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

Amplifying Immunoassay Sensitivity

Microfluidics-Based Microplate Technology Offers Benefits Over Traditional ELISA Methods

  • Click Image To Enlarge +
    Figure 1. Microfluidics microplate showing enlarged view of a microchannel associated with its microplate well

    Enzyme-linked immunosorbent assays (ELISA) are commonly used in research and clinical applications to quantify analytes from complex matrices such as serum and plasma/cell lysates. Excellent selectivity is provided by the use of a high affinity capture antibody and multiple wash steps. Good sensitivity is achieved through direct labeling of an antibody to a different epitope on the analyte or the use of a labeled secondary antibody. The basic ELISA workflow and sensitivity have not changed significantly in decades.

    Siloam’s Optimiser microplate offers improved ELISA workflows, sample and reagent conservation, improved reaction kinetics, and the ability to fine-tune assay sensitivity through multiple analyte loading in fluorescent-based assays. The Optimiser uses an SBS-configured 96-well microplate architecture and a dedicated microfluidic channel immediately below each well (Figure 1) where binding occurs.

    Each 200 x 200 μm spiral microchannel offers approximately 50 times larger surface area-to-volume ratio for capture of antibodies compared to a standard ELISA microplate, which relies on the microplate well bottom for capture. This allows for more efficient capture antibody binding and highly efficient antibody-antigen reactions, which decrease overall assay time.

  • Principle of Operation

    Click Image To Enlarge +
    Figure 2. Key steps in the Optimiser workflow

    The Optimiser workflow (Figure 2) mirrors standard ELISA assay steps, however, the volumes used are significantly smaller, and washing steps are reduced.

    Sample/reagent volumes as small as 5–10 μL are added to each well and drawn through the microchannel via capillary forces. Each microchannel, where binding occurs, holds 4.5 μL of liquid, and excess liquid is channeled to an absorbent pad under the microplate.

    The sequence of reagent/sample additions is demonstrated in Figure 2. For each successive addition, the capillary barrier is broken at the microchannel inlet, and all previous reagents are flushed into the absorbent pad. Flushing excess liquid effectively removes unbound materials and also prevents cross-contamination of reagents within the microchannel. By making multiple additions of sample containing analyte, sensitivity can be tuned up to 100-fold higher than single additions of analyte.

    Automating the process further enhances ELISA assay efficiency and increases throughput. An automated pipetting station, such as BioTek’s Precision™ Microplate Pipetting System, automatically loads analytes and reagents into the microfluidics microplate, and the multistation platform allows operation of multiple Optimiser microplates. Automation increases pipetting precision compared to manual methods, especially at the low volumes used in the Optimiser workflow, and also allows the user to attend to other tasks while the instrument is in operation.

    As the Optimiser plate conforms to SBS recommended microplate specifications, it can be read in any standard fluorescence microplate reader.

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