Time Resolved Fluorescence

Fluorometric detection typically relies on the use of an antibody that has been labeled with a fluorophore. Once the antibody has bound to its target, a light source is used to excite the fluorophore, which then produces a transient light emission as it returns to its ground state. The light is emitted at a higher wavelength than that which was used for excitation and is detected with a specialized reader. Fluorometric detection is central to many applications, including, but not limited to, flow cytometry, ELISA, Western blotting, immunohistochemistry (IHC) and immunocytochemistry. Its popularity can be attributed to the high levels of sensitivity that it affords, in addition to the fact that it is quantitative and, with a wide range of fluorophores available, provides the opportunity for multiplexing.

What is Time Resolved Fluorescence?

Time Resolved Fluorescence (TRF) is very similar to standard fluorometric detection. The main difference between the two measurements is the timing of the excitation / emission process. During standard fluorometric detection, excitation and emission are simultaneous; the light emitted by the sample is measured while excitation is taking place. In contrast to this, TRF relies on the use of very specific fluorescent molecules, called lanthanide chelate labels, which allow detection of the emitted light to take place after excitation has occurred. The most commonly used lanthanide chelate label is the Europium ion (Eu3+).

Although conventional fluorophores are extremely popular, they share several limitations. Firstly, the simultaneous excitation / emission process can result in high background signal. Secondly, the Stokes shift (the difference between the maximum absorbance and emission wavelengths) of many commercially available fluorophores is relatively small, meaning that these reagents can suffer from self-quenching due to overlap between their absorption and emission spectra. Thirdly, biological matrices such as serum or tissue samples often contain autofluorescent substances; these can particularly be a source of background signal in homogeneous assays, where such components are not washed away prior to measurement. Finally, in High Throughput Screening formats, false positives can occur due to the fluorescent nature of certain chemical classes of test compounds.

Lanthanides therefore offer several key advantages:

  1. A large Stokes shift greatly increases the signal:background (S:B) ratio
  2. A sharp emission peak allows different lanthanides to be easily distinguished from one another, and contributes to improved S:B
  3. High fluorescence intensity significantly improves assay sensitivity
  4. A long fluorescence lifetime (µseconds–milliseconds), several orders of magnitude greater than any nonspecific background fluorescence (typically nanoseconds), and stable fluorescent signal, enables the fluorescent emission to be read at a time well after any background fluorescence has decayed, delivering a greater dynamic range.
Absorbance and emission spectra of Europium

Absorbance and emission spectra of Europium. Europium has a large Stokes shift, a wide excitation spectrum and a narrow emission spectrum, typical of lanthanide chelates.

Europium Conjugation Kit from Expedeon

Our Europium Conjugation Kit allows quick and easy conjugation of antibodies, proteins, peptides or any other biomolecule with primary amine groups to specially treated 200nm Europium chelate microspheres. This unique product may be used within a microplate based assay or in an immunochromatographic assay, and provides significantly higher sensitivity in comparison with other particle based assays.

The Europium Conjugation Kit labeling process

The Europium conjugation kit labeling process. The Europium particles are supplied freeze dried. The conjugation reaction is initiated by reconstituting the lyophilized mixture with the antibody, which then becomes covalently bound (via lysine residues) to the proprietary surface of the Europium particles.

In addition to enhanced sensitivity, our Europium particles offer several further advantages. The bond that is formed between the antibody and the surface of the particle is covalent, resulting in conjugates that are highly stable. The particles are also resistant to aggregation and require no harsh resuspension methods. Furthermore, the conjugation process takes just 35 minutes from start to finish and, unlike passive methods of conjugation to particles, does not involve extensive optimization at different pH values.

Microplate Based Detection Using Europium Particles

Microplate-based assay for evaluation of an antibody-Europium particle conjugate

Microplate based assay for evaluation of an antibody-Europium particle conjugate. The schematic represents the assay format: the wells of a microplate were coated with an antiCRP antibody, and solutions containing known concentrations of CRP were then added, and incubated. For detection, an antiCRP antibody conjugate prepared using Expedeon’s Europium Conjugation Kit was used. The plate was read using a microplate reader with TRF settings; the data demonstrates high sensitivity (~10pg / ml) and low background.

Europium-Streptavidin Conjugates

In addition to our Europium Conjugation Kits, we also offer Europium-Streptavidin conjugates. These are manufactured using covalent attachment of streptavidin to our specially treated 200nm Europium particles, and are ideal for use in TRF applications that exploit the streptavidin-biotin interaction.

Microplate-based assay for evaluation of a Europium-Streptavidin conjugate

Microplate based assay for evaluation of a Europium-Streptavidin conjugate. The wells of a microplate were coated with biotinylated ovalbumin, and varying dilutions of Expedeon’s Europium-Streptavidin conjugate were then added, and incubated. The plate was read using a microplate reader with TRF settings.

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