Antibody reproducibility and validation

For three decades, Miltenyi Biotec has been providing high-quality biotechnological products to basic research, translational, and clinical laboratories.  In recent years, the topic of irreproducible results has become increasingly important, and antibodies have been identified as one of the main reasons for non-reliable results1-4

Because of this, Miltenyi Biotec has committed to providing high quality antibodies you can count on. So that it’s even easier for our customers to make the choice that is right for them, we have begun to share our in-house validation efforts, and are continuously extending the availability of such data to demonstrate our rigorous production, quality control, and validation standards. In doing so, we hope to give them the confidence they need to trust their precious research with our high-quality products, as well as help them minimize their own validation efforts. 

For detailed information on related content, please follow the links below: 

Antibody validation

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Antibody development, production, and quality control

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REAfinity™ Recombinant Antibodies

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Reproducibility crisis

For several years now, there has been growing concern in the scientific community regarding the reproducibility of experiments. This has been reflected in multiple publications and articles1-4, and in this context antibodies have been identified as one of the key sources of inconsistent results5.

Reliable antibodies do not only lead to reproducible results, but also save a lot of money in the long-run for several reasons: First, there is no need to buy several reagents when the initially chosen reagent performs well. Second, when using lot-to-lot consistent reagents, studies or experiments that are repeated or that run for several years will result in consistent findings, saving labor-costs, resources, and time.
Several types of antibodies exist on the market and encompass different features. Polyclonal antibodies might be beneficial for some applications, for example where the need for a stronger signal exceeds the need for lot-consistent reagents, but this is not true for flow cytometry experiments. Hybridoma-derived monoclonal antibodies have a higher lot-to-lot consistency, but are often contaminated with antibody impurities from two sources: myeloma derived–Ig light chains, and serum-derived IgGs. 
Eliminating both lot-consistency and impurity problems, recombinant antibodies represent the current best bet to tackle the reproducibility crisis6. For flow cytometry applications in particular, they provide several benefits compared to other antibody types (see table below).

Different types of antibodies

 Polyclonal antibodiesMonoclonal/Hybridoma-derived antibodiesRecombinant monoclonal antibodies
DefinitionMixture of heterogenous antibodiesMixture of homogenous antibodiesGenetically defined antibodies
FeaturesRecognize several epitopes -> stronger signalRecognize one epitope -> higher specificityDefined on DNA level, recognize one epitope -> highest specificity
How are they producedPurified from serum of immunized animalsPurified from single clones of immortal hybridoma cell linesGenetically engineered and produced in a defined expression system
Performance consistencyLow lot-to-lot consistencyHigher lot-to-lot consistencyHigher lot-to-lot consistency
Good fit for flow cytometry?-++++

Miltenyi Biotec’s methodology

As a manufacturer of antibodies, we take the topic of irreproducible results and high quality reagents very seriously, and recognize we have a responsibility to serve the scientific community with validated and consistent tools. This recognition led to our decision in 2012 to produce monoclonal recombinant antibodies instead of focusing on hybridoma-derived monoclonal antibodies. 

Portfolios based on recombinant technologies

REAfinity Recombinant Antibodies - designed for flow cytometry and imaging experiments

REAlease® Releasable Antibodies - tailored for sequential flow sorting

REAdye_lease™ Releasable Fluorochromes - designed for multiplex/cyclic immunofluorescence experiments

Resources

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Webinar
Antibody validation for reproducible flow cytometry analysis

Mona Al-Maarri highlights how our antibody validation efforts improve quality and reproducibility in flow cytometry experiments.

Biocompare documentary on reproducibility issues

Watch the video in which Biocompare examines the problem through interviews with stakeholders and presents interesting potential solutions. Find out how you can be part of the solution.

Biocompare article “Improving Antibody Selection for Flow Cytometry”

Read this article by Biocompare on selecting the right antibodies for flow cytometry experiments. The author gives insights on common challenges and provides useful tips on what to look for when deciding which reagents suit best.

References

1. Goodman, S.L. (2018) The antibody horror show: an introductory guide for the perplexed. N Biotechnol. 45: 9-13. PMID: 29355666
2. Baker, M. (2015) Reproducibility crisis: Blame it on the antibodies. Nature 521(7552): 274-6. PMID: 25993940
3. Bradbury, A. and Plückthun, A. (2015) Reproducibility: Standardize antibodies used in research. Nature 518(7537): 27-9. PMID: 25652980
4. Journals unite for reproducibility. (2014) Nature 515(7525): 7. PMID: 25373636
5. The Second Annual State of Translational Research, 2014 Survey Report, conducted by the Sigma-Aldrich Corporation. 
6. Bradbury, A.R.M. et al. (2018) When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. Mab 10(4): 539-546. PMID: 29485921