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Tutorials : Dec 1, 2009 (Vol. 29, No. 21)

"Crude Sample Analysis" Instrumentation

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

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

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.

Hybridoma Supernatants

Often, antibody-containing hybridoma supernatants have to be purified and, in some cases, labeled before off-rate determination; this is time-consuming, labor-intensive, and introduces complications such as loss of yield or aggregation. To save time and labor, we have screened unpurified hybridoma supernatants containing serum directly against an antigen on the sensor surface (referred to as a direct approach) with good results using the Attana A200 system.

Hybridoma supernatants are often screened using a capture approach, where an antibody is first captured before the antigen is injected and the antibody-antigen interaction studied. However, this approach consumes a large amount of sample and is time-consuming due to the extra antibody-capturing step. The direct approach saves both time and sample, thereby decreasing the cost of expensive antigens while increasing throughput.

A key requirement of the direct approach is that it has to be performed on surface chemistries that have low, nonspecific binding. To address this need, we have developed a low, nonspecific binding surface. A tagged antigen was immobilized on one surface, and the tag without the antigen was immobilized on the reference surface.

Hybridoma supernatants were then injected and screened directly against the antigen. The system’s response over the antigen-coupled surface is shown in Figure 3A, and the reference surface response in Figure 3B. Reference subtraction is then made to correct for bulk effects and drift (Figure 3C). Off-rate constants were subsequently determined directly from the resulting curves and are displayed either in a comprehensive list (Figure 3D) that can be printed or exported or as a report also containing graphical data of the interaction.

Screening E.coli Lysates

As with hybridoma supernatants, lysates often have to be diluted or purified before off-rate screening. To save time and labor, and to increase sensitivity, we have screened unpurified, undiluted E. coli lysates containing scaffold proteins with good results using the Attana A200 system.

Due to debris, nonspecific binding, and poor referencing, lysates are frequently  diluted and then screened using a capture approach. Diluting the samples, however,  decreases the sensitivity of the assay and introduces errors, directly affecting the results. Capturing surfaces could be an alternative, but for a variety of reasons, including the fact that they cannot be made generic since proteins, apart from antibodies, are very diverse. Again, the direct approach saves both time and sample consumption, thereby decreasing the cost of expensive antigens while increasing throughput.