As an extension of fluorescence spectroscopy, time-resolved fluorescence assays are very robust (limited sensitivity to several types of assay interference) and are easily miniaturized. Methods based on enzyme labels have been developed for time resolved fluorescence immunoassays in Creative Biolabs. We offer a wide range of ELISA kits for specific detection of disease-related proteins. Our professional team is optimized to help you with high-quality and cost-effective testing service to make your project a success.

How does it work?

Time-resolved spectroscopy uses spectroscopic techniques to study dynamic processes in materials or chemical compounds. With the help of pulsed lasers, the time scale can be studied as short as 10-16 seconds. This technique can be applied to any process which causes changes in the properties of the material. Time-resolved fluorescence spectroscopy is an extension of fluorescence spectroscopy. Here, the fluorescence of the sample is monitored as a function of time after being excited by the flash. The time resolution can be obtained in various ways (depending on the required sensitivity and time resolution), including:

  • With fast-detection electronics (nanoseconds and slower)
  • With Time Correlated Single Photon Counting, TCSPC (picoseconds and slower)
  • With a streak camera (picoseconds and slower)
  • With intensified CCD (ICCD) cameras (down to 200 picoseconds and slower)
  • With optical gating (femtoseconds-nanoseconds)

Fig.1 The sensitivity of TRFIA anisotropy curves, r(t), was assessed to evaluate the conjugation state between the nanocarrier and the fluorescent drug (mimetic).Fig.1 Sensitivity of time-resolved fluorescence anisotropy curves r(t) for the conjugation state between nanocarrier and fluorescent drug (mimetic).1,4

Advantages of Our Time Resolved Fluorescence Immunoassay (TRFIA) Platforms

Creative Biolabs has been devoted for the development of highly sensitive, more flexible, quantitative, and easy-to-use time resolved fluorescence immunoassay-based kits. TRF relies on the use of very specific fluorescent molecules, the lanthanide. They have the unusual property of emitting over long periods of time (measured in milliseconds) after excitation when most standard fluorescent dyes (e.g. fluorescein) emit within a few nanoseconds of being excited. As a result, the lanthanide element can be excited using a pulsed light source (such as a xenon flash lamp or a pulsed laser) and measured after the excitation pulse. This leads to a lower measurement background than the standard FI assay. The disadvantage is that the instruments and reagents are generally more expensive and the application must be compatible with the use of these very specific lanthanide dyes.

Creative Biolabs is a world leader in the development of ELISA based kits and offers a wide range of assay kits for specific detection of disease-related proteins. If you are interested in our ELISA kits development, please feel free to contact us for more details.

Published Data

1. Dual-Label Time-Resolved Fluorescence Immunoassay for Simultaneous Detection

Fig.2 The dual-label TRFIA for the simultaneous detection of BLCA-4 and NMP52.Fig.2 Schematic representation of the dual-label TRFIA for BLCA-4 and MNP52 detection.2,4

This study aimed to develop a dual-label time-resolved fluoroimmunoassay (TRFIA) capable of simultaneously detecting BLCA-4 and NMP52 within a single run. The assay employed a sandwich immunoassay format, in which anti-BLCA-4 and anti-NMP52 antibodies were immobilized on microtiter wells to capture BLCA-4 and NMP52 from the urine sample. These targets were then bound by europium (III) Sm3+ and samarium (III) Eu3+-labeled antibodies, followed by fluorescence measurement using time-resolved fluorometry. The assay's performance was evaluated using clinical urine samples and compared with commercial kits. The sensitivity of this method demonstrated 2 U/mL for BLCA-4 and 1 μg/mL for NMP52. The dual-label TRFIA demonstrates high sensitivity, specificity, and accuracy, having good prospects for clinical applications.

2. A Novel Time-Resolved Fluoroimmunoassay for Quantitative Detection

Fig.3 A schematic of TRFIA for quantitative detection of anti-PLA2R antibodies in serum.Fig.3 A schematic representation of the anti-PLA2R-IgG-TRFIA.3,4

In this work, researchers established an ultrasensitive time-resolved fluoroimmunoassay (TRFIA) to measure anti-phospholipase A2 receptor (PLA2R) antibodies (anti-PLA2R-IgG) in serum for the membranous nephropathy differential diagnosis. Recombinant PLA2R (rPLA2R) was immobilized on 96-well plates for antigen capture, and a europium-chelate-conjugated goat anti-human IgG tracer was used for detection. After washing to separate bound/free components, fluorescence measurements quantified anti-PLA2R-IgG levels. A purified anti-PLA2R-IgG calibrator ensured consistent results. The assay had a detection limit of 0.03 mg/L and a linear range of 0.03–340 mg/L, with intra- and inter-assay coefficients of variation (CVs) of 3.8% and 6.2%, respectively. The method successfully detected anti-PLA2R-IgG concentrations in lupus nephropathy, IgA nephropathy, idiopathic membranous nephropathy patients and healthy volunteers. This quantification method enhances the diagnostic utility of serum anti-PLA2R-IgG for idiopathic membranous nephropathy.

References

  1. Boreham, Alexander, et al. "Time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy for characterization of dendritic polymer nanoparticles and applications in nanomedicine." Molecules 22.1 (2016): 17.
  2. Liu, X., et al. "Development of a Dual-Label Time-Resolved Fluorescence Immunoassay (TRFIA) for Screening of Bladder Cancer based on Simultaneous Detection of BLCA-4 and NMP52 in Urine." J Bioprocess Biotech 7.303 (2017): 2.
  3. Huang, Biao, et al. "A novel time-resolved fluoroimmunoassay for the quantitative detection of antibodies against the phospholipase A2 receptor." Scientific reports 7.1 (2017): 46096.
  4. Distributed under Open Access license CC BY 4.0, without modification.

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