Developments to Support Alphalisa Detection in Array Tape™
ELISA is widely used for screening and quantification of substances in biological samples. While advances have been made in delivery of partial automation for ELISA applications through the use of workstations, the multiple wash steps inherent to the traditional ELISA process complicate adaption to a fully integrated automation environment. The AlphaLISA®* chemistry available from PerkinElmer greatly simplifies the ELISA process by eliminating the repetitive wash steps. Although the standard protocol requires either a two or three step incubation process, additional work completed by Prasad et al suggests that a single incubation step will generate similar results for some assays2. The resulting streamlined process increases the opportunity for application and integration of fully automated, inline equipment platforms to achieve lower cost in high throughput screening applications.
In response to the high throughput screening needs of protein chemistry, the Array Tape Platform from Douglas Scientific presents an effective and efficient inline automation solution for laboratory processes. Well proven for SNP genotyping, the platform’s unique consumable, Array Tape, replaces microtiter plates with a flexible, continuous strip of embossed, low volume reaction wells (Figure 2).
In this article, results of AlphaLISA assays performed in Array Tape are described and compared with the published results from PerkinElmer1.
Materials and Methods
The AlphaLISA Process
The AlphaLISA chemistry is a bead-based assay. A biotinylated anti-analyte antibody binds to the streptavidin-coated donor beads while another anti-analyte antibody is conjugated to AlphaLISA acceptor beads. When exposed to an analyte of interest, the beads come into close proximity. Excitation of the donor beads then provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the associated acceptor beads resulting in a sharp peak of light emission at 615 nm1 (Figure 1).
Figure 1: Excitation of donor beads results in a sharp peak of light emission
A specialized Array Tape was designed to accommodate the requirements of the AlphaLISA assay (Figure 2). Array Tape is a continuous polypropylene strip, serially embossed with 384- well arrays. A 7.5 µL reaction well volume was used in the testing; however, reaction well volumes of 2-20 µL are possible.
Araya ® Detection System for Alpha Chemistry
Chemistry excitation and signal detection in the Araya are accomplished through the use of a specialized optical reader designed to capture and detect the signals associated with the smaller volumes.
Figure 2: Array Tape™ is a thin and flexible microplate replacement, serially embossed with reaction wells.
AlphaLISA-based insulin assays were performed using commercially available samples (Human Insulin Kit AL 204 C) using a series dilution ranging from 1 – 345,000 pg/mL. Assays were performed in Array Tape using a final reaction volume of 5.0 µL with three concentrations of both acceptor and beads. The 5 µL reaction volume was achieved by adding 0.5 µL of analyte from the dilution series, 2.0 µL of acceptor beads and 2.5 µL of donor beads. The 5 µL reaction volume represents a deviation from the 10 µL minimum volume recommended in the PerkinElmer technical data sheet1. Donor and acceptor beads were used in three concentrations, including PerkinElmer’s recommended 2.5X preparation1 (referred to as 1X in this paper) with additional dilutions of 0.6X and 0.3X of the recommended preparation. All other methods followed to published protocols1. After 90 minutes of incubation, signals were captured from assay mix using the Araya technology.
Results and Discussion
The signal acquired was plotted against the standard concentration (Figure 3). The LDL was calculated using the standard curve and background mean + 3 SD. The dynamic range and limit of detection was compared with published report of an Insulin AlphaLISA assay using a dilution series of insulin standards. The Array Tape, in combination with Araya, delivers similar results reported by PerkinElmer using a microplate system for limit of detection and dynamic range of the assay, including the lower limit of detection of 3 pg/mL1 (Figure 3) .The dynamic range (Figure 3) demonstrates the relationship between insulin concentration and AlphaLISA signal captured from the Array Tape containing assay mix. The dynamic range was equal to the reported results from the kit provider.
Figure 3: Insulin Dilution Curves at standard and reduced bead concentrations
ELISA is the most widely used detection platform for analysis and quantification of analytes in biological samples. Array Tape offers unique opportunity to perform the assay at a miniaturized volume. Data quality generated from miniaturized reactions and Araya detection technology closely matches the dynamic range of published results. Detection of signal from assays using reduced bead concentrations of both donor and acceptor beads was achieved with. Dynamic range and sensitivity of the reduced bead concentrations was similar and comparable with PerkinElmer’s reported results. These results clearly demonstrates that AlphaLISA assays can be performed with lower concentration of beads and miniaturized reaction volumes in Array Tape using Araya detection technology.
Combining a single incubation step2 with an optimized Array Tape Platform for homogeneous AlphaLISA featuring in-line, modular liquid handling provided by the Nexar system combined with inline incubation and Araya detection is credible and can be expected to provide walk-away automation for miniaturized, HTP AlphaLISA assays. Furthermore, published literature by PerkinElmer2, suggests that opportunities exist to shorten incubation times for selected assays, which allows for even greater opportunities in continuous systems such as Array Tape.
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