High Throughput Screening: AlphaLISA (Part I)

Following our recent discussion on ELISA we’re now highlighting AlphaLISA - a powerful alternative with enhanced sensitivity, no wash steps, and scalability for high-throughput screening.

What is AlphaLISA?

AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay-Linked Immunosorbent Assay) is a bead-based assay technology used for the detection and quantitation of biomolecules such as proteins, peptides, and small molecules; all in a highly sensitive, specific, and time efficient manner. This technique is particularly well suited for applications in the biotech industry including drug discovery, biomarker research, and life sciences due to its robustness, flexibility, and scalability.

How Does AlphaLISA Work?

AlphaLISA relies on the proximity-based interaction between donor and acceptor beads. When these beads are brought close together by a specific interaction between an analyte and the biomolecules conjugated to the beads, excitation of the donor beads by a laser or LED light sources causes the release of singlet oxygen molecules. These singlet oxygen molecules can diffuse a short distance, and if they reach the acceptor beads, trigger a chemiluminescent reaction that results in a bright fluorescent signal. The intensity of this signal is directly proportional to the concentration of the target analyte in the sample. Simply, AlphaLISA is a streamlined workflow that allows the detection and quantification of both simple and complex sample types.

A biotinylated antibody binds the streptavidin-coated donor beads, while a second antibody is conjugated to the AlphaLISA acceptor beads. In the presence of the analyte, the beads come together, and excitation at 680 nm generates singlet oxygen, triggering light emission at 615 nm.

Components

• Donor Beads: Contain a photosensitizer that upon excitation produces singlet oxygen.
• Acceptor Beads: Contain thioxene derivatives that emit light upon interaction with singlet oxygen.
• Capture and Detection Reagents: These are typically antibodies or other molecules with high specificity for the target analyte; and are conjugated to the donor and acceptor beads.
  
  

AlphaLISA Advantages

• High Sensitivity: The technology is capable of detecting very low levels of analytes, making it suitable for applications where the target molecule is present at low concentrations.
• Reduces Background Signal: The requirement for proximity between the donor and acceptor beads for signal generation minimizes background noise, leading to high signal to noise ratios.
• Flexibility and Versatility: AlphaLISA assays can be designed for a wide range of targets, including proteins, peptides, small molecules, and even cellular targets.
• No Wash Steps: Unlike traditional ELISA, AlphaLISA is a homogeneous assay that doesn’t require separation or wash steps, simplifying the workflow and reducing the total assay time.
• Scalability: The assay can be easily scaled from small to high throughout formats, accommodating the needs of both research and development and large-scale screening applications.

Standard Curve For IL-6

The graph above shows a representative standard curve for IL-6 generated using an AlphaLISA immunoassay. This data highlights the assay's sensitivity, with a lower detection limit (LDL) of 1.3 pg/mL, and its strong correlation (r² = 0.9948) in the linear range.

Partnering For Success with AlphaLISA

AlphaLISA is a powerful tool for the biotech industry, offering high sensitivity, specificity, and throughput capabilities for the quantitative analysis of biomolecules. Its adaptability across a range of applications from drug discovery to biomarker research makes it an invaluable asset in the biotech toolkit.

SBH Sciences can perform any of the dozens of developed AlphaLISA assays to support your research and development needs. Please contact SBH Sciences to discuss how our AlphaLISA expertise can accelerate your research.

Sources:

https://www.cisbio.eu/content/alphalisa-technology-basics/

https://www.revvity.com/