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

Abstract

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.

Extracellular Vesicle Flow Cytometry Analysis and Standardization

“…This could potentially enable comparisons between results from low resolution and high-resolution instruments. Fluorescence calibration and resolution is a relatively well-defined area in flow cytometry with many widely-accepted methods of quantification, that are discussed in detail elsewhere (Steen, 1992 ; Chase and Hoffman, 1998 ; Wood, 1998 ; Wood and Hoffman, 1998 ; Schwartz et al, 2002 , 2004 ; Graves et al, 2005 ; Hoffman, 2005 ; Kantor et al, 2016 ). Quantifying the number of antibodies (or other ligands) bound to EVs, and reporting fluorescent trigger thresholds in molecules of equivalent soluble fluorophore (MESF), rather than arbitrary units potentially provides a standardized way of EV detection using fluorescent threshold triggering, allowing readers to determine if it is likely the detected particles were EVs, and method of reporting flow cytometer settings that can be reproduced.…”

Bone Marrow Mast Cell Antibody-Targetable Cell Surface Protein Expression Profiles in Systemic Mastocytosis

“…Expression of individual markers on BMMC was assessed on gated CD117 hi CD45 int CD34 − events BMMC and expressed both as MFI values (arbitrary units scaled from 0 to 262,144) (Table 2) and SI (Figure 1). SI is the difference in signal width between the positive and the negative population divided by the signal spread of the negative population [81,82]. For each individual marker, the SI threshold for positivity was set at ≥1.5, after specifically gating on BMMC (Figure 2).…”

Basic Multicolor Flow Cytometry

“…and intracellular biomarkers can be quantified by antibodies bound per cell using hard-dyed calibration beads and biological reference calibrators (Kantor, Moore, Meehan, & Parks, 2016;Wang et al, 2016).…”