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October 15, 2011 (Vol. 31, No. 18)

Amplifying Immunoassay Sensitivity

Microfluidics-Based Microplate Technology Offers Benefits Over Traditional ELISA Methods

  • Basic Comparison to Standard ELISA

    When single aliquots of samples containing analyte are added to the Optimiser plate, detection and sensitivity performance, defined as a linear calibration curve slope, are similar to ELISAs. This analytical performance is suitable for many applications quantifying analyte in the pg/mL range. The benefit of using the Optimiser microplate for these applications is a significant reduction in reagent and sample consumption. Additionally, as wash steps are reduced, so is overall assay time, and sample throughput may be increased.

  • Increased Sensitivity

    Click Image To Enlarge +
    Figure 3. Human IL-4 assay in Optimiser ELISA microplate and sensitivity gains, defined as the slope of the calibration curves, is attributable to multiple sample loadings using the Optimiser plate.

    Some applications require higher sensitivity than standard ELISA detection limits. For example, cytokine secretions may be locally produced in high concentrations for autocrine and paracrine signaling, but in the supernatant of a microplate well, this concentration is greatly reduced and requires extremely sensitive detection.

    For these applications, and others where the sample is dilute or at a very low concentration, the Optimiser technology and flow-through principle allow multiple repeat samples to be added per well to increase detection sensitivity.

    To demonstrate this ability, interleukin-4 (IL-4) was used as an analyte and single concentrations spiked into cell culture media to simulate samples. These samples were loaded onto the Optimiser plate once at 10 μL, 20 times at 5 μL, and 100 times at 5 μL to construct calibration curves. The fluorescent-based assay was read on BioTek’s FLx800™ Fluorescence Microplate Reader with 528/20 nm excitation and 590/35 emission settings.

    Left shifting of the calibration curves evident in Figure 3 implies increased analyte sensitivity. Each reagent addition, including sample, requires a five-minute incubation, so the overall assay time for a sample loaded once onto the plate is approximately 90 minutes. Samples loaded 20 times increase the overall assay time to approximately 3 hours, and samples loaded 100 times increase the overall assay time to approximately 10 hours.

    The single addition of 10 μL of sample provides an operating range of 1–125 pg/mL, which is typical of a conventional ELISA-based assay. Yet this single load reduces the sample volume by 10-fold relative to a standard ELISA while retaining the ELISA’s operating range. Multiple small sample loads can significantly increase the sensitivity 10- to 100-fold and provide detection to fg/mL levels as shown in the Figure 3 table insert.

    The novel microfluidics in the Optimiser microplate offers tangible benefits over traditional ELISA techniques for fluorescence- based assays. In addition to improved efficiencies and compatibility with existing assay kits and instrumentation, researchers can choose to use the plate to conserve sample and reagents, or to greatly enhance assay sensitivity. Both choices are further enhanced by automating the Optimiser-based assay to increase throughput, reduce operator error, and improve overall assay efficiency.

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