Antibody validation for improved reproducibility

For 30 years, Miltenyi Biotec has been committed to providing quality antibodies you can count on. Whether the antibodies are used for our market-leading MACS® MicroBead Technology or for flow cytometry applications, we apply rigorous production and quality control standards to make sure we deliver reliable products.

The reproducibility crisis

In the past years, there has been a growing concern in the scientific community regarding reproducibility of experiments. This has been reflected in multiple publications and articles¹^–⁴. In this context, antibodies are considered as one of the key sources of inconsistency in results.

As a manufacturer of antibodies, we take this topic very seriously and see our responsibility in serving the scientific community with validated and consistent tools.

How does Miltenyi Biotec address the need for reproducible and validated antibodies?

With the introduction of recombinant REAfinity™ Antibodies in 2012, we made a significant investment into improving quality and consistency of our REAfinity Recombinant Antibodies. The standardized antibody production process, starting from a defined DNA sequence, ensures high purity and lot-to-lot consistency.

In addition, these recombinant antibodies do not display any undesired mixtures of heavy and light immunoglobulin chains, which is often the case with conventional hybridoma-derived antibodies⁵. These advantages make REAfinity Antibodies ideal tools for improving experimental reproducibility.

Model of a REAfinity™ Recombinant Antibody

Three pillars of antibody validation

REAfinity Recombinant Antibodies are based on three pillars of validation: reproducibility, specificity, and sensitivity. Please find more detailed information for each validation method below:

1. Antibody reproducibility and consistency

Mass spectrometry analysis of purified antibodies shows that REAfinity Antibodies are defined products, while hybridoma generated antibodies can be a mixture.

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Pure antibody products

Recombinant antibodies ensure high lot-to-lot consistency as compared to traditional hybridoma technology⁵. Mass spectrometry analysis of the purified recombinant antibodies confirms the improved purity of antibody products.

Human PBMCs from a single donor were stained with CD56 antibodies conjugated to FITC, PE, APC, APC-Vio® 770, or PE-Vio® 770 from two different production lots, and analyzed on the MACSQuant® Analyzer.

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Lot-to-lot consistent performance

In addition, all antibodies are tested for lot-to-lot consistency at two stages, during the antibody raw material production as well as the fluorochrome-conjugation process.

This includes purification steps to remove unconjugated fluorochromes and antibodies from the mixture, as well as side-by-side comparisons with previous batches.

2. Antibody specificity

To validate the specific binding of REAfinity Antibodies to their target antigens, we have introduced several methods for validation of antibodies.

Epitope competition assay

In order to compare the epitope specificity of REAfinity Clones with other known clones in the market, competition assays are performed.

Cells are incubated with an excess of purified unconjugated REAfinity Antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker.

Experimental setup of an epitope competition assay.

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Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Other clones	Overlap in epitope recognition with REA196
NCAM16.2	++
TULY56	++
5.1H11	++
AF12-7H3	-
C5.9	++
N901/NKH1	++
CMSSB	+
B159	-
HCD56	-
MEM188	-

_{REAfinity Clone CD56 (REA196) was tested in a competition assay against different known clones to identify whether they recognize completely overlapping (++), partially overlapping (+), or completely different epitopes (-).}

Fluorescence microscopy image of CD53 knockout cells

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Knockout validation via targeted genome editing

For this approach, the target gene is knocked out in a suitable cell line using site specific nucleases and the knockout is confirmed by sequencing of the target locus.

The antibody is considered to bind specifically to the intended epitope, if no antibody binding to the knockout cells can be detected.

The antibody staining is controlled by fluorescence microscopy as well as flow cytometry.

Overlay histogram showing flow cytometric analysis of CD53 knockout cells

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Knockdown of the target antigen by RNAi

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RNAi knockdown

In this approach, the target antigen is knocked down using RNA interference or RNAi. The translation of the target RNA is inhibited by transfecting cells with small non-coding RNA oligonucleotides.

A comparison between the transfected cells with the control cells reveals the specificity of the tested antibody to its antigen.

3. Antibody sensitivity

Sensitivity of an antibody in an intended application is important for reliable identification of target cells. The following approaches are taken at Miltenyi Biotec to ensure that only functional and validated antibodies are part of the flow cytometry portfolio:

Functional testing of every product prior to release

All conjugated antibodies, including multiple conjugates of the same clone, are tested on primary samples. Whenever possible, antibodies are tested in multicolor panels.

In addition, antibodies are routinely tested on cells derived from tissues using enzymatic treatment. This allows for validation of antibody sensitivity to epitopes that have undergone enzymatic processing.

High staining index with REAfinity Recombinant Antibodies

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Performance comparison

We routinely compare the performance of conjugated antibodies from Miltenyi Biotec to conjugated antibodies from other vendors to ensure similar or better performance in flow cytometry applications.

Each lot is validated by a series of dilutions to make sure the product is working within the expected antibody titer range.

Comparison data for clone REA196

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Compatibility with fixation

Our antibodies are also tested for their stability during fixation. This test verifies that the epitope of an antigen can still be detected after the fixation process. Compatibility information can be found on the data sheet.

Want to learn more about our antibody validation program and how to increase experimental reproducibility?

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Selected references

Goodman, S.L. (2018) The antibody horror show: an introductory guide for the perplexed. N Biotechnol. 45: 9-13.
Baker M., (2015) Reproducibility crisis: Blame it on the antibodies. Nature 521(7552): 274-6.
Bradbury A. and Plückthun A., (2015) Reproducibility: Standardize antibodies used in research. Nature 518(7537): 27-9.
Journals unite for reproducibility. (2014) Nature 515(7525): 7.
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.