Chimeric Antigen Receptor (CAR) cell therapies continue to advance, but the high cost and lengthy timeline required to develop and characterize CAR-engineered cells remain bottlenecks.
CST® CAR linker recombinant monoclonal antibodies target the widely used G4S or Whitlow/218 peptide linker sequences in scFv-based CARs instead of the variable heavy, variable light domains, or hinge region. In doing so, the antibodies simplify the CAR characterization workflow by delivering a versatile, flow cytometry-based method that detects and confirms the presence of your CAR, regardless of its antigen specificity. To further streamline CAR detection and increase reproducibility, CST has developed Posibeads™ control beads—easy-to-use, consistent, and affordable positive controls for anti-CAR linker assays, designed to increase confidence in your CAR detection results.
Posibeads Control Beads: Confirm Your CAR Detection Experiment Is Working
Positive controls help confirm your CAR detection assay is performing as expected. You can use a known CAR+ cell line as a positive control, but maintaining the cell culture is both time-consuming and costly. Plus, you’ll need to consider that the CAR expression may change over time.
Posibeads control beads are a unique positive control for your CAR detection experiments—they are easy to use, behave like cells in suspension, and function consistently regardless of CAR composition or expression. The 5nm non-fluorescent polystyrene beads are coated with either a G4S linker peptide or a Whitlow/218 linker peptide and are intended for use as a positive control for your G4S linker or Whitlow/218 linker antibodies, respectively. The beads can be used as a standalone sample or mixed into your experimental sample, where they can undergo the same immunostaining and wash protocol as your cells. The Posibeads control beads are labeled with the anti-CAR linker antibody and serve as a positive control for both antibody binding functionality and fluorophore performance. This enables you to be confident that a lack of signal on your cells of interest is due to a lack of CAR expression, rather than an issue with your detection reagent.
When Posibeads control beads are added directly to your sample of interest, the anti-CAR linker antibody will bind to both CAR+ cells and the Posibead control beads, fluorescently labeling the CAR cells and increasing the fluorescent signal for the peptide-conjugated beads relative to unlabeled beads. The cell and bead populations can be distinguished and gated based on size differences using forward scatter (FSC) and side scatter (SSC) data.
Posibeads G4S Linker Positive Control Beads #88651 were added to a mixed population of live cells containing wild-type Jurkat cells and Jurkat cells engineered to express an scFv-based Anti-CD19 CAR containing a G4S linker. The cells and Posibeads control beads were then immunostained with either Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 or G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor® 647 Conjugate) #69782. Cells and beads were differentiated by forward scatter (FSC) and side scatter (SSC) as shown (left), and fluorescence data (FL6) are displayed for cells (blue) and Posibeads control beads (green) for both immunostaining conditions. CAR cell line was provided by the Lohmueller Lab, University of Pittsburgh.
Posibeads reagents are reproducibly generated and validated, so skewing your data interpretation due to variables in your positive control is no longer a concern.
Learn more about anti-CAR linker antibodies for Whitlow/218 and G4S
Here are three reasons why Posibeads control beads can enhance your CAR detection flow cytometry experiments:
#1 Reliable, Cost-Effective Positive Controls
Using a CAR+ cell line as a positive control for your flow cytometry experiment is not ideal, as maintaining the cultures is time–consuming and costly, and CAR expression can vary over time. Posibeads reagents are a simple, cost-effective positive control for the linker antibody conjugate that gives you confidence that your detection reagents are working as expected. Posibeads control beads integrate easily into your flow cytometry workflow and will maintain consistent signal over time. All you have to do is add a few microliters of evenly suspended beads to your sample or to an independent control tube, and then proceed with the immunostaining protocol as usual.
If you currently don’t use any positive control in your CAR detection flow cytometry experiments, adding them in is easy and won’t complicate your workflow—especially when compared to the potential time and risks associated with repeating an experiment due to uncertainty about whether a negative signal reflects poor immunostaining or the true absence of CAR+ cells in the sample.
#2 Standardize CAR Expression Across Experiments
When developing a CAR, you may explore different CAR structures to optimize cell surface expression in order to fine-tune the cellular response to your target or interest. However, comparing results can be challenging due to inter-assay variability introduced by differences in cytometer settings, instruments, operators, or testing locations. Posibeads control beads can be used to standardize the CAR+ cell fluorescence signal for each experiment, allowing for accurate data comparison across multiple experiments.
#3 Data Interpretation Confidence Even When Results Come Up Blank
Without a positive control, it can be difficult to determine whether the absence of a fluorescence signal in your flow cytometry data is due to an immunostaining technical issue or simply because there aren’t any circulating CAR-T cells or properly expressing CARs in the sample. By mixing Posibeads control beads into your sample, you can distinguish between the two possibilities, as a positive control signal confirms the linker antibody conjugate is functioning as expected. When used at the recommended amounts—20,000 beads mixed with a cell sample or 100,000 as a stand-alone sample—they do not interact with other cells and antibodies, nor do they interfere with the CAR linker labeling, ensuring you will have no trouble differentiating your CAR+ cells.
Posibeads Control Beads for Flow Cytometry Keep Your CAR Running Smoothly
As CAR-T therapies continue to evolve, new tools will be developed to enhance and accelerate their development and characterization. Posibeads control beads serve as an ideal companion to CST CAR linker antibodies, providing a reliable positive control for the immunostaining process. They help you confidently interpret your data—whether a signal is present or not—and make comparing fluorescent signal across different experiments easier and more accurate.
Learn more about CST CAR Linker products, including available conjugates and positive control beads:
Additional Resources
- Explore the CAR Signaling Networks Interactive Pathways.
- Watch the CAR-T video series to learn more about how CAR-T cells are engineered and current research challenges.
