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December 01, 2009 (Vol. 29, No. 21)

"Crude Sample Analysis" Instrumentation

Biosensor-Based System Bypasses Need for Purified or Highly Diluted Molecules

  • Click Image To Enlarge +
    Figure 1. The Attana 200 system is shown attached to a C-Fast pipetting robot for automation.

    Measuring the kinetic properties of biomolecules has become increasingly important in drug discovery and biomanufacturing. In areas such as immunization monitoring, clonal selection, and expression analysis, biosensors offer a clear advantage. In the past, it has been necessary to obtain purified or highly diluted molecules for such studies since crude samples have posed challenges for nonspecific binding, referencing, and system microfluidics.

    The ability to analyze crude samples directly saves time, labor, and money. In this article, we show how the Attana 200 system (Figure 1) can be used directly with crude samples for off-rate screening of antibodies in hybridoma supernatants containing serum as well as for scaffold proteins in crude E. coli lysates.

    Attana’s biosensors are based on the quartz crystal microbalance (QCM) technology. By applying an alternating potential to a piezoelectric quartz crystal, the crystal can be controlled to oscillate at its resonance frequency. A change in mass on the crystal surface results in a proportional change in resonance frequency.

    This means that when a ligand is initially immobilized on the crystal surface, the added mass can be measured in real-time without the need for labeling. The analyte is then injected, and binding of the analyte to the surface-bound ligand increases the mass further, whereupon a new shift in the resonance frequency is registered. Flowing buffer through the sensor chamber enables detection of the release of bound analyte.

    Using different surface coatings provides the option of capturing or immobilizing molecules and, accordingly, QCM can be used to study molecular interactions in real time. The QCM technology enables not only the study of biomolecules of varying species such as proteins, nucleic acids, and carbohydrates, but also of vastly different sizes, ranging from peptides to cells.

  • Clonal Selection

    Click Image To Enlarge +
    Figure 2. The clonal selection process is used in phage display as well as hybridoma screening.

    Researchers often need to select clones for expression of a biomolecule either secreted from or accumulated inside cells. Hybridoma and phage display are the two predominant techniques used to create a repertoire of clones (Figure 2). 

    The Attana 200 system can be used through the entire hybridoma screening process. It offers a better methodology than ELISA for monitoring immunization, detecting the earliest antibodies of weak affinity. It also offers a powerful tool for screening of important properties such as off-rate constants early in the process, cutting sample numbers, and saving time. In addition, it is also widely used for establishing titers, due to its low coefficient variation.

    In downstream processes such as characterization it can be used to establish specificity, kinetics, active concentration, cross reactivity, and for performing epitope mapping. Similarly, the Attana 200 system is used in the phage-display process to check specificity and correct folding of proteins in E. coli lysates after panning.

    After re-cloning into the most favorable expression system, the Attana 200 system can be used under biologically appropriate conditions such as physiological temperatures to ensure that more relevant clones are chosen for expansion early in the process.