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February 01, 2010 (Vol. 30, No. 3)

Minimizing Protein-Detection Expenses

Multiplex Systems that Conserve Time, Money, and Samples Are Now Available

  • Advantages of the System

    The advantages of the Q-Plex ELISA include time, sample, and cost savings. Multiplex ELISA is an evolutionary step from a traditional ELISA involving similar steps of antigen/sample incubation, secondary or detection antibody incubation, IR fluorescent or chemiluminescent label incubation, wash steps, and plate reading resulting in a 2.5 hour assay time.

    Nonsandwich ELISAs such as competitive (a single capture antibody is placed on the plate and a labeled antigen is used that  competes with the antigen in the sample for binding with the antibody) or direct ELISAs (the antigen is on the plate rather than a capture antibody) have been designed and multiplexed that require as little as 30 minutes total assay time.

    When the time it takes to run a single multiplex ELISA is compared to the time required by multiple standard ELISAs, the time savings is apparent. Similar logic applies to sample volumes. Running 16 simultaneous ELISAs in a single well (the sample amount required for a Q-Plex ELISA is 30 µL per well) greatly reduces the amount of sample required to obtain the same results.

    While multiplexing technologies for protein detection are useful for screening which proteins are in a specific sample, a less obvious but potentially more significant use is for the establishment of protein-expression patterns. Such patterns might eventually lead to the discovery of better biomarkers, especially in circumstances where a single suitable biomarker has yet to be found.

    For example, cytokines are generally low-level transitory signaling molecules that are tightly regulated. However, they are differentially upregulated and released depending upon the stimulus. Using a mouse cytokine Q-Plex ELISA measuring 16 different cytokines and chemotactic factors researchers have found that influenza virus stimulates a rather consistent pattern of cytokine release in mice.

    The major upregulation of RANTES, interleukins 1α, 6, 12, and monocyte chemotactic protein-1 in mice following influenza virus infection is shown in the Figure, with lesser alterations in the other proteins detectable by the array. While this is far from sufficient for diagnostic purposes it does demonstrate that protein-expression patterns are elicited from a virus, and that even patterns of low-abundance transitory proteins are detectable.

  • Other Uses

    Other uses of multiplex protein-detection technologies include the reverse-phase array. Analogous to miniaturized dot blots, homogenized protein is arrayed and probed with antibodies. With reverse-phase arrays the protein can be spotted at differing concentrations and EC50 values can be calculated to determine antibody binding efficiency.

    Alternatively equal concentrations of different protein homogenates can be spotted and probed with antibodies to specific proteins, for example, tissue homogenates from cancer biopsies and healthy tissue or cell lysates from treated and untreated cells can be arrayed next to each other and examined for relative protein expression.

    There remain many areas in the still immature multiplex protein-detection market where improvement is warranted. Expanded use of nonlinear regression analysis allows for better modeling of the type of standard curves generated by antibody-based technologies, but not all of the standard curves in an array are best explained by a single model.

    As the number of standard curves generated in a protein array increases, the need for automated regression of multiple curves and determination of optimal curve fit becomes more apparent. Also, large amounts of data are generated with protein arrays and much of the sifting, prioritizing, and interpreting of the data remains in the hands of the researcher using many of the same tools that would be used to analyze standard ELISA data.

    There are few software options for protein-array analysis that have the sophistication of those used for genomic arrays offering features such as pathway and statistical analysis. These shortcomings will likely be addressed as the multiplex protein-detection market expands and bioinformaticians notice its relatively untapped potential.

    As there is no single dominant technological platform for multiplex protein detection, researchers are presented with multiple alternatives. With the establishment and proven functionality of lower cost multiplex technologies, the value of multiplex protein detection will likely be realized by more researchers. Most biological science laboratories will probably adopt multiplex protein detection, whether in their own laboratories or through the use of off-site sample testing services, within the next few years. 

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