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Tutorials : Jun 15, 2009 (Vol. 29, No. 12)

Real Time Multiplexed Protein Measurement

Strategies to Address the Difficulties Associated with Endpoint Multiplex Assays
  • Wei Hu
  • ,
  • Jean-Francois Houle
  • ,
  • Paul Smith

The ability to measure multiple protein markers simultaneously is attractive to clinical researchers for many reasons. Combining protein markers into panels has been increasingly necessary to achieve an acceptable level of sensitivity and specificity. Specimen conservation is critical because it is often expensive and difficult to obtain well-characterized cohorts. Analytical performance may be improved as a result of reduced sample handling with the added benefit of saving time and cost.

Unfortunately, the development of robust multiplex assays using endpoint measurements is challenging. Finding a common assay format that is suitable for each protein component in terms of binding, cross reactivity, and, most importantly, detection can be difficult and time-consuming.

The assay must accommodate a single dilution factor for the sample while providing separate calibration curves for each analyte’s expected physiological range. This is particularly constraining for the broad range of protein concentrations experienced in blood, where it may become necessary to group analytes in a panel by the expected detection level and to split the sample for multiple analysis.

A new approach was recently introduced to solve these problems. The Axela dotLab® System employs real-time detection in combination with a flow-based system to simplify multiplex protein measurement. Utilizing diffractive optics technology to detect binding to discrete capture regions in disposable plastic panelPlus™ Sensors, the system individually tracks the performance of each analyte in the sample. Sequential steps of capture antibody immobilization, washing, analyte binding, and detection are monitored simultaneously, immediately highlighting any issues with cross reactivity, nonspecific interactions, or sample integrity.

This information is valuable in speeding the development of an assay and can also serve as ongoing quality control to improve performance. The need for a common endpoint is removed, allowing multiple detection methods and incubation times for analytes within the same sample.

For example, high-abundance proteins can be detected label-free in real-time during the sample binding step while an additional signal amplification step may be added for low-abundance markers. Combining formats in this fashion significantly extends the effective dynamic range, eliminating the need to split the sample and group analytes based on concentration.

The value of this approach is illustrated in Figure 1. Here, two cardiac markers that occur at widely different concentrations are measured together in a 25 µL serum sample from a research subject with an acute myocardial infarction (AMI). C-reactive protein (CRP) is normally measured between 0.2 and 20 µg/mL, while cardiac Troponin (cTn) levels may be clinically relevant anywhere between 0.01 and 100 ng/mL.

The panelPlus Sensor is automatically washed, the capture antibodies are sequentially immobilized on each analysis spot using oligonucleotide-addressing reagents, CRP binding is detected directly during serum incubation, and then cTn is measured by amplification using an alkaline phosphotase-coupled detector antibody and a precipitating substrate. The entire assay is complete within an hour of sample introduction and can be shortened for routine use.

This ability to sequentially add reagents or probe-captured analytes can be further exploited to extract more than concentration information from a sample. For example, the capture of the intact Troponin protein complex in the serum of AMI patients followed by sequential measurement of the C, T, and I subunits has been previously demonstrated. Serology assays can be designed to yield both titer and isotype distribution or avidity for each of the multiplexed antigens. A single analysis can replace as many as 20 separate traditional plate ELISA measurements.

Relevant Panels

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.