High Throughput Screening: HTRF (Part II)

We previously spoke about AlphaLISA, a powerful assay technology with enhanced sensitivity, no wash steps, and scalability for high-throughput screening. This week, we’re focusing on HTRF (Homogeneous Time-Resolved Fluorescence) a versatile and robust technology widely utilized in the biotech industry for various applications including drug discovery, biomarker identification, and life sciences research.

How Does HTRF Work?

HTRF combines fluorescence resonance energy transfer (FRET) technology with time-resolved measurement (TR-FRET) to offer a powerful tool for detecting molecular interactions in a homogeneous (no-wash) format, which is highly beneficial for high-throughput screening and complex assay development.

HTRF assays rely on the energy transfer between a donor fluorophore and an acceptor fluorophore when they are in close proximity, typically within a few nanometers of each other. The donor molecule is excited by a light source, and if the acceptor molecule is close enough, energy is transferred from the donor to the acceptor. This transfer causes the acceptor to emit light at a longer wavelength. The time resolved aspect involves measuring the fluorescence signal after a short delay, allowing the short-lived background fluorescence to decay, which significantly improves the signal to noise ratio.

This diagram demonstrates the principle of HTRF, showing that excitation of the donor at 337 nm causes emission at 620 nm in the absence of interaction (A). When interaction occurs, FRET enables the acceptor to emit at 665 nm (B).

Components

• Donor Fluorophore: Typically, a europium or terbium cryptate complex, which has a long lived fluorescence emission, allowing for time resolved detection.
• Acceptor Fluorophore: Usually, a dye with an emission spectrum that overlaps with the emission spectrum of the donor.
• Capture and Detection Reagents: These can be antibodies, peptides, or other molecules that specifically bind to the target analyte; and are conjugated to the donor and acceptor fluorophores.
  
  

Advantages

• High Sensitivity and Specificity: The time resolved aspect reduces background fluorescence, which enhances the sensitivity and specificity of the assay.
• Homogenous Assay: HTRF does not require washing steps, which simplifies the assay process and minimizes the sample handling.
• Versatile and Adaptable: Can be used to study a wide variety of molecular interactions, including protein-protein, protein-peptide, protein-DNA/RNA, and receptor-ligand interactions.
• Suitable for High-Throughput Screening: The no wash, homogenous format makes HTRF ideal for high-throughput applications.

The graph above shows a representative standard curve. The HTRF has a dynamic range between 4-5 logs. This is greater than a traditional ELISA, allowing for additional samples to have data in the linear range of the assay.

Partnering for Success with HTRF

Like AlphaLISA, HTRF is a powerful and flexible assay technology that offers significant advantages for researchers. Its high sensitivity, specificity, and adaptability to high-throughput formats make it a valuable tool for a wide range of applications. Please contact us today to discuss your AlphaLISA and/or your HTRF needs. We would be happy to assist you.

Sources:

https://www.bmglabtech.com/en/blog/htrf/

https://www.revvity.com/category/htrf