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No-wash biomarker assay protocol with improved sensitivity and dynamic range, plus low variability
Sponsored,vendor-submitted protocol    Sponsored by PerkinElmer    Published in November 2009 (p.13) DOI: 10.2144/000113283

Abstract

The AlphaLISA® homogenous (i.e., no-wash) immunoassay platform employs a very simple assay protocol that is much faster than a traditional ELISA assay. When compared to competing TR-FRET homogeneous assays, AlphaLISA biomarker assays provide superior performance in terms of sensitivity, dynamic range, and variability. Because these biomarkers are often studied using cell-based assays, homogeneous (no-wash) assays could potentially be affected by the presence of serum or other components in the sample matrix. In our recent study of five analytes, assays were performed in serum or in culture medium with fetal bovine serum (FBS from 1% to 10%).

AlphaLISA assays use “donor” and “acceptor” beads coated with a layer of hydrogel providing functional groups for bioconjugation. Streptavidin-coated donor beads capture an analyte-specific biotinylated antibody. The acceptor beads are conjugated with a second antibody that recognizes a different epitope of the analyte. The streptavidin-coated donor beads are excited with a laser at 680 nm resulting in the release of singlet oxygen, which excites an amplified fluorescent signal in the acceptor bead, producing an emission of light at 615 nm, only if the beads are brought within 200 nm proximity by the capture of the analyte by both the antibodies. The amount of light emission is proportional to the amount of analyte present (Figure 1).

Materials

Figure 1. Schematic of the AlphaLISA assay.

EnVision® Multilabel Plate Reader (PerkinElmer)

AlphaLISA® IL-1β assay kit (PerkinElmer)

IL-1β TR-FRET assay kit (Competitor C)

OptiPlate® 384-well microplates (PerkinElmer)

Protocols

AlphaLISA assay protocol

test 1. Mix 5 L standard or sample with 10 L of anti-analyte acceptor beads and 10 L of biotinylated anti-analyte antibody (final volume = L).

2. Incubate at room temperature for 60 min.

3. Add 25 L of streptavidin-donor beads.

4. Incubate in subdued light at room temperature for 30 min.

5. Read the assay at room temperature with an Alpha-enabled EnVision or EnSpire® Multilabel Plate Reader.

The TR-FRET assays were performed as described by the assay supplier.

Data analysis

test The dynamic range and lower detection limit (LDL) for the IL-1β assay were determined by preparing a dilution series of the standards in DMEM-F12 media + 10% FBS. The signal obtained was plotted against the standard concentrations tested with each assay. The LDL was determined from the calibration curve using the calculation of background mean ± 3 × SD.

Results and discussion

test Please download the online protocol for complete assay details and full data sets for the five biomarkers studied. These included biomolecules involved in inflammation (TNFα, IL-1β, IL-6), metabolic disorders (insulin), and Alzheimer’s disease (amyloid bβ1-40).

The dynamic range and lower detection limit (LDL) for the IL-1β AlphaLISA and TR-FRET assays are shown in Figure 2, A and B, respectively, and in Table 1. The AlphaLISA assay for this analyte delivered >100-fold greater sensitivity and a significantly wider dynamic range (3.8 logs vs. 1.9 logs).

The intra-assay and inter-assay variability of the AlphaLISA and TR-FRET IL-1β biomarker assays are shown in Figure 3. The percentage of coefficient of variation (CV) was determined for sets of nine samples at a low, medium, and high analyte concentration for each assay. The AlphaLISA assay showed lower variability overall, with the most significant benefit being apparent at the lower analyte concentrations.

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