The reproducibility of published research has emerged as an urgent topic in today’s scientific community. From funding agencies to researchers to manufacturers and publishers, it is critical for all of these groups to align themselves to ensure that research is done with rigor to acheive reproducible results. BioLegend is committed to taking extensive measures to ensure our quality products meet reproducibility requirements both today and into the future.

In 2012, a landmark study conducted by Glenn Begley and his team concluded that the majority of cancer research studies were not reproducible, demonstrating that a startling 47 out of 53 published findings could not be replicated (Nature 483, 531-533; 2012). The implications for biomedical research are potentially staggering considering the billions spent on research every year, some suggesting that as much as $28 billion dollars are wasted on irreproducible pre-clinical research every year (Freedman LP, et. al. PLoS Biology 13: e1002165). While many factors are likely to be involved, research reagents, particularly antibodies used in a wide variety of applications, have been at the center of the discussion. 

In 2016, BioLegend in cooperation with key stakeholders from academia, antibody producers, pharma, funders, and journal publishers came together to discuss possible solutions for developing antibody validation standards. 

Through our rigorous quality standards and continuous improvement initiatives, we are dedicated to supporting scientific research by continuing to provide high quality, validated antibodies.

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NIH Requirements


A key element of the NIH guidelines for rigor and reproducibility includes the authentication of key biological and/or chemical resources in research grants. This includes antibodies that are key to any proposed research applications.


What can you do to fulfill these requirements? Prepare an Authentication Plan.


  • Refer to BioLegend’s datasheets that provide quality testing methods, validated applications, reported applications, usage instructions, application notes, publications using the reagent, and other relevant data.
  • Inquire with us about quality control testing data for the specific lot of product that you have and obtain a Certificate of Analysis.
  • Ensure that the lot of product that you have (or will obtain) does not expire before the end of your research plan.
  • Consider your own methods for validating the reagent using orthogonal methods in addition to BioLegend’s quality testing.
  • Consider additional controls, such as knockdown/knockout cells, positive and negative cells, or transfected cells.
  • Consider using Veri-Cells™ lyophilized control cells as targets to ensure consistent performance of your antibodies over time.
  • If using a commonly sold antibody clone, consider testing across multiple manufacturers to ensure consistent expected performance.

The points above should serve as a roadmap to a successful plan for authentication of your proposed reagents. Learn more about Steps to Improve Your Reproducibility. Also read some FAQs provided by the NIH.




Watch Biocompare’s documentary on Antibody Manufacturing Demystified, featuring Craig Monell, our VP of Business Operations.

New Antibody Development

BioLegend spends a considerable amount of effort in creating new antibodies for research. The majority of these new antibody products are monoclonal antibodies (mAb) produced from hybridomas. Clones of these hybridomas are carefully selected based on a number of criteria including robust growth and efficient production of a single clone of antibody that is specific to the intended target. The best clones move on to applications testing.

As manufacturers are starting to produce recombinant antibodies, these would need to be validated and addressed in the same ways as hybridoma-derived antibodies. A mAb produced from an engineered DNA source is not inherently more specific than one produced from a traditional hybridoma. However, genetic modifications to the overall structure could potentially convey improved performance in assays.

Applications Testing

All newly developed clones at BioLegend undergo validation testing for multiple applications. This serves as a cross-check for specificity and provides clarity for research uses. Typically, antibodies are tested by two or more of the below methods.

As an example, our newly developed clone, 13A3-1, for phosphorylated STAT3 (Tyr705) demonstrated excellent performance in flow cytometry, western blot, and chromatin immunoprecipitation. Thus, the clone cross-validates itself, by demonstrating functionality across orthogonal testing methods. Additionally, the biological induction of the phosphorylated state using IL-6 further validates the specificity of the antibody.

Clone 13A3-1 Application Validation

Clone 13A3-1 Application Validation

Gene Knockdown Testing

Knockout or knockdown of gene expression, such as with siRNA, is also an excellent tool for target validation. Our SIRT5 antibody, clone O91G9 was verified by western blot using HeLa cells treated with SIRT5 targeting siRNA. Lane 1 indicates untreated HeLa cells, lane 2 contains scrambled siRNA control treated cells, and lane 3 contains SIRT5-specific siRNA treated cells.

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BioLegend antibodies undergo an extensive series of testing to ensure quality at every step in the manufacturing process, as well as maintaining quality after the sale.


Maintaining lot-to-lot consistency is vital for reproducibility. It simply is not enough for antibody manufacturers to validate antibodies just once. Pass/fail specification requirements are essential for quality control testing of every lot of product. BioLegend maintains records for all lot-specific testing, so inquire with our Technical Support if you would like to see the data. 

An ISO 13485:2016 certified quality management system, which meets the requirements for medical device manufacturers, means that processes are well-defined, adhered to, and undergoing constant improvements. It also ensures that there is a robust system for customer notifications in case of non-conforming products. BioLegend’s 100% satisfaction guarantee provides peace of mind for researchers, so that they can be confident in the results every time.

Researchers are central to the endeavors of maintaining research reproducibility. Here are some steps you can take today to improve reproducibility for the long term.


  • When finding commercial antibody products, confirm the methods of validation and quality control. Is every lot of product tested? How is it tested? Ask for data if needed.
  • Is the manufacturer well known and well respected in the research industry? Ask colleagues who might be familiar. Biocompare's Antibody Market Report is a good resource.
  • Does the reagent manufacturer have a product guarantee?
  • Does the reagent manufacturer have a defined quality management system? Look for ISO certification to ensure that the manufacturer meets international standards.
  • Is the antibody product published and peer reviewed? Look for third party independent reviews like those on or
  • Contact technical support before buying if you have any questions.
  • If you're not familiar with a technique, get trained on it by a qualified expert before you start. BioLegend and other companies can also provide applications training as needed.


  • When starting with a new antibody reagent, verify that it is the most appropriate product for your application. Most antibodies don't work across all applications.
  • Regardless of the manufacturer's validation, confirm that the reagent works for your application. Some ideas are below:
    • Cross-compare antibodies from multiple manufacturers.
    • Confirm specificity by orthogonal methods, ie. use ELISA to confirm that your flow cytometry anti-IL-2 antibody is correctly recognizing IL-2.
    • Always use positive and negative controls, such as isotype controls for flow cytometry. Also, consider validating with positive and negative cell lines where you would expect the target to be expressed or not expressed.
    • Perform titrations of the antibody to verify that your antibody binding is dosage-specific.
    • Use knockout or knockdown cell lines to confirm specificity.


  • When new products come into the lab, document the manufacturer, catalog numbers, and lot numbers.
  • Always document dilutions or concentrations of reagents used in assays.
  • When publishing, always indicate the manufacturer and catalog numbers or RRIDs of products used.
  • Submit supplemental data whenever possible.

External Resources


BioLegend Libraries


Publications certainly contribute to the validity of a research product. BioLegend provides an extensive library of publications citing BioLegend products. The library is updated monthly with hundreds of new citations each month. According to a study by Pivotal Scientific, BioLegend had the largest percent increase in citations among all companies with 10,000+ citations, between 2010 and 2014, and again led the way in citation growth between 2015 and 2016.

Browse the Publications…


In addition to seeing published data, you can also find first-hand customer reviews of our products. Search results can also be filtered to view reviewed products. Our reviews library displays a brief protocol and data for each submitted review. Where necessary, our technical team comments on the application. The reviews library also includes reviews of BioLegend products from other websites such as Biocompare, OneDegreeBio, and AntibodyResource.

Committed to Functional Quality


BioLegend embraces all methods of antibody production without losing focus on what matters most: the functional quality of our end product. We understand that the “reproducibility crisis” cannot be solved by improving just a single aspect of the antibody pipeline, which is why we are committed to having the highest quality standards for all stages of production.


Learn about the approaches we take to eliminate variation, and how our validation process ensures consistent quality of all our antibodies:

Recombinant antibodies

Recombinant DNA allows for full control of the antibody protein, which makes it easier to create antibody fragments, switch antibody classes, and minimize nonspecific binding.


Hybridoma-derived antibodies

Hybdridomas are not genetically engineered, but can be screened to exclude antibodies with high binding variability, e.g. gene/transcript sequencing can identify clones producing multiple light chains.


Downstream stages of production. A 2019 market survey found that many scientists believe recombinant antibodies have improved specificity relative to their hybridoma-derived counterparts. Controlling the protein sequence is one potential method to limiting variability, but other downstream procedures can affect all antibodies. This is why BioLegend performs rigorous structural and functional validation of each antibody batch as the final step, regardless of its source.


Representative validation data


Structural validation ensures antibody purity



HPLC analysis of anti-mouse CD8a hybridoma antibody (clone 53-6.7) (left) and anti-mouse CD8a recombinant antibody (clone QA17A07) (right) post-SEC purification.


Functional validation ensures consistent lot-to-lot performance


Alexa Fluor® 647 anti-human Granzyme B Hybridoma Antibody

Two lot comparison of Alexa Fluor® 647 anti-human Granzyme B hybridoma antibody (clone GB11) staining on human peripheral blood lymphocytes. Cells were co-stained with FITC anti-human CD8 (clone RPA-T8).


PE anti-human CD56 (NCAM) Recombinant Antibody

Two lot comparison of PE anti-human CD56 (NCAM) recombinant antibody (clone QA17A16) staining on human peripheral blood lymphocytes (different blood donor than above). Cells were co-stained with APC anti-human CD16 (clone 3G8).



Two lot comparison (red, black lines) of PE/Cyanine7 anti-human CD66b hybridoma antibody (clone G10F5) staining on human peripheral blood granulocytes. Dotted line indicates isotype control  (Mouse IgM, κ) staining.

Two lot comparison (red, black lines) of PE/Cyanine7 anti-human CD66b recombinant antibody (clone QA17A51) staining on human peripheral blood granulocytes. Dotted line indicates isotype control  (Mouse IgG1, κ) staining.

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