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  3. CAR (Whitlow/218 Linker) Antigen Density Quantitation Kit (PE)

CAR (Whitlow/218 Linker) Antigen Density Quantitation Kit (PE) #33709

    Product Information

    Protocol

    Product Description

    The CAR (Whitlow/218 Linker) Antigen Density Quantitation Kit (PE) enables the quantitative analysis of chimeric antigen receptor (CAR) surface expression on cells that are engineered to express a Whitlow/218 linker-containing single-chain variable fragment (scFv)-based CAR. The kit contains bead populations surface labeled with increasing amounts of phycoerythrin (PE) calibrated in molecules of equivalent soluble fluorochrome (MESF) units. Analyzing the beads on a flow cytometer enables the generation of a calibration curve that relates instrument geometric mean or median fluorescence intensity (MFI) to standardized MESF units. Because the beads are surface labeled with the actual fluorochrome conjugated to the antibodies used to stain cells, the standards are environmentally responsive (e.g., to pH, temperature, etc.). Quantitative assignments are truly relevant when run on the same day, at the same instrument settings (e.g., PMT voltages, compensation), and in the same buffer formulation as the cell samples. The PE-conjugated antibody included in this kit was purified to have an average degree of label (DOL) of approximately 1.0.

    Assuming monovalent antibody-to-CAR binding, the MESF values after subtracting background will equate to the number of CAR molecules on the cell surface.

    This Cell Signaling Technology® antibody is conjugated to PE under optimal conditions and tested in-house for direct flow cytometric analysis in human cells. This antibody conjugate is expected to exhibit the same reactivity as the unconjugated Whitlow/218 Linker (E3U7Q) Rabbit mAb #57710, which is expected to react with cell surface expressed CARs of varying specificity containing a Whitlow/218 linker within the scFv of the extracellular domain.

    Bead Specifications

    Calibration beads are polystyrene microspheres with a mean diameter of 6-9 μm, unlabeled or labeled with increasing amounts of PE calibrated in MESF units. The beads are supplied in dropper bottles at a concentration of approximately 2 x 106 beads/mL.

    Specificity / Sensitivity

    Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) recognizes exogenously expressed levels of scFv-based CARs containing a Whitlow/218 linker.

    Species Reactivity:

    All Species Expected

    Background

    The stoichiometric relationship between surface expressed scFv-based chimeric antigen receptors (CARs) and their cognate antigens expressed on target cells is a critical determinant of the efficacy of CAR-engineered immune cells. As such, accurate measurement of CAR density on the cell surface is of paramount importance to assess how changes to CAR structure and cell engineering protocols affect CAR expression (1-15).

    The Whitlow/218 peptide is a commonly used linker that connects the variable heavy (VH) domain and variable light (VL) domain of single-chain variable fragments (scFvs) and CARs that utilize an extracellular domain scFv for target antigen recognition. The Whitlow/218 linker affords the scFv with enhanced resistance to proteolysis and aggregation (16).
    1. Gratama, J.W. et al. (1997) Cytometry 30, 10-22.
    2. Kay, S. et al. (2006) Cytometry B Clin Cytom 70, 218-26.
    3. Schwartz, A. et al. (1996) Cytometry 26, 22-31.
    4. Iannone, M.A. et al. (2003) Methods Mol Biol 226, 123-34.
    5. Mizutani, K. et al. (2007) Am J Transplant 7, 1027-31.
    6. Baerlocher, G.M. et al. (2006) Nat Protoc 1, 2365-76.
    7. Wood, J.C. and Hoffman, R.A. (1998) Cytometry 33, 256-9.
    8. Chan, K.W. et al. (2004) Anal Biochem 334, 227-33.
    9. Vaidya, S. (2008) Transplantation 85, 1046-50.
    10. Schwartz, A. and Fernández-Repollet, E. (1993) Ann N Y Acad Sci 677, 28-39.
    11. Zagursky, R.J. et al. (1995) Biotechniques 18, 504-9.
    12. Lenkei, R. and Andersson, B. (1995) J Immunol Methods 183, 267-77.
    13. Denny, T.N. et al. (1996) Cytometry 26, 265-74.
    14. Borowitz, M.J. et al. (1997) Blood 89, 3960-6.
    15. Schwartz, A. et al. (1998) Cytometry 33, 106-14.
    16. Whitlow, M. et al. (1993) Protein Eng 6, 989-95.

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    For Research Use Only. Not For Use In Diagnostic Procedures.
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