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Aug 1, 2012 (Vol. 32, No. 14)

High-Throughput AlphaLISA Analysis

Inline Automation Solution Demonstrates Improved Sensitivity and Reduced Assay Cost

  • Immunoassays have been widely adopted for analysis and quantification of low concentration proteins. The immunoassay is selective and sensitive; however, its utility has been limited in high-throughput systems due to a cumbersome process involving multiple incubation and wash steps.

    AlphaLISA® was specifically designed to overcome the challenges of traditional enzyme-linked immunosorbent assays (ELISA) and to provide opportunities for high-throughput automation. Although microplate-based automation has yielded some success, it failed to meet the requirement of continuous automation for high-throughput (HT) applications.

    The ideal system should provide: continuous automation, reproducible and rapid analysis of biological samples, easy setup and use, potential for broad deployment, and cost savings per data point. To address these needs, Douglas Scientific has developed modules that optimize the Array Tape™ platform for automated inline homogenous ELISA processing.

    Array Tape is ideal for high-throughput nonwash homogenous ELISA assays. Recently, PerkinElmer developed such assays, founded on an amplified luminescent proximity, or AlphaScreen® assay. The fundamental principle rests on the use of oxygen channeling chemistry for ELISA analysis, commonly known as AlphaLISA. Based on a published report from PerkinElmer, almost any standard sandwich ELISA can be converted to AlphaLISA, thus providing opportunity to deploy Array Tape for inline high-throughput automation.

    In the AlphaLISA assay, acceptor and donor beads are coated with hydrogel to provide a functional group for bioconjugation. Streptavidin-coated donor beads bind with a biotinylated anti-analyte antibody while another anti-analyte antibody is conjugated to the acceptor beads. In the presence of the analyte, the biomolecular reaction brings the beads into close proximity. The excitation of the donor beads provokes the release of singlet oxygen molecules that trigger a cascade of energy transfer in the acceptor beads, resulting in a sharp peak of light emission at 615 nm.

    In response to the high-throughput screening needs of protein chemistry, the Array Tape platform from Douglas Scientific presents a highly effective and efficient inline automation solution for laboratory processes. This paper describes performing AlphaLISA assays such as TNF-α, IgG, and insulin from PerkinElmer using Array Tape and detection system specially developed for AlphaLISA assays.

  • Material and Methods

    Click Image To Enlarge +
    Figure 1. Array Tape is a thin and flexible microplate replacement, serially embossed with reaction wells.

    Array Tape: A specialized Array Tape (Figure 1) was designed to perform AlphaLISA assays in low reaction volume. The Array Tape, thin and bendable, is spooled onto compact reels (90 mm wide by 560 mm in diameter) holding in excess of 200 microplate equivalents for assay processing. Indexing holes running along the edge of Array Tape guide the fully automated movement of the plastic strip through discrete, inline modules where individual arrays of reaction wells are independently processed.

  • Araya Detection

    Araya® Inline Detection: The system provides fully automated, HT signal detection with integrated reporting features. Customized Araya scanning instrumentation provides inline and stand-alone detection capabilities for diverse applications. The Araya engineered for Homogeneous ELISA detection supports PerkinElmer’s AlphaLISA and AlphaScreen technology with less than three-minute scan times per 384-well array.

    AlphaLISA Kit 
    Human Insulin (AL 204 C)
    Human Immunoglobulin G (AL 205 C)
    Human Tumor Necrosis Factor Alpha (AL 208 C)

  • Assay Protocol

    The AlphaLISA standard curve was performed in accordance with the kit protocol in a miniaturized reaction volume. Briefly, serial dilution of analytes were prepared in the AlphaLISA buffer. The assays were performed in 384-well Array Tape designed for the protein assays. Experimental runs were completed using 5 µL reaction volumes by adding 0.5 µL of analyte from the dilution series, 2.0 µL of acceptor beads, and 2.5 µL of donor beads. Additional assays were completed using bead dilutions of .6x and .3x. All other methods were identical to published methods.

  • Results and Discussions

    Click Image To Enlarge +
    Figure 2. Insulin dilution series (A), human immunoglobulin dilution series (B), and human TNF alpha dilution series (C): at standard and reduced bead concentrations.

    The AlphaLISA assay, human insulin, Immunoglobaline G, and human necrosis factor TNF-α were successfully performed in Array Tape. The signal count obtained from the Araya was plotted against the analyte concentration (Figure 2). The lower detection limit (LDL) was calculated by the mean of background plus three times Standard Deviation. The LDL obtained for human insulin, IgG, and TNF-α 3 pg/mL, 300 pg/mL, and 3 pg/mL, respectively, were equal to reported results in the kit insert.

    In addition, these results were compared with the published report of these kits for LDL and dynamic range. The results obtained from the experiments shows that the performance of the assays were very similar to the reported results.

  • Conclusion

    The Array Tape system is a proven automation platform ideally suited for AlphaLISA assays. The assay sensitivity was successfully achieved in the miniaturized volume in Array Tape. In this case, the sensitivity appears slightly better in small assay volume. Findings from the experiments clearly show the sensitivity of the assay has been achieved in Array Tape at reduced volumes, thus offering a significant reduction in the assay cost.

    The Array Tape platform has been successfully deployed to automate SNP genotyping. The modularity of the platform provides a unique opportunity to automate AlphaLISA processing. The Array Tape Platform can be configured as a turnkey operation to generate the highest quality data in a miniaturized volume without interference. Sample in, data out.



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