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

Laser-Scanning Fluorescence Microplate Cytometer Aims to Improve upon Results

The study of antibodies has been one of the focal points in biology and medicine for over 20 years. Monoclonal antibodies now constitute the most rapidly growing class of human therapeutics and have profoundly modified the treatment of a number of indications including cancer and autoimmune diseases.

One of the most established methods for the production of monoclonal antibodies is through the generation of myeloid myeloma cells lines, although more recently the selected lymphocyte antibody method has also become increasingly popular. One problem associated with these techniques is the rapid and accurate identification of the resultant antibodies. The screening of cell-culture supernatants for specific antibodies can be a tedious, time-consuming process and especially problematic when screening for low-abundance antigens, e.g., cell-surface proteins.

Traditionally, ELISA has been used for antibody screening but with numerous wash and incubation steps these assays are also time-consuming. Quantification of cell-surface proteins is also difficult using this method, primarily due to their low abundance and dependence on cellular expression.

Homogenenous mix-and-read assays overcome these problems. These simplified protocols involve the simultaneous addition of all assay constituents to one well, with the analysis performed once equilibrium has been reached, making these assays rapid, robust, and suitable for automation.

Automated mix-and-read assays for antibody screening were originally enabled by a cellular detection instrument that was based on fluorometric microvolume assay technology. Although this instrument has been discontinued, there remains a requirement for a system that is able to perform high-throughput, robust assays for antibody discovery.

Accelerating Antibody Discovery

TTP LabTech’s Mirrorball™ is a new laser-scanning fluorescence microplate cytometer. The high-performance, low-loss optics enable this system to perform high-sensitivity, mix-and-read assays for applications such as antibody screening. Its simultaneous scanning functionality permits higher throughput, single-pass scanning that allows direct correlation of data across lasers; this results in better multiplexing and analytical capabilities.

The laser-scatter channel permits identification of objects with little or no fluorescence, unlike most laser-scanning cytometers that utilize the fluorescence of an object for both recognition and assay readout. Combining independent object recognition with the concurrent collection of up to four channels of fluorescence data gives improved sensitivity in multiplexed assays and this is advantageous when using mix-and-read protocols.

This combination also enables both beads of different sizes, and beads with different fluorescence encoding to be detected at the same time resulting in many levels of multiplexing (Figure 1). These properties facilitate the detection of antigens by reducing the number of false negatives from wells by using bead count.

The versatility of the Mirrorball system permits both bead- and cell-based assays, as demonstrated by the following two applications.

Figure 1. Label-free object recognition of beads coupled with simultaneous reporting of multicolor fluorescence detection enable high levels of multiplexing on Mirrorball.

Bead-Based Assays

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 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.

Figure 2. Fluorescence intensity and bead count for a bead-based sandwich immunoassay for rabbit IgG

Cell-Based Assays

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

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.


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.

Wayne Bowen, Ph.D. ([email protected]), is CSO, and Joanne Franklin, Ph.D., is a science writer at TTP LabTech.

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