The Latest on Antibody Validation

As a researcher in the life sciences, antibodies are your main tools used in experiments. Reliable and reproducible data can be generated when high-quality primary antibodies are properly used.  To be of “high quality” antibodies should be validated by methods that demonstrate the following antibody properties:

  • Sensitivity – “on target” binding properties
  • Specificity – “off target” binding properties  
  • Reproducibility – produced to ensure lot-to-lot consistency. 

Our highest priority has always been the quality and performance of antibodies produced by Rockland.  By trusting your experiments to us, your data will reflect increased antibody specificity, low background staining, increased assay sensitivity, reduced incubation times and you will see consistent results from lot-to-lot. 



August 2018 | Benefits of Polyclonal Antibodies


Dr. Carl Ascoli, Chief Science Officer at Rockland, and Dr. Birte Aggeler, Director of Antibody Development at Bio-Techne, composed a peer-reviewed article using their combined experience of more than 50 years. The review article was published online on August 3, 2018 and will be printed in the September 2018 issue by the journal BioTechniques. The review article, entitled “The Overlooked Benefits of Polyclonal Antibodies,” discusses the pros and cons of polyclonal, conventional monoclonal, and recombinant monoclonal antibodies, while presenting procedures for experimental design, the inclusion of relevant controls, and validation strategies for polyclonal antibodies.


View the full press release here



June 2018 | Committee on Reproducibility in Science


NAS logo

The National Academy of Science and Medicine acting on a Congressional mandate commenced activities in October of 2017 to explore the issues of reproducibility and replication in scientific and engineering research. By forming a committee composed of 15 subject matter experts inclusive of members of the National Academies, issues affecting reproducibility in science are to be explored, with conclusions and recommendations to be made to Congress in early 2019. This committee has held open sessions to hear testimony of experts from diverse disciplines including physical and earth sciences, industry perspectives, and economics. The industry perspective for life science research tools was presented to the committee by Dr. Carl Ascoli, Chief Science Officer of Rockland Immunochemicals.


Six open session meetings are planned for the committee. During the third meeting held on April 18, 2018, testimony to the committee focused on the following causes of irreproducibility: (1) biases in experimental design, (2) biases in statistical analysis, (3) inaccuracies of standards and constants, and (4) variability of life science research tools, specifically biological reagents. Biological reagents include cell lines, antibodies and animal models.


Ascoli testified to the committee that “despite published claims of the lack of specificity of antibodies the clear majority of research performed using antibodies results in the collection of high quality data sufficient to support and reproduce scientific claims." 


For instance, the pace at which new treatments have accelerated from the bench to the clinic has resulted in tremendous breakthroughs in medicine, and antibodies have been central to the discovery, diagnosis and cure of many diseases that just a decade ago were considered to have very poor patient outcomes. Antibodies, unlike any other life science research tool, are saving lives in ways that were not possible before the development and application of this new technology.


Never-the-less, leading antibody manufacturers and resellers, along with funding agencies and some journals, are embracing change rather than the status quo. For instance: (1) better use of standards and controls can be made when collecting experimental data; (2) the ‘knowledge gap” that has resulted from outsourcing antibody technology can be addressed; (3) funding agencies can require precise description of reagents and methods to foster reproducibility; (4) journals can publish all necessary details of reagents and protocols critical for reproducibility; and (5) antibody manufacturers and resellers can adopt immunoassay specific validation guidelines for validation and provide greater transparency when describing antibody production, screening methods and release criteria.


Ascoli and others are currently working on antibody validation guidelines to be published in early 2019.



View the full press release here.



September 2017 | IWGAV "A Proposal for Validation of Antibodies"


A recently published report of the International Working Group on Antibody Validation (IWGAV) titled A Proposal for Validation of Antibodies established five conceptual pillars for assay specific antibody validation.  A framework for immunoassay specific validation guidelines was discussed at a subsequent international meeting of key opinion leaders held at Asilomar, California in 2016. Most recently the National Academy of Sciences commenced activities to explore issues of reproducibility. Rockland Immunochemicals supports these concepts and is actively working with others to establish guidelines for antibody validation that can be applied globally.



June 2017 | Reproducibility Issues in Life Science Research: Antibody Validation Challenges Documentary


In this Biocompare documentary, researchers discuss what is being done in their labs to improve reproducibility, what’s being done by the manufacturers to decrease lot-to-lot variation, what’s being demanded by journals and funding agencies to ensure that certain standards are adhered to, and finally what role organizations and societies are playing to disseminate information and increase awareness of the problem:

Related content: Click here to read Biocompare's Q&A with Rockland's CSO, Carl Ascoli, Ph.D.


February 2017 | Antibody Guidelines


We propose some basic guidelines for how researchers use antibodies. Key questions must be answered by researchers on the intended outcome of antibody based experiments. These issues should be resolved before experimental work commences, ideally before an antibody is produced or purchased. If these guidelines are followed the likelihood of obtaining valuable, reproducible data dramatically increases. 






January 2017 | The Antibody Crisis

Transparency, shared responsibility and partnership are essential elements of any plan to standardize antibody validation if the goal is to affect researchers worldwide. A 2017 article in Biocompare titled The Antibody Crisis: An Ongoing Discussion engaged thought leaders from around the globe including Rockland's CSO, Dr. Carl Ascoli who stated, “It is important for transparency that the nature of the immunogen, how an antibody was screened during development and specific details pertaining to lot-specific validation are communicated." Appropriately transparent data should be provided by the manufacturer to the user to allow replication of data and to gain confidence in antibody performance.    



December 2016 | GBSI Antibody Validation Workshop




The Global Biological Standards Institute (GBSI) and The Antibody Society recently organized the Antibody Validation: Standards, Policies, and Practices workshop that included more than 100 key stakeholders from academia, antibody producers, pharma, funders, and journals to share perspectives and contribute to tangible solutions for validating antibodies. A workshop report was issued by GBSI that summarized the conceptual framework agreed upon at the meeting for affecting change including a call for detailed assay-specific antibody validation standards.


 profile-carl   Rockland's CSO, Dr. Carl Ascoli spoke as a panelist during the Producers and Service Providers in a Pivotal Role: QC/QA and Certification discussion. 



Additional Information:

Who should validate antibodies?  


All manufacturers should validate all antibodies by at least a minimum criteria. Antibody validation data should be lot specific and the data should be communicated to the researcher in the form of a datasheet or certificate of analysis, which would accompany each unit of product shipped to the researcher. Data should demonstrate transparency and include information regarding the nature of the immunogen, how the antibody was screened during development and the final release validation data. It should also include properties like appropriate dilutions for use.


Validation data should be lot specific and assay specific. Researchers should carefully review provided information and, when necessary, independently validate an antibody before starting with research to accommodate any changes in buffers used, equipment or other parameters specific to your laboratory.




 Antibody Application   Rockland scientists validate the performance of antibodies by assays commonly used by most researchers including ELISA, western blotting and immunohistochemistry.

Many Rockland products are validated in a variety of assays and are guaranteed to work as per the applications stated on the data sheet. 




  Antibody Cross-Reactivity   Rockland antibodies are tested at every stage of production using positive and negative controls to ensure the antibody targets the right protein. 

Rockland quality control standards require the highest possible specificity for antibodies. Reactivity to other species is listed on the data sheet.



 Antibody Variability   Rockland manufacturing processes deliver unsurpassed repeatability in antibody production and minimal lot-to-lot variation.

Rockland adheres to QSR/cGMP quality standards (CFR: 21H part 820) with full reporting and traceability. A product data sheet is provided with each order.

Assay-specific Methods for Validating Antibodies


Validating an antibody is not the same as characterizing an antibody.  A data image of an antibody used in an immunoassay with or without controls could be described as characterizing an antibody.  But validating an antibody means that you have determined the antibody’s sensitivity for the target, its specificity for off target binding and its reproducibility, especially to demonstrate that the antibody’s properties are consistent from lot-to-lot.


Legitimate methods for validating antibodies are immunoassay specific, meaning that the methods used for Western blotting would not necessarily apply to other applications such as ELISA, immunoprecipitation or immunohistochemistry. 

Selected Readings: Antibody Quality and Validation

  • Bordeaux, J., Welsh, A.W., Agarwal, S., Killiam, E., Baquero, M.T., Hanna, J.A., Anagnostou, V.K, and Rimm, D.L. Antibody ValidationBiotechniques. 2010 Mar; 48(3): 197–209.
  • M. Uhlen, A. Bandrowski, S. Carr, A. Edwards, J. Ellenberg, E. Lundberg, D. L. Rimm, H. Rodriguez, T. Hiltke, M. Snyder, T. Yamamoto.  A proposal for Antibody ValidationNature  Methods 13:10; 823-827.


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