體驗證工作組

字號:T|T|T
摘要:國際抗體驗證工作組關于抗體特異性檢測提議的常見問題及回答是什么
抗體特異性檢測提議問答-國際抗體驗證工作組
The International Working Group on Antibody Validation:
國際抗體驗證工作組
Proposal for Antibody Specificity Testing Q&A
關于抗體特異性檢測提議的常見問題及回答

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. 
國際抗體驗證工作組(IWGAV)是一個專門成立的委員會,由研究興趣各異的各國科學家組成,共同致力于優化抗體的使用和驗證標準。就其規模和影響力而言,IWGAV是首家為抗體生產商和使用者制定抗體驗證策略的組織。Thermo Fisher Scientific公司作為科學服務領域的世界領導者在2015年為IWGAV提供了資金支持,以推進行業標準的建立,并幫助解決抗體特異性和可重復性方面面臨的挑戰。
2.Who is part of the IWGAV?
2.IWGAV的成員都有誰?
Dr. Mathias Uhlen, Chair of IWGAV, Royal Institute of Technology
Mathias Uhlen博士,IWGAV主席,瑞典皇家工學院
Dr. Anita Bandrowski, University of California at San Diego
Anita Bandrowski博士,加州大學圣地亞哥分校
Dr. Steven Carr, Broad Institute?and MIT
Steven Carr博士,布洛德研究所和麻省理工學院
Dr. Aled Edwards, Structural Genomics Consortium
Aled Edwards博士,結構基因組協會
Dr. Jan Ellenberg, European Molecular Biology Laboratory
Jan Ellenberg博士,歐洲分子生物學實驗室
Dr. Emma Lundberg, Royal Institute of Technology
Emma Lundberg博士,瑞典皇家工學院
Dr. David Rimm, Yale University
David Rimm博士,耶魯大學
Dr. Henry Rodriguez, NCI and NIH
Henry Rodriguez博士,美國國家癌癥研究所和美國國立衛生研究院
Dr. Michael Snyder, Stanford University
Michael Snyder博士,斯坦福大學
Dr. Tadashi Yamamoto, Niigata University
Tadashi Yamamoto博士,新瀉大學

3.Why was this proposal developed?
3. 為什要出臺這份提案?
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主要針對抗體特異性檢測而提出這些建議,以強調在確定抗體有效性和結合特異性時,缺乏一個由最佳方案和標準化方法所組成的整體框架的問題。因為缺少上述框架,導致抗體在質量和性能一致性方面存在巨大的差異,并對生物醫學研究的嚴謹性和可重復性造成了顯著的不良影響。(2018第一屆中國抗體驗證大會)

4.What does the IWGAV hope to achieve by developing the recommended strategies?
4. IWGAV希望通過出臺這些策略而達到什么樣的目標?
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.
這些驗證策略的主要目標是為常見應用建立驗證抗體特異性的最優方法,或者稱之為“支柱策略”。次要目標是就長期而言為確保抗體性能的可重復性提供建議。

5.What are the key “conceptual pillars”?
5. 關鍵的“支柱策略”是什么?
The five validation pillars in the IWGAV’s proposal include:
IWGAV提案中的五個支柱驗證策略包括:
  • 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等技術,將對照細胞或組織中的目標基因敲除或敲低,并檢測這些對照細胞或組織中的相關信號。
  • 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:使用非抗體依賴方法對大量樣品進行定量,并檢測基于抗體和非抗體定量間的相關性。
  • Independent antibody strategies: that recognize different epitopes on the target protein and confirm specificity via quantitative comparisons.
  • 獨立抗體策略:使用2種或多種可識別目標蛋白表面不同抗原決定簇的獨立抗體,并通過對比或定量分析確定特異性。
  • 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.  
  • 標簽蛋白的表達:為內源性目標基因添加親和標簽或熒光蛋白標簽。利用抗體檢測方法可對來自標簽蛋白的信號進行校正。
  • 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). 
  • 免疫捕獲及質譜分析(MS):免疫沉淀(IP)技術可通過使用與目標蛋白特異性結合的抗體分離出目標蛋白,其與MS分析結合(IP-MS)可識別與純化抗體直接發生相互作用的目標蛋白以及可能與目標蛋白形成復合物的蛋白。

6.What is next step for this proposal?
6. 該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.
這份建議將作為建立通用抗體驗證標準的基礎,同時IWGAV鼓勵所有利益相關方通過各種渠道給出反饋以便建立抗體使用的統一指南。

7.Who is this proposal for?
7. 提案的受眾是誰?
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.
這份IWGAV提案的目的是支持科研相關業界,包括出版機構、資助機構、抗體生產商及研究人員。在確定抗體有效性和結合特異性時缺少一個由最佳方案和標準化方法所組成的整體框架,最終將對生物醫學研究和科學進步造成顯著的不良影響,且帶來巨大的損失。因此,這些提議和業界每個人都有關。


8.Do you think these strategies will be widely adopted?
8. 你認為這些策略會被廣泛采用嗎?
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. 
該組織提出的綜合驗證策略的廣泛實施需要全球研究界、出版機構、資助機構和抗體生產商的持續投入。每個利益相關方都將在這些建議的采納和實施過程中扮演重要角色。

9.What do you recommend antibody users do?
9. 你們對抗體使用者有何建議?
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.
我們建議抗體使用者堅持進行抗體驗證檢測和抗體目標鑒定,尤其是當抗體生產商沒有提供相關數據時。可以在論文中發表盡可能多的信息,例如確定抗體特異性的方法、所使用抗體的貨號以及批號,以及可能的資源標識(Resource Identifiers,RRIDs)1以確保研究所用試劑如抗體可以被清晰的識別。

10.What do you recommend antibody producers do?
10. 你們建議抗體生產商如何行動?
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建議生產商使用任何可用的技術(如CRISPR、免疫捕獲后進行MS分析、標記蛋白表達、siRNA、獨立抗體驗證及正交策略)驗證抗體與目標的結合,并對每一新批次的抗體重復驗證以確保驗證數據仍然有效。資源標識(RRIDs)也可以提供(I)貨號和(II)批號1。IWGAV還建議生產商提供盡可能多的有關抗體的信息,包括鑒定實驗中的抗體濃度、抗體生產所用的免疫原相關細節、抗體-抗原表位親和力、抗體亞型、緩沖液配方以及物料數據表。由于實驗參數的改變有時候會改變試劑的性能,因此盡可能實行標準操作程序。

11.What are the strategies’ potential impact on biomedical research?
11. 這些策略對于生物醫學研究有哪些潛在影響?
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.
抗體可以在不同類型的應用中使用,因此在特定使用背景下對抗體進行檢測和驗證非常重要。對抗體的驗證可以確保抗體特異性識別其目標抗原、其性能不受時間影響,并且最終所有使用者都可以根據正確的實驗方案得到相同的結果。 IWGAV提出的這些策略向著驗證抗體特異性和可重復性邁出了堅實的一步。

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
缺少充分驗證的抗體可能會導致不可靠的實驗結果,造成時間、精力、材料和金錢的浪費,并且最重要的是可能導致錯誤的科學結論以致明顯的研究停滯。據估算,使用缺乏驗證的抗體造成的損失,僅在美國就高達3億五千萬美元,而在世界范圍內由于失敗的或不可靠的結果而造成的損失甚至高達8億美元。

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. 其它特異性驗證方法(如親和測定、DNA測序、亞型確定等)為什么沒有被包括在提案中?
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.
其它驗證方法如親和測定、DNA測序、亞型確定、抗原表位定位、吸附實驗以及蛋白芯片可以產生非常有用的信息,但它們并不直接測定抗體特異性和交叉活性。因此,不推薦使用上述方法對抗體特異性進行驗證。

13.How do these strategies differ from strategies being developed by other groups?
13. 這些策略和其它組織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.
目前還沒有廣泛被接受的抗體驗證和特異性評估指南。就其規模和影響力而言,IWGAV是首家為抗體生產商和使用者制定抗體驗證策略的組織。

14.Do the IWGAV strategies take specific use of the antibody into consideration when validating?
14. IWGAV的策略在進行抗體驗證時考慮抗體的特殊應用了嗎?
The intended use of antibodies are taken into account when different pillars are used to validate specificity for antibodies in different applications. 
當用不同策略驗證不同應用中的抗體特異性時,會充分考慮該抗體的目標應用。

15.How will the IWGAV address application specific techniques in the future?
15. IWGAV未來將如何解決應用特異性技術的問題?
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希望整合來自學會成員的反饋,重新審視這些原則并進而提出針對抗體使用的綜合指南,這一指南可以獨立提出也可以通過其它正在進行的項目而提出。

16.What is Thermo Fisher Scientific’s relationship with the IWGAV?
16. Thermo Fisher Scientific和IWGAV之間是什么關系?
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.
作為科學服務領域的世界領導者,Thermo Fisher Scientific公司在2015年為IWGAV提供了資金支持,以推進行業標準的建立并幫助解決抗體特異性和可重復性方面面臨的挑戰。IWGAV是一個獨立組織,Thermo Fisher對IWGAV保持其獨立觀點沒有任何影響。

17.Did Thermo Fisher Scientific help to develop the strategies?
17. Thermo Fisher Scientific在策略制定過程中起了幫助作用嗎?
No, the IWGAV is an independent group. The strategies were created by the members of the IWGAV.
沒有。IWGAV是一個獨立組織。這些策略由IWGAV的成員制定。

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