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  3. CAR (G4S Linker) Antigen Density Quantitation Kit (PE)

CAR (G4S Linker) Antigen Density Quantitation Kit (PE) #93513

    Product Information

    Protocol

    Product Description

    The CAR (G4S 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 G4S 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. This antibody conjugate is expected to exhibit the same reactivity as the unconjugated G4S Linker (E7O2V) Rabbit mAb #71645, which is expected to react with cell surface expressed CARs of varying specificity containing a G4S 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

    G4S Linker (E7O2V) Rabbit mAb (PE Conjugate) recognizes exogenously expressed levels of scFv-based CARs containing a G4S 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 poly-Glycine-Serine (G4S) linker is a type of flexible, unstructured synthetic peptide linker sequence often leveraged to connect 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 linker itself consists of a core pentapeptide sequence, Gly-Gly-Gly-Gly-Ser, that is repeated and commonly found as either a 15-mer (G4S)3 or 20-mer (G4S)4 within scFv-based CARs and scFv fragments. The linker sequence length plays a role in controlling scFv stability and the noncovalent association between the VH and VL domains (16,17).
    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. Huston, J.S. et al. (1988) Proc Natl Acad Sci USA 85, 5879-83.
    17. Chen, X. et al. (2013) Adv Drug Deliv Rev 65, 1357-69.

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