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June 15, 2010 (Vol. 30, No. 12)

Mix-and-Read Assays for Antibody Discovery

Laser-Scanning Fluorescence Microplate Cytometer Aims to Improve upon Results

  • Bead-Based Assays

    Click Image To Enlarge +
    Figure 2. Fluorescence intensity and bead count for a bead-based sandwich immunoassay for rabbit IgG

    Mirrorball enables a mix-and-read, bead-based assay for screening antigens. One application of this is in the screening for antibodies raised against soluble antigens. To demonstrate this capability a bead-based sandwich immunoassay for rabbit IgG was established.

    Beads coated with antirabbit Fc antibody were mixed with a range of rabbit IgG concentrations and Cy5-labeled anti-rabbit F(ab´)2 antibody. After a period of incubation, plates were scanned using Mirrorball and bead-bound fluorescence quantified. In addition, beads were identified using laser scatter.

    High sensitivity detection of rabbit IgG was achieved with a limit of detection of <1 ng/mL rabbit IgG (Figure 2). A reduction in bead fluorescence was observed at concentrations above 150 ng/mL due to the well-documented “hook effect”. The variability of assay replicates was low (4% CV) suggesting stable measurement of bead fluorescence despite a solution background of 0.67 nM AlexaFluor® 647 antibody conjugate.

    Independent recognition of beads using label-free scatter detection permitted detection of beads in all wells, including zero control wells. This capability improves detection sensitivity and eliminates false negatives from wells containing reduced bead number.

  • Cell-Based Assays

    Click Image To Enlarge +
    Figure 3. Dual-laser scanning: whole wells of the emitted fluorescence were created from the raw PMT readings and pseudocolored. Top: cells were stained with live cell stain DiO and scanned with a 488 nm laser. Bottom: corresponding data for anti-EGFR antibody simultaneously detected using 640 nm laser excitation.

    Homogeneous mix-and-read assays are routinely used for antibody screening against cell-surface receptors. To demonstrate the capability of the Mirrorball for performing these assays, we established an assay for human epidermal growth factor receptor (EGFR) binding in A549 cells, which are known to express high levels of EGFR.

    A549 cells were incubated with mouse anti-EGFR antibody and AlexaFluor 647 labeled antimouse IgG antibody. To allow detection of all cells they were labeled with Vybrant™ DiO (Invitrogen), a lipophilic tracer ideal for labeling cell membranes. Cells were labeled with 30 nM DiO by adding the dye to a suspension of cells/anti-mouse IgG AlexaFluor 647 solution.

    Without washing away unbound antibodies, plates were scanned using Mirrorball. Cell identification (DiO) and the amount of anti-EGFR labeling (AlexaFluor 647 fluorescence) were determined by scanning the whole well concurrently with 488 nm and 640 nm lasers. Whole well images of the emitted fluorescence were created from the raw PMT readings (Figure 3).

    Simultaneous scanning demonstrated that the cell count was reasonably stable across the concentration range of the antibody and that anti-EGFR antibody binding sensitivity was <5 ng/mL.

  • Conclusion

    This tutorial demonstrates that Mirrorball is ideally suited for use with mix-and-read assays for the discovery of antibodies. The versatility of this instrument enables cell-based assays that can be performed on live or dead cells, in adherent or suspension cultures. The ability to perform bead-based assays permits the detection of soluble antigens with high sensitivity. Independent object recognition means that the user can be confident that false negatives are no longer a problem and the simultaneous scanning enables superior multiplexing for high-throughput, robust data generation.

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