TR-FLISA Assay Principle
Fluorescence-Linked Immunosorbent Assay (FLISA) is a fluorescence-based immunoassay derived from the conventional ELISA format. It enables the detection and quantification of specific proteins, biomolecules, peptides, cytokines, hormones, antibodies, and other analytes in samples such as serum, plasma and cell culture supernatants.
Bright-Dtech™ technology combines high signal intensity, long-lived fluorescence, and excellent photostability to support sensitive and reliable fluorescence-based detection. Compared with conventional fluorescence detection, the use of lanthanide nanoparticles with time-resolved fluorescence helps reduce short-lived background fluorescence, supporting improved sensitivity and lower detection limits.
In Bright-Dtech™ TR-FLISA, antibodies or other detection molecules are coupled to fluorescent lanthanide nanoparticles. Following excitation, the long-lived fluorescence emitted by the nanoparticles is measured after a defined time delay using a compatible time-resolved fluorescence (TRF) microplate reader. This time-resolved detection helps reduce background fluorescence and supports sensitive quantitative analysis.
Required material : Black microplates, plate shaker, and compatible Time-Resolved Fluorescence (TRF) microplate reader.
Why use Bright-Dtech™ for FLISA applications?
Bright-Dtech™ technology provides several advantages for fluorescence-based immunoassays:
- High signal intensity: Bright-Dtech™ fluorescent lanthanide nanoparticles provide strong fluorescence signals, supporting sensitive detection.
- Low background fluorescence: Time-resolved detection reduces interference from short-lived background fluorescence.
- Long-lived fluorescence: Lanthanide emission occurs on the millisecond timescale, enabling time-resolved measurement.
- Excellent photostability: Bright-Dtech™ nanoparticles maintain stable fluorescence over extended periods and are resistant to photobleaching.
- Low LOD and LOQ: High signal intensity combined with low background supports improved analytical sensitivity.
- Reduced hands-on time: FLISA eliminates the need for enzymatic substrates and substrate development steps used in conventional ELISA.
Direct and Indirect FLISA
Direct and Indirect FLISA formats can be used to detect antibodies against an antigen immobilized on the surface of a microplate. In indirect FLISA, antibodies present in the sample bind to the immobilized antigen and are detected using a Bright-Dtech-conjugated secondary antibody specific to the host species of the primary antibody. In direct FLISA, the antibodies used for detection is directly conjugated to Bright-Dtech™ nanoparticles.
Example: Indirect FLISA for Anti-SARS-CoV-2 IgG Antibody Detection
Samples and standards are added to microplate wells coated with SARS-CoV-2 nucleoprotein. Anti-SARS-CoV-2 antibodies present in the samples bind to the immobilized antigen. After incubation and washing, a Bright-Dtech-conjugated secondary antibody is added to detect the bound antibodies. Following incubation and washing, the fluorescence signal is measured using a compatible TRF microplate reader. Within the assay range, the measured signal increases with the concentration of anti-SARS-CoV-2 IgG antibodies present in the sample, allowing quantitative analysis using a standard curve.
Bright-Dtech™ technology enables sensitive detection of anti-SARS-CoV-2 IgG antibodies through the high signal intensity and TRF of the fluorescent lanthanide nanoparticles.
Figure 1. Indirect FLISA for anti-SARS-CoV-2 IgG antibody detection.
Bright-Dtech™ technology supports sensitive detection of anti-SARS-CoV-2 nucleoprotein IgG antibodies through high signal intensity and TRF detection. The assay range was 20-100,000 pg/mL, with a reported limit of detection (LOD) of approximately 30 pg/mL, calculated using 3 standard deviations.
Sandwich FLISA
Sandwich FLISA is commonly used for the sensitive detection and quantification of specific antigens in biological samples. The assay uses a pair of antibodies recognizing different epitopes of the target analyte. The capture antibody is immobilized on the microplate and binds the target antigen present in the sample. A second detection antibody recognizes a different epitope of the captured antigen. The detection antibody can be directly conjugated to the Bright-Dtech™ nanoparticles or detected using a Bright-Dtech-conjugated secondary antibody, depending on the assay design. This format provides specific target detection while the high signal intensity and TRF of Bright-Dtech™ support sensitive quantitative analysis.
Example: Sandwich FLISA for SARS-CoV-2 Nucleoprotein Detection
This assay enables the detection and quantification of human SARS-CoV-2 nucleoprotein in samples such as serum, plasma, or cell culture supernatants. Samples and standards are added to microplate wells coated with anti-SARS-CoV-2 nucleoprotein capture antibody. The nucleoprotein present in the samples binds to the immobilized capture antibody. After incubation and washing, a Bright-Dtech-conjugated detection antibody recognizing a different epitope of the nucleoprotein is added to form the sandwich complex
Following incubation and washing, the fluorescent signal is measured using a compatible TRF microplate reader. The measured signal increases with the amount of SARS-CoV-2 nucleoprotein captured in the assay, allowing quantitative determination using a standard curve.
Bright-Dtech™ technology enables sensitive detection of SARS-CoV-2 nucleopritein through high signal intensity, low background, and TRF detection.
Figure 2. Sandwich FLISA for SARS-CoV-2 nucleoprotein detection.
Bright-Dtech™ technology supports sensitive detection of SARS-CoV-2 nucleoprotein through high signal intensity and TRF detection. The assay range was 20-20,000 pg/mL, with a reported limit of detection (LOD) of approximately 18 pg/mL, calculated using 3 standard deviations.
Our Link-Dtech™ kits and custom Bright-Dtech™ conjugation service enable the adaptation of matched antibody pairs developed for conventional sandwich ELISA formats, facilitating the adaptation of established immunoassays to fluorescence-based detection with Bright-Dtech™ nanoparticles.