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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

  • 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

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

    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.

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