Antibody validation

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.

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. 

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.

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.  

Knockdown of target antigens can be used to validate antibody specificity and their use in flow cytometry applications.

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. 

To be able to verify specificity of an antibody, it is often necessary to stimulate cells to ensure that the target antigen is expressed.

Jurkat cells were either left unstimulated (left peak) or stimulated with 5 mM H₂O₂ for 15 minutes. Cells were then fixed and permeabilized using the Cell Signaling Buffer Set A followed by intracellular staining with Anti-Syk pY348 antibodies. Flow cytometry was performed using the MACSQuant® Analyzer. Dead cells and cell debris were excluded from the analysis based on scatter signals

To verify specific recognition of human Lgr5 by clone 22H2.8, we transfected cell lines showing no intrinsic Lgr5 expression to generate cells that expressed this protein stably. In addition, cell lines that expressed human Lgr4 or Lgr6 stably were generated to prove that the Lgr5 antibodies do not cross-react with close homologs. Using the cell lines that expressed Lgr5 stably, high-affinity binding to Lgr5 was confirmed. Using the cell lines that expressed human Lgr4 or Lgr6 stably, we demonstrated that the antibodies are specific for Lgr5 and do not cross-react with the close homologs. As a reference, another commercially available antibody was analyzed using the same settings.

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. 

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 product page and within the application section and the extended validation tab.