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June 15, 2009 (Vol. 29, No. 12)

Real Time Multiplexed Protein Measurement

Strategies to Address the Difficulties Associated with Endpoint Multiplex Assays

  • Relevant Panels

    Click Image To Enlarge +
    Figure 2. Use of panelPlus Sensor reagents to create a tumor marker research panel

    Researchers developing new multiplex protein assays need the ability to easily customize panel content to accommodate the specific clinical question under study and the ability to incorporate novel biomarkers with existing clinically relevant assays to speed the validation process. To greatly simplify these requirements, the panelPlus Sensors for the dotLab System uses a unique approach to addressing reagents.

    Each assay location within the sensor is provided precoupled with a unique oligonucleotide-addressing reagent. Capture reagents are supplied pre-addressed with the complementary oligonucleotide sequence. When introduced to the sensors, they rapidly hybridize to their location without cross reacting to other spots. Simple conjugation kits allow users to address their own affinity reagents as required. This approach gives complete flexibility in designing panels and is made practical by the removal of endpoint restrictions on the assays.

    Figure 2 illustrates the use of these self-addressing reagents to create a custom triplex tumor marker panel. If desired, the sensors can be regenerated by chemical disruption of the address pair and automatically replaced with a fresh capture surface. This eliminates the need to optimize regeneration for each assay and maximizes analytical performance. 

  • Conclusion

    The combination of a flow-based system with real-time detection has been shown to significantly shorten the development time and complexity of creating multiplex protein assays. The removal of endpoint restrictions can provide extended dynamic range and allows the generation of significant secondary information from each analyte in the multiplex panel. Sample consumption, handling, time to result, and cost are all reduced with this approach, representing a significant advantage to the clinical researcher.

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