Quantitative Flow Cytometric Analysis of Membrane Antigen Expression

D'Hautcourt, Jean-Luc

doi:10.1002/0471142956.cy0612s22

Cited by 11 publications

(8 citation statements)

References 21 publications

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“…The ALK48 antibody was conjugated using a Dylight 650 Antibody Labeling Kit (Thermo Fisher Scientific). Staining for ALK expression on tumor lines was performed using methods described above, and ALK expression was quantified using the same antibody with the Quantum Simply Cellular kit 62 (Bangs Laboratories).…”

Section: Antibodies and Flow Cytometry Analysesmentioning

confidence: 99%

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase

et al. 2017

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We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.

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Section: Antibodies and Flow Cytometry Analysesmentioning

confidence: 99%

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase

et al. 2017

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“…Multiple methods for titrating antibodies, usually on cells, have been published and are referenced here (Kantor and Roederer, 1996;Stewart and Stewart, 2001;D'hautcourt, 2002 andHulspas, 2010). Specific recommendations are present here.…”

Section: Preliminary Reagent Titrationsmentioning

confidence: 99%

A Quantitative Method for Comparing the Brightness of Antibody‐dye Reagents and Estimating Antibodies Bound per Cell

et al. 2016

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We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

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“…To allow inter-laboratory comparisons, calibrated reference beads are available to standardize procedures. The individual steps of quantitation experiments have been standardized and guidelines are available (Schwartz and Fernandez-Repollet, 2001; D'hautcourt, 2002; Schwartz et al , 2004; Mittag and Tarnok, 2009). …”

Section: Introductionmentioning

confidence: 99%

Quantification of cell surface proteins with bispecific antibodies

Panke

Weininger

Haas

et al. 2013

Protein Engineering Design and Selection

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Flow cytometry is an established method for fast and accurate quantitation of cellular protein levels and requires fluorescently labeled antibodies as well as calibration standards. A critical step for quantitation remains the production of suitable detection antibodies with a precisely defined ratio of antigen-binding sites to fluorophores. Problems often arise as a consequence of inefficient and unspecific labeling which can influence antibody properties. In addition, the number of incorporated fluorophores necessitates a special normalization step for quantitation. To address these problems, we constructed different mono- and bivalent bispecific antibodies with binding site(s) for the cell surface antigens, cMET, EGFR1/HER1, ErbB2/HER2 or ErbB3/HER3 and with an additional digoxigenin-binding single-chain Fv fusion. The fluorophore Cy5 was covalently coupled to digoxigenin and quantitatively bound by the bispecific antibody. A panel of tumor cell lines was assessed under different culture conditions for absolute receptor expression levels of the indicated antigens and the data were set in relation to mRNA, gene count and immunoblot data. We could reproducibly quantify these receptors, omit the otherwise required normalization step and demonstrate the superiority of a 1 + 1 bispecific antibody. The same antibodies were also used to quantify the number of proteins in intracellular vesicles in confocal microscopy. The antibodies can be stored like regular antibodies and can be coupled with different digoxigenin-labeled fluorophores which makes them excellent tools for FACS and imaging-based experiments.

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Quantitative Flow Cytometric Analysis of Membrane Antigen Expression

Cited by 11 publications

References 21 publications

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase

A Quantitative Method for Comparing the Brightness of Antibody‐dye Reagents and Estimating Antibodies Bound per Cell

Quantification of cell surface proteins with bispecific antibodies

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