How does Miltenyi Biotec address the need for reproducible and validated antibodies?
With the introduction of recombinant antibodies in 2012, we made a significant investment into improving the quality and consistency of our antibodies. The standardized antibody production process, starting from a defined DNA sequence, and the nature of recombinant antibodies ensure high purity and lot-to-lot consistency.
In addition, recombinant antibodies do not display any undesired mixtures of heavy and light immunoglobulin chains, which is often the case with conventional hybridoma-derived antibodies⁵ (PMID: 29485921). Furthermore, our REAfinity™ Recombinant Antibodies have a mutated Fc region that abolishes any binding to Fcγ receptors, resulting in a background-free analysis. These advantages make REAfinity Recombinant Antibodies ideal tools for improving experimental reproducibility.
In 2020, we began the further step of providing antibody validation data directly on our product pages. We do this in order to make it even easier for our customers to choose the antibodies that best match their needs, and to decrease the validation efforts required on their researcher side. With well over 10,000 antibodies in our portfolio, this is an ongoing project and information is updated regularly, so please do check back from time to time. In addition, below you can find some insights on how we conduct our antibody validation process.
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Extended validation stamp
All our antibodies are rigorously tested and validated before release. In the application section on the product page, you can find examples of typical performance data. In addition, we provide extended validation data highlighting details of antibody performance, specificity, and fixation compatibility. All antibodies for which any of these datasets are already available will be indicated with the extended validation stamp. That list will be continuously expanded, so be sure to check back soon if you don’t see what you need yet.
Three pillars defining antibody quality and 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:
Recombinant antibodies
The nature of our REAfinity Recombinant Antibodies ensures reproducibility since they don't have any Immunoglobulin impurities and don't show background signal due to a mutated Fc region.
Highly purified antibody products
Purification of final product
After conjugation of antibodies, any unbound fluorochromes and antibodies are removed to purify the final product.
Lot-to-lot consistent performance
During development of an antibody, a suitable test to verify specificity of the clone is performed. Several approaches are possible. Below you will find a list of methods we typically perform.
Counterstaining
To validate the specificity of an antibody, a suitable counterstaining is performed, which verifies the target population.
Knockout of target protein
Epitope competition assay
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 (-).
siRNA knockdown
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Stimulation of cells
Overexpression of target protein
Binding to purified antigen (latex bead coating)
Cross-reactivity
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 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.
How to validate an antibody after purchasing?
Validation is of course not solely the responsibility of the supplier because antibody validation methods and antibody validation protocols are highly dependent on the intended application and experimental setup. For this reason, we recommend thoroughly researching the most suited validation methods for your application. Several articles have been published providing antibody validation guidelines, as well as publication guidelines. A selection is given below:
- Uhlen et al. (2016) A proposal for validation of antibodies. Nat Methods,13(10):823-7 PMID: 27595404
- STAR methods from CellPress
- Lucas et al. (2020) MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments. Cytometry A, 97(2):148-155 PMID: 31769204
- Roncador et al. (2016) The European antibody network's practical guide to finding and validating suitable antibodies for research. mAbs, 8(1), 27–36 PMID: 26418356
